MECHANISMS OF GNAO1-ASSOCIATED NEUROLOGICAL DISORDERS
By
Huijie Feng
A DISSERTATION
Michigan State University
in partial fulfillment of the requirements
Submitted to
for the degree of
Pharmacology and Toxicology—Doctor of Philosophy
2019
ABSTRACT
MECHANISMS OF GNAO1-ASSOCIATED NEUROLOGICAL DISORDERS
By
Huijie Feng
Tremendous advances in the genetics of neurodevelopmental disorders have
markedly improved the understanding of disease mechanisms. This project will focus on
understanding the mechanisms of GNAO1 encephalopathies, a devastating but complex
disorder, which exhibit multiple neurological symptoms. These include developmental
delay and variable components of early onset epilepsy and/or hyperkinetic movement
disorders (MDs). These symptoms are associated with mutations in the GNAO1 gene,
which encodes the Gαo protein. GNAO1 mutation-associated neurological disorders
include neurodevelopmental delay with involuntary movements (NEDIM, OMIM#617493)
and early infantile epileptic encephalopathy (EIEE17, OMIM#615473). The number of
identified patients and mutant alleles for EIEE17 or NEDIM is increasing rapidly.
Gαo is the most abundant membrane protein in the mammalian central nervous
system. It couples to multiple G protein-coupled receptors (GPCRs) including GABAB, α2
adrenergic, D2 dopamine, and adenosine A1 receptors; all are associated with both MDs
and epilepsy. In addition, GPCRs are readily targetable by agonists and antagonists.
This provides the possibility of treating GNAO1-associated neurological disorders.
This project revealed a fundamental mechanistic distinction among these GNAO1
mutations: Loss-of-function (LOF) GNAO1 alleles are associated with epilepsy, while
gain-of-function (GOF) GNAO1 alleles are associated primarily with MDs. However, this
simple model is insufficient to explain all clinical observations. To explore this correlation,
we have created mouse models carrying two of the most common human GNAO1
mutant alleles (G203R and R209H). They largely share the human pathophysiology; the
G203R mouse model exhibits both MD and enhanced seizure propensity, while the
R209H mutant results in MD alone, as seen in children with those mutations. Using
these models, we can further explore mechanisms that lead to distinct patterns in human
GNAO1 encephalopathies. To explore electrophysiological alterations in the Gnao1
G203R mutant mouse model, I performed patch clamp studies on cerebellar Purkinje
cells. The results show a decreased frequency of both miniature inhibitory postsynaptic
currents (mIPSCs) and spontaneous
inhibitory postsynaptic currents (sIPSCs),
suggesting a reduced presynaptic GABA release.
This study provides a molecular and physiological understanding of different GNAO1
alleles in vitro, and identifies key candidate alleles for further analysis in in vivo mouse
models and in human GNAO1-associated neurological disorders. Furthermore, our study
may serve as a prototype for other correlations between reported monogenic mutations
and human neurological disorders.
ACKNOWLEDGEMENTS
I would like to thank my mentor Dr. Richard R. Neubig. I consider myself fortunate to
have had this opportunity to work with him. Through Dr. Neubig’s mentorship I have
become not just a better scientist but also a better writer and presenter. I appreciate
Rick’s constant attention to details and his enthusiasm in pushing my project forward. His
suggestions have been invaluable to the results presented in this dissertation. I also
would like to thank him for his encouragement and trust for me to freely explore and
decide what I deem the best in science and in my career development. This helps me to
become an independent thinker and researcher.
I also highly value the mentorship of Dr. Yukun Yuan, who has unique and extensive
experience and knowledge in doing patch clamp recordings. I deeply appreciate his
generous sharing of his research equipment and his help in troubleshooting failed
experiments. My other committee members have also played an important role in
guiding the development of the work presented in this dissertation. I would like to thank
Dr. Colleen Hegg, Dr. James Galligan and Dr. Charles Lee Cox for their insights and
suggestions during every committee meeting I had. Their questions and perspectives
have helped me assess my experimental results from alternative viewpoints and have
enhanced the work presented here. I would also like to thank any clinical knowledge and
detailed explanations regarding movement disorders and epilepsy given by Dr. Christos
Sidiropoulos and Dr. Florian Kagerer.
iv !
Within the Neubig lab, I would like to thank Jeff Leipprandt who managed with all my
orders and animal subjects. In addition, I would also like to thank former research
associate Dr. Benita Sjögren, who patiently helped me through my first year in the lab
and whose detailed lab records provided me invaluable resources when in need. I am
thankful for our Masters student Cassie Larrivee, who helped with a majority of
behavioral experiments. I also would like to express my gratitude to the current members
of the laboratory. Dr. Erika Lisabeth, Behirda Karaj, Dr. Vincent Shaw, Sean Misek,
Yajing Ji, and Zhe Zhang have been great friends and colleagues during my time in the
lab. I also value the assistance of our undergraduate student, Nils Wellhausen, who has
been important to my ability to conduct this research.
I would like to thank the MSU Transgenic Core headed by Dr. Elena Demireva and
Dr. Huirong Xie. Their work provided me with animal subjects to work with. I also would
like to thank Dr. Thomas Dexheimer from the Assay Development and Drug
Repurposing Core, who gave me valuable suggestions for my experiments.
I would like to thank the Department of Pharmacology and Toxicology. Dr. Anne
Dorrance offered mentorship and support during my time as a graduate student at MSU.
In addition, I would like to thank the department office staff for their patience and
assistance with administrative issues during my completion of the department’s
academic requirements.
The research presented in this dissertation would not have been possible without the
v !!
funding received from numerous sources. I would like to express my thanks to the Bow
Foundation, whose grants supported my research, and to American Epilepsy Society
(AES), for the pre-doctoral fellowship that supplied my stipend for a year. I would also
like to thank the Neuroscience Program for providing me with a teaching assistantship
and the Department of Pharmacology & Toxicology for a dissertation completion
fellowship.
I have also received
financial support
from
the AES, Society of
Neurosciences (SfN) and MSU Council of Graduate Students to attend scientific
meetings.
A heartfelt thanks also goes to all the GNAO1 patients and families. Their support is
a big driving force of the advancement of this project. I sincerely hope this project will
benefit the development of new therapeutics for treating the GNAO1-encephalopathies.
I reserve special thanks for my friends and family. I owe them a great deal of credit
for my success.
vi !
TABLE OF CONTENTS
LIST OF TABLES ............................................................................................................ xi
LIST OF FIGURES ........................................................................................................ xiii
KEY TO ABBREVIATIONS .......................................................................................... xviii
CHAPTER 1: AN OVERVIEW OF GNAO1-ASSOCIATED NEUROLOGICAL
DISORDERS .................................................................................................................... 1
1.1 Abstract ................................................................................................................... 2
1.2 Introduction ............................................................................................................. 2
1.3 Gαo (GNAO1) mechanisms ..................................................................................... 5
1.4 The clinical spectrum of GNAO1 mutation-associated movement disorders .......... 7
1.4.1 GNAO1 encephalopathy displays a variety of neurological symptoms ............ 7
1.4.2 Movement disorders related to GNAO1 encephalopathy show limited
response to pharmacological treatments ................................................................... 9
1.5 Potential pathogenic mechanisms of GNAO1-associated movement disorders ... 13
1.5.1 Role of cAMP regulation in movement disorders ........................................... 14
1.5.1.1 GNAL ....................................................................................................... 17
1.5.1.2 GNB1 ....................................................................................................... 18
1.5.1.3 ADCY5 ..................................................................................................... 19
1.5.1.4 PDE10A ................................................................................................... 21
1.5.1.5 Summary of cAMP regulation in movement control ................................. 22
1.5.2 Role of neurotransmitter release and synaptic vesicle fusion in movement
disorders .................................................................................................................. 23
1.5.2.1 SYT1 ........................................................................................................ 25
1.5.2.2 PRRT2 ..................................................................................................... 26
1.5.2.3 SNAP25 ................................................................................................... 27
1.5.2.4 KCNMA1 .................................................................................................. 28
1.5.2.5 CACNA1A ................................................................................................ 29
1.5.2.6 CACNA1B ................................................................................................ 30
1.5.2.7 GPR88 ..................................................................................................... 31
1.5.2.8 Summary of neurotransmitter release and synaptic vesicle fusion in
movement control ................................................................................................ 32
1.6 Developmental defects may also contribute to movement disorders .................... 32
1.7 Conclusion ............................................................................................................ 34
1.8 Organization of the thesis ..................................................................................... 35
APPENDIX .................................................................................................................. 37
REFERENCES ............................................................................................................ 46
vii !
CHAPTER 2: MOVEMENT DISORDER IN GNAO1 ENCEPHALOPATHY
ASSOCIATED WITH GAIN-OF-FUNCTION MUTATIONS ............................................ 66
2.1 Abstract ................................................................................................................. 67
2.2 Introduction ........................................................................................................... 68
2.3 Materials and Methods .......................................................................................... 70
2.3.1 Materials ......................................................................................................... 70
2.3.2 DNA constructs and mutagenesis ................................................................ 70
2.3.3 Cell culture and transfections ....................................................................... 71
2.3.4 SDS-PAGE and Western blot ....................................................................... 72
2.3.5 cAMP measurements .................................................................................. 73
2.3.6 Data analysis and statistics ......................................................................... 73
2.4 Results .................................................................................................................. 74
2.4.1 Most pathogenic GNAO1 mutations cause reduced Gαo protein expression
................................................................................................................................. 74
2.4.2 Validation of an in vitro assay to assess function of GNAO1 mutations ......... 76
2.4.3 Nine GNAO1 mutations result in loss- (LOF) or partial loss-of-function (PLOF)
................................................................................................................................. 77
2.4.4 Six GNAO1 mutations result in gain-of-function (GOF) or normal function (NF)
................................................................................................................................. 80
2.4.5 Clinical correlation with biochemical behavior of mutant GNAO1 alleles ....... 86
2.4.6 Location of mutations linked to GNAO1 encephalopathies in the Gαo protein
................................................................................................................................. 87
2.5 Discussion ............................................................................................................. 88
APPENDICES ............................................................................................................. 93
APPENDIX A: SUPPLEMENTAL DATA .................................................................. 94
APPENDIX B: VALIDATION OF THE GENOTYPE-PHENOTYPE CORRELATION
OF GNAO1-ASSOCIATED NEUROLOGICAL DISORDERS ................................ 103
REFERENCES .......................................................................................................... 112
CHAPTER 3: BEHAVIORAL ASSESSMENT OF MOUSE MODELS WITH
GNAO1-ASSOCIATED MOVEMENT DISORDER AND EPILEPSY ........................... 119
3.1 Abstract ............................................................................................................... 120
3.2 Introduction ......................................................................................................... 121
3.3 Materials and Methods ........................................................................................ 126
3.3.1 Animals ......................................................................................................... 126
3.3.2 Generation of Gnao1 mutant mice ............................................................... 126
3.3.2.1 Generation of Gnao1+/G203R mouse model ............................................. 126
3.3.2.2 Generation of Gnao1+/R209H mouse model ............................................. 130
3.3.3 Genotyping and Breeding ............................................................................. 132
3.3.3.1 Genotyping Gnao1+/G203R mice .............................................................. 132
3.3.3.2 Genotyping Gnao1+/R209H mice ............................................................... 133
3.3.4 Behavioral Studies ........................................................................................ 134
viii !
3.3.4.1 Open Field ............................................................................................. 134
3.3.4.2 RotaRod ................................................................................................. 135
3.3.4.3 Grip Strength .......................................................................................... 135
3.3.4.4 DigiGait .................................................................................................. 136
3.3.5 PTZ Kindling Susceptibility ........................................................................... 137
3.3.6 Data Analysis ................................................................................................ 137
3.4 Results ................................................................................................................ 138
3.4.1 The growth patterns of the three newly developed Gnao1 mutant mouse
models (G203R, R209H and ΔT191F197) ............................................................ 138
3.4.1.1 Gnao1+/G203R mice showed normal viability and growth. ........................ 138
3.4.1.2 Gnao1+/R209H mice have expected frequency and normal viability ......... 138
3.4.1.3 Gnao1+/ΔT191F197 mice developed spontaneous seizures at P7 and died
before P16 ......................................................................................................... 139
3.4.2 Behavioral assessment of mutant Gnao1 mouse models (G184S, G203R,
R209H and KO) for movement patterns ................................................................ 141
3.4.2.1 Female Gnao1+/G184S and male Gnao1+/G203R mice show similar movement
abnormalities and gait disturbances .................................................................. 141
3.4.2.2 Gnao1+/R209H mouse model exhibits unique hyperactive behavior in the
open field arena but no abnormal motor coordination in other behavior tests and
minor disturbance in gait analysis ...................................................................... 149
3.4.2.3 Previously described Gnao1+/- mouse model did not show any
abnormalities in the behavioral test battery ....................................................... 152
3.4.3 PTZ kindling study of G203R and R209H mice. ........................................... 155
3.4.3.1 Male Gnao1+/G203R mice are sensitized to PTZ kindling. ........................ 155
3.4.3.2 R209H mutant mice are not hypersensitive to PTZ kindling .................. 157
3.5 Discussion ........................................................................................................... 158
APPENDIX ................................................................................................................ 165
REFERENCES .......................................................................................................... 183
CHAPTER 4: MICE WITH GNAO1-ASSOCIATED MOVEMENT DISORDER EXHIBIT
REDUCED INHIBITORY SYNAPTIC INPUT TO CEREBELLAR PURKINJE CELLS ......
...................................................................................................................................... 193
4.1 Abstract ............................................................................................................... 194
4.2 Introduction ......................................................................................................... 195
4.3 Materials and Methods. ....................................................................................... 198
4.3.1 Tissue preparation and solutions .............................................................. 198
4.3.2 Electrophysiological recording .................................................................. 199
4.3.3 Pharmacology ........................................................................................... 201
4.3.4 SDS Page and Western Blots ................................................................... 201
4.3.5 Statistical Analysis .................................................................................... 203
4.4 Results ................................................................................................................ 204
4.4.1 Presynaptic GABA release is suppressed in the cerebellar Purkinje cells of
ix !
Gnao1+/G203R mice .............................................................................................. 205
4.4.2 Gαo blockers can reverse the enhanced inhibition of mIPSC frequency in
G203R mice ....................................................................................................... 207
4.4.3 Presynaptic glutamate release is not affected by the G203R mutation in Gαo
protein ................................................................................................................ 211
4.4.4 Effects of G-protein coupled GABAB receptors on AP-independent GABA
release onto Purkinje cells ................................................................................. 213
4.4.5 Effects of G-protein coupled α2A adrenergic receptors on AP-dependent
GABA release onto Purkinje cells ...................................................................... 215
4.4.6 α2A adrenergic receptor-induced inhibition of sIPSC frequency depends on
activation of voltage-gated calcium channels .................................................... 217
4.4.7 G203R mice exhibit decreased Gαo protein expression but no change in Gβ
levels .................................................................................................................. 221
4.5 Discussion ........................................................................................................... 223
APPENDIX ................................................................................................................ 230
REFERENCES .......................................................................................................... 236
CHAPTER 5: CONCLUSION AND FUTURE DIRECTIONS ....................................... 244
5.1 General conclusion ............................................................................................. 243
5.2 Testing the functional changes of a growing variety of GNAO1 mutations ......... 248
5.2.1 Do GNAO1 mutations affect Go’s inhibition of high-voltage gated calcium
channels (N- type & P/Q- type calcium channels)? ............................................... 248
5.2.2 Do GNAO1 mutations affect Go’s activation of G protein-regulated inward
rectifying potassium (GIRK) channels? ................................................................. 251
5.2.3 How do GNAO1 mutations affect G protein-regulated neurite outgrowth? .. 252
5.3 Comparison between R209H and G203R mouse models .................................. 254
5.3.1 Do G203R and R209H mouse models exhibit delayed development? ........ 255
5.3.2 How does the G203R mutation in Gαo lead to epileptogenesis? ................. 258
5.3.3 How do G203R and R209H mice differ in movement disorder phenotypes?
............................................................................................................................... 260
5.3.4 How is sex involved in abnormalities of the Gnao1 mutant mice? ............... 262
5.4 Development of a high-throughput assay
for drug repurposing or drug
development .............................................................................................................. 267
5.5 Impact of work in this thesis on the field ............................................................. 270
APPENDIX ................................................................................................................ 272
REFERENCES .......................................................................................................... 281
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LIST OF TABLES
Table 1.1 Most Common GNAO1 Mutant Alleles Associated With Movement Disorders 8
Table 1.2 Drugs showing beneficial effects to control involuntary movements of GNAO1
encephalopathy patients ................................................................................................... 9
Table 1.3 Patients responding positively to deep brain stimulation (DBS) ..................... 12
Table S1.1 A complete summary of clinical information regarding GNAO1 patients ..... 38
Table 2.1 Functional data for loss-of-function (LOF) and partial loss-of-function (PLOF)
mutants. .......................................................................................................................... 79
Table 2.2 Functional data for normal and gain-of-function (GOF) mutants. ................... 82
Table 2.3 Correlation between cAMP inhibition and clinical diagnosis. .......................... 83
Table S2.1 Primer sequences for mutagenesis to create GNAO1 mutants. .................. 95
Table S2.2 All non-functioning GNAO1 mutations tested with GNAS KO cells ............ 110
Table S2.3 All functioning GNAO1 mutations tested with GNAS KO cells ................... 110
Table S2.4 Genotype-phenotype correlation of the newly reported GNAO1 mutations
...................................................................................................................................... 111
Table 3.1 The status of Gnao1 mutant mice ................................................................. 125
Table 3.2 Location, sequence and genotyping of gRNA targets in the Gnao1 locus. .. 128
Table 3.3 Location and sequence of gRNA and ssODN template for CRISPR-Cas
targeting Gnao1 locus; primers and genotyping method for Gnao1+/R209H mice ........... 131
Table 3.4 Phenotypes of Gnao1 mutant mice .............................................................. 160
xi !
Table S3.1 Gait analysis parameters of male Gnao1 G203R mutant mice ................ 173
Table S3.2 Gait analysis parameters of female Gnao1 G203R mutant mice ............. 174
Table S3.3 Gait analysis parameters of male Gnao1 G184S mutant mice ................ 175
Table S3.4 Gait analysis parameters of female Gnao1 G184S mutant mice ............. 176
Table S3.5 Gait analysis parameters of male Gnao1 R209H mutant mice .................. 177
Table S3.6 Gait analysis parameters of female Gnao1 R209H mutant mice ............. 178
Table S3.7 Gait analysis parameters of male Gnao1 KO mutant mice ........................ 179
Table S3.8 Gait analysis parameters of female Gnao1 KO mutant mice ................... 180
Table S3.9 Benchling off-target list for Gnao1 G203R gRNA ..................................... 181
Table 5.1 Comparison of clinical patterns of G203R and R209H patients ................. 254
xii !
LIST OF FIGURES
Figure 1.1 Summary of reported cases of GNAO1 encephalopathy ................................ 5
Figure 1.2 Genes regulating the cAMP pathway are related to movement disorders .... 16
Figure 1.3 Pathogenic mutations in genes that regulate neurotransmitter release ........ 25
Figure S1.1 Correlation between seizure frequency and a severe EEG/MRI result ....... 44
Figure 2.1 Location and protein expression levels of human GNAO1 mutations related to
epileptic encephalopathy. ............................................................................................... 75
Figure 2.2 Effect on α2A AR-mediated cAMP inhibition by GNAO1 mutants. ................ 78
Figure S2.1 Alignment of the human and mouse Gαo protein sequences. ..................... 97
Figure S2.2 Validation of the Lance Ultra cAMP assay with transient transfection of α2A
adrenergic receptor (α2A AR) and Pertussis toxin (PTX)-insensitive Gαo ....................... 98
Figure S2.3 Assessment of dominant-negative effect of complete LOF mutants G40R,
N270H, D174G, T191_F197del, L199P, and F275S. ..................................................... 99
Figure S2.4 Paired t-test analysis of LogEC50 values for normal and GOF mutants .... 101
Figure S2.5 Mapping of mutations on the structure of Gαo-GDP bound to RGS16 ...... 102
Figure S2.6 Comparison of validation of the Lance Ultra cAMP assay between HEK293T
and GNAS KO HEK293 cells with transient transfection of α2A adrenergic receptor (α2AR)
and Pertussis toxin (PTX)-insensitive Gαo .................................................................... 107
Figure S2.7 GNAO1 mutations’ functionalities correlate to their protein expression
patterns. ........................................................................................................................ 108
Figure S2.8 GNAO1 mutations’ functionalities correlate to their protein expression
patterns (assays done by Nils Wellhausen with a different group of mutations from those
xiii !
in Figure S2.7). ............................................................................................................. 109
Figure 3.1 Development of Gnao1+/G203R mouse model. .............................................. 127
Figure 3.2 Targeting of the mouse Gnao1 locus. ......................................................... 131
Figure 3.3 The timeline for utilizing animals in this study. ............................................ 134
Figure 3.4 Gnao1+/ΔT191F197 mice developed spontaneous seizures at P7 and died before
P16. .............................................................................................................................. 140
Figure 3.5 Female Gnao1+/G184S mice and male Gnao1+/G203R mice show reduced time on
RotaRod and reduced grip strength. ............................................................................. 143
Figure 3.6 G184S mutant mice showed reduced activities in Open Field Test but G203R
mutants do not. ............................................................................................................. 145
Figure 3.7 DigiGait Imaging System reveals sex-specific gait abnormalities in
Gnao1+/G184S mice and Gnao1+/G203R mice. ................................................................... 148
Figure 3.8 Gnao1+/R209H mice show significant hyperactivity and reduced time in center in
the open field arena. ................................................................................................... 150
Figure 3.9. Male and female Gnao1+/R209H mice shows gait abnormalities in different
tests on the DigiGait imaging system. .......................................................................... 151
Figure 3.10 Male and female Gnao1+/- mice do not show any abnormalities in the
behavioral tests including open field, Rotarod, and grip strength. ................................ 154
Figure 3.11 DigiGait Imaging System reveals the decreased stride length in male
Gnao1+/- mice. .............................................................................................................. 155
Figure 3.12 Gnao1+/G203R male mice have an enhanced Pentylenetetrazol (PTZ) Kindling
response. .................................................................................................................... 157
Figure 3.13 Gnao1+/R209H mice do not have an enhanced pentylenetetrazol (PTZ)
kindling response .......................................................................................................... 158
xiv !
Figure S3.1 RotaRod test was conducted with 5 training sessions and 1 test session
over two consecutive days. ........................................................................................... 166
Figure S3.2 Time spent at the center in the Open Field Test. ...................................... 167
Figure S3.3 RotaRod learning curve was collected in 10 consecutive tests with a 5-min
break between each test. ............................................................................................ 168
Figure S3.4 False discovery rate (FDR) calculation probed of significantly different
parameters from the DigiGait data in Gnao1+/G184S mice. ............................................. 169
Figure S3.5 False discovery rate (FDR) calculation probed of significantly different
parameters from the DigiGait data in Gnao1+/G203R mice. ............................................. 170
Figure S3.6 False discovery rate (FDR) calculation probed of significantly different
parameters from the DigiGait data in Gnao1+/R209H mice. ........................................... 171
Figure S3.7 False discovery rate (FDR) calculation probed of significantly different
parameters from the DigiGait data in Gnao1+/- mice. ................................................... 172
Figure 4.1 Cerebellar Purkinje cells in brain slices from 4-6 week-old G203R mice
display reduced GABAergic spontaneous synaptic currents (sIPSCs) and reduced
miniature synaptic currents (mIPSCs). ......................................................................... 206
Figure 4.2 The frequencies of mIPSCs were sensitive to NEM, an inhibitor of Gαi/o
proteins. ........................................................................................................................ 209
Figure 4.3 A selective inhibitor of Gi/o, pertussis toxin (PTX), increased the frequency of
mIPSCs in G203R but not WT mice. ............................................................................ 210
Figure 4.4 G203R mutant slices show no difference in either spontaneous excitatory
postsynaptic currents (sEPSCs) or miniature excitatory postsynaptic currents (mIPSCs).
...................................................................................................................................... 212
Figure 4.5 Activating GABAB receptor with baclofen reduces mIPSC frequency but not
amplitude. PTX incubation eliminates baclofen-induced inhibition of mIPSC frequency in
WT and G203R mice. ................................................................................................... 214
Figure 4.6 The frequency of sIPSCs is modulated by α2AR receptors. ......................... 216
xv !
Figure 4.7 Cadmium-block of extracellular calcium influx suppresses the frequency of
sIPSCs in both WT and G203R mice. .......................................................................... 219
Figure 4.8 Cadmium-block of extracellular calcium influx does not affect the frequency
and the amplitude of mIPSCs in both WT and G203R mice. ....................................... 220
Figure 4.9 G203R mice showed a significant decrease in Gαo protein expression. ..... 222
Figure 4.10 G203R mice did not show any significant changes in Gβ expression in the
brain. ............................................................................................................................. 223
Figure 4.11 Models of GABABR and α2AR mediated inhibition of GABA release. ........ 225
Figure 4.12 Gβγ may play a major role in the regulation of IPSCs. .............................. 227
Figure 4.13 GNAO1 mutations may have region specific effects, which cause an
imbalance between excitatory and inhibitory neurotransmitters. .................................. 229
Figure S4.1 Despite the hypothesis that α2A receptor antagonist yohimbine (10µM) could
reverse the inhibition of sIPSC frequency induced by UK14, 304, the application of
yohimbine further reduced the sIPSC frequency with the application of UK14, 304. ... 231
Figure S4.2 Baclofen does not affect either frequency or amplitude of sIPSCs, and
UK14,304 does not affect mIPSC frequency or amplitude. .......................................... 232
Figure S4.3 Heterozygous G184S (GOF) mice also showed a low Gαo protein
expression level. ........................................................................................................... 233
Figure S4.4 Female Gnao1+/G184S mice showed reduced sIPSC frequency in
hippocampal pyramidal cells, cortical layer II/IV pyramidal cells but not in cerebellar
Purkinje cells. ................................................................................................................ 234
Figure S4.5 Brain Gαo expression does not change in R209H mice’s brain lysates. ... 235
Figure S5.1 α2AR activates Gαo, which inhibits N-type calcium channels in G1A1 cells.
GOF mutation G184S enhance the inhibition of calcium currents. ............................... 273
Figure S5.2 Both G1A1 and SH-SY5Y cells are good candidates to study mutant Gαo’s
xvi !
effects on N-type calcium channels with Fluo-4-NW dye in a Hamamatsu µCELL plate
reader. .......................................................................................................................... 274
Figure S5.3 Neurite outgrowth in rat pheochromocytoma cells (PC12) can be induced by
50 ng/mL Nerve Growth Factor (NGF) in normal growth medium with reduced serum.
...................................................................................................................................... 275
Figure S5.4 Neurite outgrowth can be induced by 10 µM retinoid acid (RA) in normal
growth medium with reduced serum (30% FBS) in human neuroblastoma cells
(SH-SY5Y). ................................................................................................................... 276
Figure S5.5 Representative traces of modulations in calcium oscillations with different
ion concentration in mixed cortical cultures from a WT mouse. ................................... 277
Figure S5.6 High-throughput assessment of neural excitability in cortical cultures. ..... 278
Figure S5.7 Nissl staining compares gross morphological changes in cerebellum from
Gnao1+/G184S and Gnao1+/+ mice at age 8 weeks old. .................................................. 279
xvii !
KEY TO ABBREVIATIONS
Adenylyl cyclases
Artificial cerebral spinal fluid
Attention Deficit Hyperactivity Disorder
AC
ACSF
ADHD
ANOVA
Analysis of variance
AP
APV
BRET
DMEM
CNQX
DOR
CREB
CRISPR
EA2
EIEE17
FBS
FDR
FFT
Action potential
2-Amino-5-phosphonopentanoic acid
Bioluminescence resonance energy transfer
Dulbecco’s modified Eagle’s medium
6-Cyano-7-nitroquinoxaline-2,3-dione
Delta opioid receptor
cAMP-response element binding protein
Clustered regularly interspaced short palindromic repeats
Episodic ataxia type 2
Early infantile epileptic encephalopathy 17
Fetal bovine serum
False discovery rate
Fast Fourier Transform
!
xviii
GABA
GDD
GIRK
GOF
GPCR
HD
ID
KO
LID
LOF
MD
M-D
ME
mEPSC
mIPSC
MOR
NAc
NEDIM
γ-Aminobutyric acid
Developmental delay
G-protein activated inward rectifying potassium channels
Gain-of-function
G protein coupled receptor
Huntington’s Disease
Intellectual disability
Knock-out
L-DOPA-induced dyskinesia
Loss-of-function
Movement disorder
Myoclonus-dystonia syndrome
Mercaptoethanol
Miniature excitatory postsynaptic current
Miniature inhibitory postsynaptic current
Mu opioid receptor
Nucleus accumbens
Neurodevelopmental disorder with involuntary movement
xix !
NEM
NF
NGF
PD
PDE
PI3-K
PLOF
PNKD3
PRRT2
PTX
PTZ
RNP
sEPSC
sIPSC
SNAP25
ssODN
SV
TH
N-Ethylmaleimide
Normal-functioning
Nerve Growth Factor
Parkinson’s Disease
Phosphodiesterase
Phosphatidyl-inositol-4,5-bisphosphate 3-kinase
Partial-loss-of-function
Paroxysmal nonkinesigenic dyskinesia 3
Proline-rich transmembrane protein 2
Pertussis toxin
Pentylenetetrazol
Ribonucleoprotein
Spontaneous excitatory postsynaptic current
Spontaneous inhibitory postsynaptic current
Synaptosomal associated protein-25
Single-stranded oligo DNA nucleotides
Synaptic vesicles
Tyrosine hydroxylase
xx !!
TTX
WT
Tetrodotoxin
Wildtype
xxi !
CHAPTER 1: AN OVERVIEW OF GNAO1-ASSOCIATED NEUROLOGICAL
DISORDERS
Modified from Feng, H., Khalil, S., Neubig, R. R., & Sidiropoulos, C. (2018). A
mechanistic review on GNAO1-associated movement disorder. Neurobiology of disease,
116, 131-141. DOI: https://doi.org/10.1016/j.nbd.2018.05.005.
With permission from the Elsevier. All rights reserved.
1 !
1.1 Abstract
Mutations in the GNAO1 gene cause a complex constellation of neurological
disorders including epilepsy, developmental delay, and movement disorders. GNAO1
encodes Gαo, the α subunit of Go, a member of the Gi/o family of heterotrimeric G protein
signal transducers. Go is the most abundant membrane protein in the mammalian central
nervous system and plays major
roles
in synaptic neurotransmission and
neurodevelopment. GNAO1 mutations were first reported in early infantile epileptic
encephalopathy 17 (EIEE17), but are also associated with a more common syndrome
termed neurodevelopmental disorder with involuntary movements (NEDIM). Here we
review a mechanistic model in which loss-of-function (LOF) GNAO1 alleles cause
epilepsy and gain-of-function (GOF) alleles are primarily associated with movement
disorders. We also develop a signaling framework related to cyclic AMP (cAMP),
synaptic vesicle release, and neural development and discuss gene mutations
perturbing those mechanisms in a range of genetic movement disorders. Finally, we
analyze clinical reports of patients carrying GNAO1 mutations with respect to their
symptom onset and discuss pharmacological/surgical treatments in the context of our
mechanistic model.
1.2 Introduction
Mutations in GNAO1 were first reported in patients with Ohtahara syndrome and
early infantile epileptic encephalopathy 17 (EIEE17, OMIM 61547) (Nakamura et al.,
2 !
2013). More recently, a syndrome of neurodevelopmental disorder with involuntary
movements without epileptic seizures (NEDIM, OMIM 617493) has been defined,
expanding the phenotypic spectrum of GNAO1 mutation-associated neurological
disorders (Ananth et al., 2016; Zhu et al., 2015). Currently, there have been published
reports on 81 patients representing 36 different GNAO1 mutations (23 missense, 1
in-frame deletion and 1 splicing site mutation, see Figure 1.1) (Ananth et al., 2016; Arya,
Spaeth, Gilbert, Leach, & Holland, 2017; Blumkin et al., 2018; Bruun et al., 2018;
Carecchio et al., 2019; Danti et al., 2017; R. Dhamija, Mink, Shah, & Goodkin, 2016;
Dietel, 2016; Epi, 2016; Epi et al., 2013; Euro, Epilepsy Phenome/Genome, & Epi, 2014;
Farwell et al., 2015; Gawlinski et al., 2016; Gerald et al., 2018; Helbig et al., 2016; Honey
et al., 2018; Kelly et al., 2019; Koy et al., 2018; Kulkarni, Tang, Bhardwaj, Bernes, &
Grebe, 2016; Law et al., 2015; Malaquias et al., 2019; Marce-Grau et al., 2016; Menke et
al., 2016; Nakamura et al., 2013; Okumura et al., 2018; Saitsu et al., 2016; Sakamoto et
al., 2017; Schirinzi et al., 2019; Schorling et al., 2017; Takezawa et al., 2018; Talvik et al.,
2015; Ueda, Serajee, & Huq, 2016; Waak et al., 2018; Xiong et al., 2018; Yilmaz et al.,
2016).
Although recent reviews on monogenic complex hyperkinetic disorders recognized
GNAO1 mutations as pathogenic (Carecchio & Mencacci, 2017; Mencacci & Carecchio,
2016), our review focuses on a mechanistic analysis illustrating the shared pathways of
pathogenic mutations across multiple movement disorder-associated genes. It is
3 !
important to consider the mechanisms that underlie the GNAO1-associated movement
disorders to rationalize the clinical heterogeneity resulting from different mutations in
GNAO1, as well as the implications for therapeutic choices. We (H.F. and R.R.N.)
recently demonstrated that GNAO1 mutations associated with movement disorders
result in a gain-of-function (GOF) biochemical behavior related to control of cAMP levels,
while epilepsy-associated mutations cause loss-of-function (LOF) behavior (Feng et al.,
2017). This is consistent with other single-gene epilepsy and movement disorders, which
also share causal genes (Batty, Fenrich, & Fouad, 2017; Szczepanik et al., 2015).
Focusing on movement disorders, there is a clear functional connection between
GNAO1 and other “movement disorder genes” related to two molecular mechanisms.
Both the cAMP pathway (GNAL, GNB1, ADCY5, PDE10A) and regulation of synaptic
vesicle fusion and neurotransmitter release (GNB1, CACNA1A, CACNA1B, KCNMA1,
SYT1, SNAP25, and PRRT2) have been implicated. In this review, we attempt to
develop models of these systems and explore how they may connect pathophysiology
with clinical patterns and therapeutic responses.
4 !
Figure 1.1 Summary of reported cases of GNAO1 encephalopathy
(A) Locations of reported mutations on the Gαo amino acid sequence. The splicing site
mutations are not included here. (B) Sex distribution among the 48 patients reported. (C)
Distribution of movement disorders and/or epilepsy symptoms
in GNAO1
encephalopathy patients (Green = movement disorder only; Red = epilepsy only; Orange
= both phenotypes).
1.3 Gαo (GNAO1) mechanisms
GNAO1 encodes the α-subunit of a heterotrimeric guanine nucleotide-binding protein
(Gαo), which is the most abundant membrane protein in the mammalian central nervous
system, constituting approximately 1% of total brain membrane protein. Gαo localizes
ubiquitously throughout the brain with relatively high expression in hippocampus,
5 !
striatum and cerebellum (Worley, Baraban, Van Dop, Neer, & Snyder, 1986). It couples
to a variety of important G protein coupled receptors (GPCRs) including GABAB, α2
adrenergic, adenosine A1 (A1R), and dopamine D2 (D2R) receptors. These play key roles
in regulating neurotransmitter release, movement, and neural development.
There are multiple downstream signaling targets of Go, as well as of the other
members of the Gi/o family. These include: inhibition of adenylyl cyclases (ACs) which
decreases cAMP production, inhibition of N-type (Cav2.2) and P/Q type calcium
channels (Cav2.1) (Colecraft, Brody, & Yue, 2001; McDavid & Currie, 2006), and direct
inhibition of neurotransmitter vesicle release by the binding of Gβγ released from active
Gαo to inhibit syntaxin 1A and SNAP25 (Zamponi & Currie, 2013). Both Gαo and Gβγ
subunits also bind to G protein-coupled inward rectifying potassium (GIRK) channels to
stimulate channel opening (Luscher & Slesinger, 2010). GIRK channels are
well-recognized as playing a role in seizure disorders (Mayfield, Blednov, & Harris, 2015;
Signorini, Liao, Duncan, Jan, & Stoffel, 1997; Torrecilla et al., 2002).
Many of these targets of Go (both Gαo and Gβγ) signaling are also implicated in
movement disorders. Mutations in ADCY5 (which encodes adenylyl cyclase type 5) have
been reported in patients with dyskinesia and dystonia (Meijer, Miravite, Kopell, & Lubarr,
2017; Mencacci, Erro, et al., 2015; Shaw, Hisama, Friedman, & Bird, 1993). Mutations in
CACNA1A (encoding Cav2.1) cause episodic ataxia type 2 (EA2) (Sintas et al., 2017;
Wan et al., 2011). In the G protein family, mutations in both GNAL (Dufke et al., 2014;
6 !
Kumar et al., 2014; Putzel et al., 2016) and GNB1 (Lohmann et al., 2017; Steinrucke et
al., 2016) are also associated with dystonic syndromes. The former encodes Gαolf which
mediates dopamine D1/5 receptor stimulation of AC and the latter encodes Gβ1 which
mediates many actions of Gi/o.
1.4 The clinical spectrum of GNAO1 mutation-associated movement disorders
1.4.1 GNAO1 encephalopathy displays a variety of neurological symptoms
To understand the molecular mechanisms underlying GNAO1 disorders, it is
important to consider the substantial clinical heterogeneity which includes both
early-onset epileptic encephalopathy (Nakamura et al., 2013) and patients with complex
movement disorders with or without epilepsy (Ananth et al., 2016; Kulkarni et al., 2016;
Menke et al., 2016; Saitsu et al., 2016; Sakamoto et al., 2017; Zhu et al., 2015). Recently,
we reported a biochemical analysis of 15 different GNAO1 mutant alleles (Feng et al.,
2017), which revealed that LOF mutations are associated with epileptic seizures while
mutations that result in GOF for inhibition of cAMP as well as mutations that show largely
normal function in this assay (p.R209 mutations) are mainly associated with movement
disorders (Feng et al., 2017).
The two most common manifestations of patients with GNAO1 mutations (Table
S1.1 and Figure S1), regardless of their clinical pattern or biochemical phenotype, are
hypotonia (68%) and developmental delay (78%, Table S1.1). Choreoathetotic
movements (44%) and dystonia (32%) are the next most common findings (Table S1.1).
7 !
Approximately 28% of patients had intellectual disability.
While many individuals have abnormal EEG or MRI findings (Table S1.1), less than
half of patients with GNAO1 mutations (50%) showed markedly abnormal EEGs and that
was primarily in epilepsy patients with LOF mutations. Approximately 64% of the
reported patients showed significant MRI findings and these were distributed across both
epilepsy and movement disorder patients (Table S1.1). This heterogeneity in both
clinical pattern and effect on brain structure/function suggests a role for both
neurodevelopmental alterations and functional signaling perturbations. The latter seems
more prominent in patients with the GOF mutants who show less evidence for brain
structural abnormalities as well as having some therapeutic responses to drug treatment
(see below).
Table 1.1 Most Common GNAO1 Mutant Alleles Associated With Movement
Disorders
No. of
Epileptic
Chorea/
Frequency of occurring
GNAO1 alleles
patients
seizures Hypotonia
athetosis Dystonia Myoclonus
Ballismus Dyskinesia Stereotypies
p. R209H/L/G/C
12
p. E246K
9
25%
22%
p. G203R 7
100%
83%
89%
14%
67%
63%
43%
33%
56%
29%
p. E237K
2
0
100%
100%
100%
8%
0
0
50%
8%
50%
0
50%
25%
25%
0
50%
8%
0
0
0
!
8 !
Table 1.2 Drugs showing beneficial effects to control involuntary movements of
GNAO1 encephalopathy patients
Drug Positive
GNAO1
Response
Mutations
Sex
Inheritance
de novo
de novo
de novo
de novo
de novo
de novo
de novo
de novo
de novo
de novo
de novo
p.E246G
p.S47G
p.R209H
p.E237K
Tetrabenazine
p.E237K
p.E246K
p.E246K
p.E246K
p.E246K
p.G45R
p.E237K
Levetiracetam
Topiramate
p.R209C
Trihexyphenidyl
p.R209H
!
F
M
M
M
M
F
F
M
F
M
M
F
M
Age of
onset
6 mo
5 mo
10 mo
4 mo
3 mo
4 y
6 mo
14 y
3 mo
NA
4 mo
Symptoms
Epileptic
Movement
seizures
Disorders
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Ref
Danti et al., 2017
Danti et al., 2017
Dhamija, 2016
This report
Waak et al., 2017
Ananth et al., 2016
Ananth et al., 2016
Ananth et al., 2016
Waak et al., 2017
Ueda, 2016
This report
Saitsu et al., 2016;
Sakamoto et al., 2017
Dhamija, 2016
de novo
11 mo
de novo
10 mo
1.4.2 Movement disorders related to GNAO1 encephalopathy show limited
response to pharmacological treatments
There are three mutation hotspots (G203, R209 and E246) in GNAO1 that
prominently result in movement disorders (Table 1.1) (Ananth et al., 2016; Arya et al.,
2017; Danti et al., 2017; R. Dhamija et al., 2016; Honey et al., 2018; Kulkarni et al., 2016;
Menke et al., 2016; Nakamura et al., 2013; Saitsu et al., 2016; Sakamoto et al., 2017;
Waak et al., 2018; Xiong et al., 2018; Zhu et al., 2015). All show GOF or normal function
(NF) phenotypes in the in vitro cAMP regulation assay (Feng et al., 2017). This
9 !
correlation raises the possibility of rationalized drug selection in treating diseases related
to GNAO1 mutations. Gαo-coupled-receptor antagonists should reduce signaling in
movement disorder patients by decreasing the signal from hyperactive GOF GNAO1
mutants. Gαo-coupled-receptor agonists might be beneficial in epilepsy. In both cases,
however, the receptors mediating the abnormal function would need to be identified.
The NF mutant alleles (in p.R209) raise questions about the simple GOF/LOF model
despite the fact that those patients show clear clinical movement disorder pathology.
Moreover, the G203R mutation results in a modest GOF biochemical effect (Feng et al.,
2017) but causes both movement disorder and frequent seizures – though the latter are
reasonably easily controlled (Table S1.1). These mutations show that there is more to
learn about the genotype-phenotype correlation. A key question will be whether another
downstream signal (e.g. calcium and potassium channel regulation) may better correlate
with clinical patterns.
Among patients with movement disorders, tetrabenazine is the most effective drug
(Table 1.2) (Ananth et al., 2016; Danti et al., 2017). This is not surprising given that
tetrabenazine’s actions on VMAT2 will deplete multiple amine neurotransmitters
(dopamine, norepinephrine, and serotonin). This should result in a wide-spread
reduction of Go signaling through, for example, α2A adrenergic receptors, D2/D4 receptors,
and 5-HT1 receptors.
Responses were also reported to trihexyphenidyl, topiramate, and levetiracetam
10 !
(Table 1.2). Trihexyphenidyl is described as a selective muscarinic M1 receptor
antagonist, but it binds to all five muscarinic receptors subtypes with similar affinity
(Dorje et al., 1991). The M4 muscarinic receptor subtype is second most potently
inhibited by trihexyphenidyl. That receptor is Gi/o coupled and strongly implicated in
striatal function (Ztaou et al., 2016). This may be a therapeutic target worth serious
consideration. Interestingly, the patient with the p.R209H mutation who responded to
trihexyphenidyl also responded to tetrabenazine (R. Dhamija et al., 2016). Topiramate
was very effective in suppressing chorea in a patient carrying a p.R209C mutation (Table
1.2) (Sakamoto et al., 2017). Levetiracetam also showed effectiveness in two patients.
Neither of these latter two drugs is known to affect G protein coupled receptors. However,
levetiracetam partly works by binding to its high-affinity binding site on a synaptic vesicle
protein to inhibit neurotransmitter release globally (Grimminger et al., 2013; Ohno &
Tokudome, 2017), which would explain its multi-functionality in suppressing both
epilepsy and movement disorders. While multiple therapies have shown some efficacy in
controlling involuntary movements, no drug seems to be able to mitigate developmental
delay.
11 !
Table 1.3 Patients responding positively to deep brain stimulation (DBS)
GNAO1
Age of
Epileptic
Movement
Positive
Mutation Sex
Inheritance
onset
seizures
Disorders
Response
Ref
Symptoms
DBS
p.E246G
F
p.E237K M
p.E246K
F
de novo
de novo
de novo
6 mo
3 mo
3 mo
p.R209H M
de novo
18 mo
p.R209H M
de novo
2 y
F
M
F
de novo
6 mo
de novo
2 y
de novo
13 mo
p.R209C
p,R209L
!
p.Q233P
+
+
+
+
+
+
+
+
+
+
Y
Y
Y
Y
Y
Y
Y
Y
Danti et al., 2017
Waak et al., 2017
Waak et al., 2017
Kulkarni et al., 2016
Kulkarni et al., 2016
Waak et al., 2017
Honey et al., 2018
Yilmaz et al., 2016
In contrast to the modest efficacy of drug treatment, in seven cases where DBS was
performed, patients all responded well and involuntary movements were suppressed
(Table 1.3) (Danti et al., 2017; Honey et al., 2018; Kulkarni et al., 2016; Waak et al., 2017;
Yilmaz et al., 2016). Consequently, DBS targeting the globus pallidus pars interna (GPi),
appears to be the most effective treatment for GNAO1 related movement disorders, at
least in medication refractory cases. There are no reports to assess the effectiveness of
DBS in other brain regions such as the subthalamic nucleus. DBS may be effective due
to
its general effects
in modulating aberrant synchronization
in
the basal
ganglia-thalamo-cortical loops (McIntyre & Anderson, 2016). There is no information yet
on long-term-sustained efficacy however. Furthermore, there may be a publication bias
12 !
towards patients who responded well and DBS can have a prominent placebo effect, as
seen for patients with Parkinson’s disease (de la Fuente-Fernandez, 2004; Mercado et
al., 2006).
Seizures in patients with GNAO1 mutations can be controlled to some degree by
multiple anti-epileptic drugs (AEDs) (Danti et al., 2017). However, no drug has been
shown to be particularly effective (Table S1.1).
1.5 Potential pathogenic mechanisms of GNAO1-associated movement disorders
The possible etiological bases of GNAO1-associated movement disorder may be
explained by examination of GNAO1 signaling. Inhibition of cAMP is a canonical
pathway of Go, which may be mediated by Gαo itself or by the released Gβγ
(Dortch-Carnes & Potter, 2003; Gill & Hammes, 2007). Mutations in ADCY5 (which
encodes an AC protein that synthesizes cAMP) also result in movement abnormalities in
human patients. Dysregulation of cAMP signaling leads to brain malfunction (Borlikova &
Endo, 2009; Guan et al., 2011). Therefore disturbances of cAMP levels could disrupt a
finely tuned neurodevelopmental system.
A second theoretical basis of GNAO1-associated movement disorder relates to Go’s
role in regulating neurotransmitter release. A close relationship has been proposed
among neurotransmitter levels, brain morphology and behavioral experience (Goldstein,
2006). Deficiency of key neurotransmitters like catecholamines (dopamine, epinephrine
and norepinephrine) and serotonin are widely studied in movement disorders or seizures
13 !
(Mercimek-Mahmutoglu et al., 2015). Go’s presynaptic role in regulating neurotransmitter
release suggests another potential mechanism in the etiology of movement disorders.
A third possibility, from a developmental view, could be alterations of neuronal
maturation, which needs to occur at appropriate stages of neurological development.
Therefore children with developmental defects would exhibit abnormal behaviors. It is
striking that most patients with GNAO1-associated movement disorders also suffer from
severe developmental delay. Morphologically, MRI scans may show global atrophy and
delayed myelination. Overall, genetic causes are responsible for about 40% of the
developmental delay cases,
including developmental delay/intellectual disability
(GDD/ID) (Miclea, Peca, Cuzmici, & Pop, 2015). Control of cAMP levels and
neurotransmitter release clearly could affect ongoing neural functions as well as
neurological development. By this concept, GNAO1-associated movement disorders
could result from perturbations of either of these processes (Leung & Wong, 2017).
Clearly, the former would be more amenable to the therapeutic intervention than the
latter.
1.5.1 Role of cAMP regulation in movement disorders
The second messenger cAMP modulates a broad spectrum of cellular functions
including gene expression, metabolism, exocytosis, and neuronal development. cAMP is
synthesized from ATP by the AC enzymes upon activation by G-protein coupled
receptors (GPCRs). An increase of cAMP levels activates protein kinase A (PKA), which
14 !
phosphorylates other kinases, transcription factors, and ion channels. cAMP can also
activate the Rap guanine nucleotide exchange factor Epac, which has important
functions in neural plasticity (Schmidt, Dekker, & Maarsingh, 2013; Tong et al., 2017).
cAMP formation is negatively regulated by phosphodiesterases (PDEs) or Gi/o family
coupled GPCRs such as these associated with GNAO1. There are many isoforms of
ACs and PDEs in specific brain regions, consistent with the need to maintain a delicate a
balance of cAMP levels for a normally functioning nervous system. cAMP signaling in the
brain is known to mediate neuronal excitability and synaptic plasticity, which further
regulates learning and memory, and motor function (Bollen & Prickaerts, 2012; Kandel,
2012; Pierre, Eschenhagen, Geisslinger, & Scholich, 2009). Also, dibutyryl-cAMP, an
analog of cAMP, promotes axon regeneration and the recovery of motor function by
inhibiting the RhoA signaling pathway (Jeon et al., 2012). The importance of cAMP in
pro-regenerative action makes it a potential therapeutic target for enhancing nerve repair
(Yu, Wang, Wu, & Yi, 2017).
In this section, we will focus on the role that cAMP plays in movement disorders.
There are many movement disorder-related genes that directly regulate cAMP levels in
the brain (D'Angelo et al., 2017; Padovan-Neto & West, 2017). Figure 1.2 provides a
schematic overview of the genes discussed including the type of mutations (LOF or GOF)
seen. However, see the text below for details, as not all results in the literature are
clear-cut. Based on functional studies associated with those genes, both up- and down-
15 !
regulation of cAMP production may contribute to the pathophysiology of movement
disorders. Here we provide a hypothetical model of the role of cAMP regulation in
movement disorders (Figure 1.2).
Unfortunately, however, there is not a simple, completely coherent model of the
relationship between predicted changes in cAMP concentration and the presence of
movement disorders. It is clear that perturbations in cAMP mechanisms are pathological.
Potential explanations for this complexity are described below after consideration of
each gene in detail.
Figure 1.2 Genes regulating the cAMP pathway are related to movement disorders
GPCRs activate Gαo (encoded by GNAO1), which may either inhibit or stimulate cAMP
production depending on the AC subtype present. Gβ1 (encoded by GNB1) forms a
complex with Gγ and this Gβγ complex, typically released from Go or Gi-family G proteins,
can also inhibit or stimulate cAMP production. Activation of Golf (encoded by GNAL)
stimulates cAMP. Phosphodiesterase 10A (encoded by the PDE10A gene) hydrolyzes
16 !
Figure 1.2 (cont’d) cAMP to the monophosphate. Both gain-of-function (GOF)
mutations in GNAO1 and loss-of-function (LOF) mutations in GNAL likely result in a
decrease of cAMP. LOF mutant alleles in PDE10A and GOF mutations in ADCY5
increase cAMP.
1.5.1.1 GNAL
GNAL encodes the alpha subunit of the guanine nucleotide-binding protein Golf. It
belongs to the Gs family and can be activated by odorant receptors in the olfactory
epithelium. It is also strongly expressed in the striatum. Gs family and Gi/o family proteins
have opposite functional effects on AC. Once activated by Gs-coupled GPCRs, Golf
activates AC enzymes to produce cAMP. In the striatum, Golf couples to D1 dopamine
(D1R) and A2A adenosine (A2AR) receptors to activate type 5 adenylyl cyclase (AC5),
which is encoded by the ADCY5 gene (Mercimek-Mahmutoglu et al., 2015).
GNAL mutations account for about 1% of all cases of focal or segmental dystonia
(Kumar et al., 2014). These include autosomal dominant, partial LOF mutations in GNAL
(Dos Santos et al., 2016; Masuho et al., 2016). As GOF mutations in GNAO1 and LOF
mutations in GNAL result in a similar functional change in cAMP production, it seems
logical that they may share similar mechanisms leading to dystonic/choreo-athetoid
disorders. Other previous functional studies of mutant GNAL also revealed deficiencies
in AC activation after D1R stimulation (Fuchs et al., 2013; Kumar et al., 2014). Typically,
patients with heterozygous GNAL mutations exhibit an adult-onset focal cervical,
laryngeal, and/or segmental dystonia (Masuho et al., 2016). Animal studies also support
17 !
the idea that LOF mutations in GNAL with a reduction in striatal cAMP lead to movement
disorders. A heterozygous mouse model Gnal+/- was reported with abnormal postures
and movements compared to WT mice after treatment with the muscarinic agonist
oxotremorine (Pelosi, Menardy, Popa, Girault, & Herve, 2017) (Zwart, Reed, Clarke, &
Sher, 2016) (Pelosi et al., 2017; Zwart et al., 2016). Note that oxotremorine-induced
movement disorders in Gnal+/- mice can be replicated with infusion of oxotremorine into
the striatum but not cerebellum (Pelosi et al., 2017), which indicates the crucial role of
cAMP signaling in striatal projection neurons and the potential role of muscarinic
receptors in modulating the motor movement.
1.5.1.2 GNB1
The GNB1 gene encodes the G protein β subunit Gβ1. In G protein signaling, Gα
binds with Gβγ and GDP in its inactive state. Upon activation, Gα binds to GTP and the
Gα-GTP and Gβγ separate, both carrying out downstream signaling.
Recently, de novo mutations in GNB1 have been identified using whole-exome
sequencing in patients with severe neurodevelopmental disability, hypotonia, and
seizures (Lohmann et al., 2017; Petrovski et al., 2016). The symptoms in GNB1 patients
share characteristics with GNAO1 encephalopathy patients. Moreover, patients with
GNB1 mutations also display early onset of movement abnormalities similar to patients
with GNAO1 mutations (Petrovski et al., 2016).
However, the functional change of the known GNB1 mutations is unclear due a
18 !
variety of assays used by different groups. Lohmann et al defines their mutant GNB1 as
LOF by using real-time bioluminescence resonance energy transfer (BRET) assays to
assess Gβγ’s ability to couple to D1R (Lohmann et al., 2017). However, other groups
describe GNB1 mutations as having a GOF effect due to enhancement of downstream
signaling pathways (Petrovski et al., 2016; Yoda et al., 2015). One unified functional
assay such as Gβγ-regulated inhibition of cAMP production or of N-type calcium
channels should be performed on all GNB1 mutants to clarify definitions of LOF or GOF.
Whether mutations in GNAO1 affect Gβγ function or not remains unknown, but the fact
that non-functioning Cav2.1 and Cav2.2 result in similar movement disorders (see below)
suggests a hypothesis that Gβγ inhibition of calcium channels may be enhanced by GOF
mutations in GNAO1 as well as GOF mutations in GNB1 itself.
1.5.1.3 ADCY5
Mutations in the ADCY5 gene, which encodes AC5 also cause early onset persistent
or paroxysmal choreic, myoclonic, and/or dystonic movements as well as alternating
hemiplegia of childhood (Carapito et al., 2015; Friedman et al., 2016; Mencacci, Erro, et
al., 2015; Westenberger et al., 2017). Patients carrying ADCY5 mutations display mixed
hyperkinetic movements including dystonia, facial myokymia, chorea, myoclonus and
tremor (D. H. Chen et al., 2015). In addition to abnormal movements, axial hypotonia
with paroxysmal exacerbations is also associated with ADCY5 mutations (D. H. Chen et
al., 2015). Delayed milestones and axial hypotonia seem to be almost universal features
19 !
in infants with ADCY5 mutations (Carecchio et al., 2017). This is very similar to patients
with GNAO1 mutations (Table S1.1).
One functional study measuring β-adrenergic agonist-stimulated intracellular cAMP
shows that two de novo mutations (c.1252C>T, p.R418W and c.2176G>A, p.A726T) in
ADCY5 are gain-of-function (GOF) mutations (Y. Z. Chen et al., 2014). More functional
studies need to be done in assessing other mutations in ADCY5 to define a clear
genotype-phenotype correlation. A GOF mutation in ADCY5 should increase cAMP
levels contrasting with expected effects of GNAL and GNAO1 mutations.
AC5 is highly expressed in striatum and nucleus accumbens (NAc). The striatum and
NAc are part of the dopaminergic system that is activated in response to stress (Carapito
et al., 2015). Chen et al reported that Adcy5-null mice developed a movement disorder,
which can be worsened by stress (D. H. Chen et al., 2015). In addition, L-DOPA-induced
dyskinesia (LID) is profoundly reduced in AC5 knockout mice and suppression of AC5 in
the dorsal striatum is sufficient to attenuate LID (Park et al., 2014). Since Go inhibits AC,
this is consistent with the fact that knockout AC5 animals show impaired movements
(Iwamoto et al., 2003). However, it remains unclear why GOF mutations in ADCY5 lead
to hyperkinetic movements in humans. These studies confirm the importance of
regulation of cAMP in the development of movement disorders and suggest that
inappropriate changes in either direction may be detrimental.
20 !
1.5.1.4 PDE10A
PDE10A (encoded by PDE10A gene) participates in signal transduction by
regulating the levels of intracellular cyclic nucleotides. PDE10A is localized in dendritic
spines proximally to postsynaptic sites in striatal medium spiny neurons (Xie et al., 2006).
It hydrolyzes both cAMP and cGMP to nucleoside 5’ monophosphate (Russwurm,
Koesling, & Russwurm, 2015). However, PDE10A is the major cellular mechanism for
degradation for cAMP but has only modest activity for cGMP in the striatum (Russwurm
et al., 2015). Recently, LOF mutations in PDE10A are found in infancy-onset
hyperkinetic movement disorders and childhood-onset chorea (Diggle et al., 2016;
Mencacci et al., 2016). Loss of striatal PDE10A associates with movement disorders like
Hungtington’s and Parkinson’s disease (Ahmad et al., 2014; Giorgi et al., 2011). Pde10a
knockout mice also show abnormalities in movements (Siuciak et al., 2008). LOF
mutations in PDE10A increase cAMP concentration, which seems contradictory to GOF
mutations in GNAO1. However, PDE10A levels differ in different striatum regions in an
animal model of dystonia. PDE10A is increased in the globus pallidus but decreased in
the entopeduncular nucleus/substantia nigra in a DYT1 model (D'Angelo et al., 2017),
which lead to opposite regulation on cAMP concentration. This result further suggests
the balance of cAMP concentration is more important in regulating neuronal functions
than shifting either way.
21 !
1.5.1.5 Summary of cAMP regulation in movement control
cAMP has complex effects on neurotransmission. It can enhance neurotransmission
through multiple actions on Ca++ concentration or on synaptic vesicle release and
trafficking (Neher, 2006). It also strongly modulates synaptic plasticity – generally
increasing neurotransmission (Nestler, Alreja, & Aghajanian, 1999), which may involve
the cAMP-response element binding protein (CREB) or Epac (Eagle, Gajewski, &
Robison, 2016; Tong et al., 2017). cAMP is also important in serotonin-mediated
enhancement of synaptic transmission where it modulates hyperpolarization-activated
cation channels (Ih channels), which underlie repetitive neuronal firing (Beaumont &
Zucker, 2000).
In addition to these relatively acute actions of cAMP on neurotransmission, it also
plays key roles in neurodevelopment. cAMP activates neurite outgrowth and facilitates
axonal guidance (Akiyama, Fukuda, Tojima, Nikolaev, & Kamiguchi, 2016; Inda et al.,
2017). It is possible that gene mutations that elevate cAMP production (i.e. LOF
mutations in PDE10A, GOF mutations in ADCY5) increase neurite outgrowth and may
result in enhanced or disordered synapse formation. This may increase excitatory
neurotransmission and neuron hyperexcitability. It is clear, however, that genetic
mutations which lead to decreased cAMP production (i.e. LOF mutations in GNAL and
GOF mutations in GNAO1) also result in movement disorders. Indeed for some
movement-disorder-associated genes (i.e. GNB1), both LOF and GOF functions and
22 !
effects have been reported (Lohmann et al., 2017; Petrovski et al., 2016).
There are several possible explanations for this highly complex picture. First, in vitro
studies used to characterize GOF/LOF behavior of mutant alleles differ between labs so
the concept of GOF or LOF may not directly correlate with in vivo cAMP production.
Second, some actions may be mediated in different neuron populations or different brain
regions. Finally, keeping an appropriate balance of cAMP levels may be the critical
underlying element for normal movement performance. So either an increase or a
decrease in cAMP could result in the observed movement disorders.
1.5.2 Role of neurotransmitter release and synaptic vesicle fusion in movement
disorders
In addition to the control of cAMP production, regulation of neurotransmitter release
is also strongly implicated in movement disorders, epilepsy, and neurodevelopmental
delays (Figure 1.3). Mutations in genes for a number of proteins directly involved in
synaptic vesicle fusion (e.g. SYT1, SNAP25, and PRRT2) have been identified.
Presynaptic calcium influx is another driving factor for neurotransmitter release and most
CNS synapses rely on Cav2.1 (CACNA1A) or Cav2.2 (CACNA1B) calcium channels for
synaptic transmission. Mutations in both of these channel genes are associated with
movement disorders.
Further tightening the connection among these various genes related to movement
disorder, Gαo activation drives Gβγ release, which mediates direct inhibition of vesicle
23 !
release (Zurawski et al., 2017) as well as indirect inhibition by suppression of
voltage-gated calcium channels (i.e. Cav2.1 and Cav2.2 encoded by CACNA1A and
CACNA1B) (Agler et al., 2005; McDavid & Currie, 2006). These mechanisms generate a
relatively consistent model in which reductions in neurotransmitter release associate with
movement disorder. GOF mutations in both GNAO1 and GNB1 would result in increased
activity of Gαo and Gβγ, which would suppress both calcium channel activity and the
synaptic vesicle release machinery (Figure 1.3). LOF mutations in the genes encoding
calcium channels (CACNA1A and CACNA1B) and three proteins involved in vesicle
release (SYT1, SNAP25, and PRRT2) also are involved in movement abnormalities.
It is well-established that calcium influx through CaV channels triggers synaptic
vesicle fusion via synaptotagmin (Sudhof, 2012). Indeed presynaptic calcium channels
form a complex with synaptotagmin and other proteins in the vesicle release machinery
such as syntaxin 1 and SNAP-25 (Leveque et al., 1994; Simms & Zamponi, 2014). Also,
Gβγ released from activated Go (Gαo/Gβγ) competes with synaptotagmin-1 for binding to
SNARE proteins to modulate vesicle fusion (Zurawski et al., 2017), providing a tight
network of mutant proteins controlling neurotransmitter vesicle release where
suppressed synaptic vesicle release is associated with human movement disorders.
24 !
Figure 1.3 Pathogenic mutations in genes that regulate neurotransmitter release
Activation of Gαo by multiple GPCRs (including GPR88) inhibits voltage gated calcium
channels (CACNA1A and CACNA1B). Calcium influx promotes synaptotagmin-1
(encoded by SYT1) and SNAP25 (encoded by SNAP25 gene) anchoring the vesicle to
the membrane in preparation for exocytosis. Reduced functions in CACNA1A,
CACNA1B, GPR88, SYT1 and SNAP25 or increased function of GNAO1, GNB1 and
KCNMA1 all reduce synaptic neurotransmitter releases.
1.5.2.1 SYT1
Synaptotagmin-1 (encoded by SYT1) is a calcium-binding synaptic vesicle protein
required for both exocytosis and endocytosis. Tucker et al., 2004 investigated the effect
of synaptotagmin-1 on membrane fusion mediated by the SNARE protein complex
SNAP25, syntaxin and synaptobrevin. In the presence of calcium, the cytoplasmic
domain of synaptotagmin-1 strongly stimulates membrane
fusion (Figure 1.3).
25 !
Stimulation of fusion is abolished by disrupting the calcium-binding activity of
synaptotagmin-1 (Rickman & Davletov, 2003). Thus, synaptotagmin-1 and SNAREs are
likely to represent the minimal protein unit for calcium-triggered exocytosis (Tucker,
Weber, & Chapman, 2004).
The study of STY1’s relation to movement disorders is limited. Only one patient has
been reported so far. A trio analysis of whole-exome sequences identified a de novo
SYT1 missense variant (I368T) in a case of human neurodevelopmental disorder
associated with hypotonia and hyperkinetic movements without seizures (Baker et al.,
2015). Functional studies showed that this mutation slows evoked synaptic vesicle (SV)
fusion and also affects SV retrieval from the plasma membrane during endocytosis
(Baker et al., 2015). It is therefore a LOF mutation. Interestingly, an equivalent STY1
mutation in Drosophila also resulted in a reduction in evoked neurotransmitter release
(Paddock et al., 2011).
1.5.2.2 PRRT2
Proline-rich transmembrane protein 2 (PRRT2) is encoded by the PRRT2 gene.
Heterozygous mutations in PRRT2 lead to epilepsy, kinesigenic dyskinesia, and
migraine. PRRT2 is enriched in presynaptic terminals. It regulates synapse number and
release of SV. Most of pathogenic mutations in PRRT2 lead to impaired PRRT2 protein
expression, which could result in impairment of neurotransmitter release (Weston, 2017).
Moreover, PRRT2 protein
interacts with
the synaptic protein SNAP25 and
26 !
synaptotagmin-1 (H. Y. Lee et al., 2012; Weston, 2017). Prrt2 expression is high in
mouse cerebral cortex, hippocampus, and cerebellum (W. J. Chen et al., 2011).
Chen et al identified 3 heterozygous truncating mutations in PRRT2 gene from eight
unrelated Han Chinese
families with episodic kinesigenic dyskinesia-1 (2011).
Independently, Wang et al identified one insertion and one nonsense mutation from 27
members of two families with autosomal dominant paroxysmal kinesigenic dyskinesias
(Wang et al., 2011). Two patients in each family also developed an infantile convulsion
and choreoathetosis syndrome (Wang et al., 2011). Law et al reported a common
PRRT2 mutation in a case series of 16 patients with familial paroxysmal kinesigenic
dyskinesia (Law et al., 2016). Heron et al identified heterozygous mutations in PRRT2
from separate families with familial infantile seizures-2 and with familial infantile
convulsions with paroxysmal choreoathetosis (Heron et al., 2012), suggesting that
mutations in PRRT2 are pathogenic for both epilepsy and movement disorders.
1.5.2.3 SNAP25
Synaptosomal associated protein-25 (SNAP25 encoded by SNAP25)
is a
component of the SNARE complex, which is essential to synaptic vesicle exocytosis. It
also negatively modulates neuronal voltage-gated calcium channels by directly
interacting with calcium channel subunits. The SNAP25 gene is associated Attention
Deficit Hyperactivity Disorder (ADHD), schizophrenia, and bipolar disorder (Antonucci et
al., 2016; Corradini, Verderio, Sala, Wilson, & Matteoli, 2009). In 2013, whole exome
27 !
sequencing identified a novel de novo mutation p.F48V (c.142G>T) in SNAP25 from a
15y old female with severe static encephalopathy, intellectual disability and generalized
epilepsy (Rohena et al., 2013). SNAP25 also plays a major role in neuronal survival.
Neuronal cultures from Snap-25 knockout mice show degenerated dendrites and
ultimately neuronal death (Delgado-Martinez, Nehring, & Sorensen, 2007).
The relationship between SNAP25 and Go has been well-studied. SNAP25 is a key
downstream target of Gβγ subunits. Gβγ binds to the extreme C terminus of SNAP25 to
inhibit vesicle release in response to Gi/o-coupled receptors activation (Zurawski,
Rodriguez, Hyde, Alford, & Hamm, 2016).
1.5.2.4 KCNMA1
KCNMA1 encodes
the pore-forming subunit of calcium-activated potassium
channels (BK channels), which are in close proximity with voltage-gated calcium
channels in neurons. Membrane depolarization activates calcium channels and
increases calcium entry, which activates BK channels to help terminate the action
potential, to produce after hyperpolarization, and to block calcium channels (U. S. Lee &
Cui, 2010). Both GOF and LOF mutations of KCNMA1 were reported in patients with
paroxysmal nonkinesigenic dyskinesia 3 (PNKD3), with or with out generalized epilepsy.
This highlights the sensitivity of developing brain to both increased and decreased BK
channel activities. Similar to GNAO1-associated movement disorders, developmental
delay is also commonly associated with PNKD3 patients (Tabarki, AlMajhad, AlHashem,
28 !
Shaheen, & Alkuraya, 2016; Yesil et al., 2018; Zhang, Tian, Gao, Jiang, & Wu, 2015).
GOF mutants of GNAO1 may work to regulate the function of BK channels both
positively and negatively. Go activates
the production of phosphatidyl-
inositol-4,5-bisphosphate 3-kinase (PI3-K), which in turn activates BK channels (Patel,
2004; Shanley, O'Malley, Irving, Ashford, & Harvey, 2002). However, the Gβγ subunit
dissociated from Gαo inhibits voltage-gated calcium channels (illustrated in 3.2.5 and
3.2.6), which would reduce intracellular calcium concentrations preventing the activation
of BK channels (Castillo et al., 2015). Therefore, it is difficult to pinpoint the relationship
between GOF mutations in GNAO1 and mutations in KCNMA1.
1.5.2.5 CACNA1A
CACNA1A encodes the P/Q type voltage gated calcium channel α1 subunit (CaV2.1).
Similar to N-type calcium channels (CaV2.2), activation of P/Q type calcium channels
promotes neurotransmitter release. Moreover, influx of calcium through P/Q-type
channels is responsible for activating expression of syntaxin-1A, a presynaptic protein
that mediates vesicle docking (Sutton, McRory, Guthrie, Murphy, & Snutch, 1999).
LOF mutations in CACNA1A also cause episodic ataxia type 2 (EA2), an autosomal
dominant neurological disease (Guida et al., 2001; Jen, Yue, Karrim, Nelson, & Baloh,
1998; Sintas et al., 2017; Wan et al., 2011) and familial hemiplegic migraine type 1
(Garza-Lopez et al., 2012; Mullner, Broos, van den Maagdenberg, & Striessnig, 2004).
There is evidence of a dominant negative effect of EA2 mutants in the CACNA1A gene
29 !
(Gao et al., 2012; Jeng, Chen, Chen, & Tang, 2006; Jouvenceau et al., 2001).
Haploinsufficiency is also pathologic (Guida et al., 2001; Wan et al., 2011).
The leaner mutation in mice affects the P/Q-type calcium channels Cav2.1 subunits,
causing a reduction in calcium currents, predominantly in cerebellar Purkinje cells
(Alonso et al., 2008). Homozygous leaner mice show severe, progressive cerebellar
ataxia from postnatal day 10 (Alonso et al., 2008). Age-dependent impairment in motor
and cognitive tasks is also observed in heterozygous leaner mice (Alonso et al., 2008).
In addition, silencing of P/Q-type calcium channels in Purkinje neurons of adult mouse
leads to a phenotype similar to episodic ataxia type 2 (EA2) (Salvi et al., 2014). Thus
P/Q-type calcium channels play an important role control of movement as well as in
neurodevelopment.
1.5.2.6 CACNA1B
A disruptive missense mutation p.R1389H (c.4166G>A) in the CACNA1B gene,
encoding neuronal voltage-gated N-type calcium channels (Cav2.2), was identified in a
new familial myoclonus-dystonia (M-D) syndrome (J. L. Groen et al., 2015). Five affected
family members were identified in a 16-member family across 3 generations (J. Groen,
van Rootselaar, van der Salm, Bloem, & Tijssen, 2011). But a genome-wide study in a
large European multicentric M-D cohort failed to detect the mutation in the 146 probands
with familial M-D (Mencacci, R'Bibo, et al., 2015). Therefore, a causal association
between the CACNA1B mutation p.R1389H (c.4166G>A) and movement disorder is still
30 !
under debate. However, a recent study linked LOF mutations in CACNA1B to the onset
of neurodevelopmental disorder with seizures and nonepileptic hyperkinetic movements
(NEDNEH; OMIM# 618497), which provides evidence for the role of Cav2.2 in human
neurodevelopment (Gorman et al., 2019).
1.5.2.7 GPR88
GPR88 (encoded by GPR88 gene) is an orphan G-protein coupled receptor (GPCR)
in the rhodopsin-like receptor family. It is widely expressed in the striatum, caudate
nucleus, putamen, nucleus accumbens, and olfactory tubercle, but is not detected in the
cerebellum (Massart, Guilloux, Mignon, Sokoloff, & Diaz, 2009). Its CNS expression is
particularly robust in the striatum, paralleling that of the dopamine D2 receptor
(Mizushima et al., 2000). Striatal GRP88 is enriched in both D1 and D2 expressing
medium spiny neurons (Jin et al., 2014) and it is emerging as a key player in the
pathophysiology of several neurological diseases. Agonists of GPR88 were developed
as potential treatment for CNS disorders such as schizophrenia (Bi et al., 2015).
One case of GPR88-associated chorea has been reported in human patients. Alkufri
et al reported a deleterious mutation p.C291X in GPR88 associated with chorea, speech
delay and
learning disabilities (Alkufri, Shaag, Abu-Libdeh, & Elpeleg, 2016).
Homozygous Gpr88 knockout mice displayed reduced striatal dependent behaviors such
as rearing, grooming, and burying (Meirsman et al., 2016). Mechanistic studies in striatal
medium spiny neurons demonstrated increased glutamatergic responses and reduced
31 !
GABAergic inhibition in the absence of GPR88 which results in enhanced neuronal firing
in vivo (Quintana et al., 2012). Studies also showed enhanced function of Gi/o-coupled
delta and mu opioid (DOR and MOR) in striatal membranes in Gpr88 knockout mice,
suggesting a functional antagonism between GPR88 and other Gi/o-coupled receptor
activities (Figure 1.3) (Meirsman et al., 2016). The increasing interest in GPR88 may
result in the design of treatments for this orphan disease, considering its unique location.
1.5.2.8 Summary of neurotransmitter release and synaptic vesicle fusion in
movement control
The disruption of neurotransmitter release leads to a broad spectrum of movement
impairments
including chorea, ataxia and dystonia but also
results
in
neurodevelopmental abnormalities and epilepsy. Apart from the relatively constrained
expression of GPR88 in striatum, the rest of the proteins mentioned above are
ubiquitously expressed throughout the brain. Therefore, it is relatively hard to determine
which brain regions are involved in the pathogenesis of the related movement disorders.
Hence, clear delineation of the neural mechanisms and relevant pathways remains
challenging. The fact that GNAO1 is involved in the regulation of many of the proteins
encoded by the above-mentioned genes makes Go-coupled receptors a possible
therapeutic target in developing treatments for patients carrying these mutations.
1.6 Developmental defects may also contribute to movement disorders
In addition to ongoing alterations in cAMP signaling and neurotransmitter release
32 !
mechanisms, it is likely that developmental abnormality that results in disordered
synaptic or neural pathway organization could contribute to the observed movement
disorders. It is estimated that genetic factors are responsible for up to 40% of
developmental disabilities (Miclea et al., 2015). Such changes in neural development
and organization could contribute to the observation that developmental delay and
epilepsy are often seen with the gene mutations discussed in this review. However, the
development effects and the physiological effects may not be mutually exclusive.
cAMP signaling affects many developmental processes including facilitation of
neuronal cell differentiation and maturation (Lepski, Jannes, Nikkhah, & Bischofberger,
2013; Sharma, Hansen, & Notter, 1990), induction of axon growth (Corredor et al., 2012),
control of guidance cue (Forbes, Thompson, Yuan, & Goodhill, 2012), and enhancement
of synaptic connections (Lessmann & Heumann, 1997). This could explain why most
patients carrying the GOF mutations in GNAO1, which would be expected to suppress
cAMP levels, exhibit severe developmental delay.
Similarly, synaptic release mechanisms are implicated in both developmental
abnormalities and movement disorders. Specifically, the individual carrying the LOF
mutation in SYT1 with hypotonia and hyperkinetic movements discussed above also
exhibited severe motor delay and profound cognitive impairment (Baker et al., 2015).
Clearly, therapeutic approaches to modulate ongoing cAMP signaling or synaptic
neurotransmitter release will be more tractable than attempting to alter developmental
33 !
abnormalities that may have already occurred by the time that the disorder is recognized.
1.7 Conclusion
A full understanding of the exact etiology of GNAO1-related movement disorders
remains elusive, however, the mechanistic clustering of GNAO1 and other movement
disorder-related genes in the pathways for control of cAMP and neurotransmitter release
strongly suggest a role for those mechanisms. For synaptic vesicle release, it appears
that loss of function is the primary alteration observed. In contrast, for cAMP control, the
picture is more confusing with mutations predicted to cause either increases or
decreases being implicated. Clearly more needs to be learned about the specific brain
regions, neuron types, and receptors that control GNAO1 signaling in these disorders.
This may be facilitated by the development of new animal models beyond the two
GNAO1 mutants already reported (Jiang & Bajpayee, 2009; Jiang et al., 1998; Kehrl et
al., 2014). In particular, it is surprising that Gnao1+/- mice, which have relatively normal
behaviors, do not mimic patients with heterozygous LOF GNAO1 mutations who exhibit
severe epileptic encephalopathy. Animal knock-in models carrying the specific GNAO1
mutant alleles that are found in human epilepsy and movement disorders may also help
clarify this conundrum.
In addition to the mechanistic analysis, our review of the clinical literature also has
implications for therapeutics. Since GOF mutations in GNAO1 cause movement
disorders (Feng et al., 2017), it is not surprising that tetrabenazine has proven to be the
34 !
most effective drug in controlling patient’s chorea (Table 1.3) (Ananth et al., 2016; Danti
et al., 2017; R. M. Dhamija, J, W.; Shah, B, B.; Goodkin, H, P., 2016; Waak et al., 2017).
The broad depletion of multiple monoamines such as dopamine, serotonin,
norepinephrine, and histamines from nerve terminals suggests that many receptors may
be involved. A better understanding of the associated upstream GPCRs that are driving
the enhanced signaling through Go may permit the use of more selective agonists or
antagonists against these receptors to alleviate symptoms with fewer side effects in
GNAO1 associated movement disorders.
1.8 Organization of the thesis
This dissertation shows a genotype-phenotype correlation model of
the
GNAO1-associated neurological disorders and how this model helps further mechanistic
study of the GNAO1-related neurological disorders. The organization of this thesis is as
follows. Chapter 1 gives a broad review of GNAO1 encephalopathies and provides
analysis of the pathophysiological mechanisms. The appendix includes a review of each
case of reported human patients carrying GNAO1 mutations. Chapter 2 introduces the
genotype-phenotype correlation model based on in vitro biochemical functional analysis.
The appendices verify and expand this model with newly reported GNAO1 mutations.
Chapter 3 provides a detailed behavioral description on the animal models carrying the
human mutations G203R, R209H and T191F197del respectively. These three animal
models are compared with the previously reported mouse model carrying a GOF Gnao1
35 !
mutant G184S and the Gnao1 KO mice. The appendix includes all the raw data collected
from
the behavioral experiments. Chapter 4 explores
the electrophysiological
mechanisms of the cerebellar Purkinje cells of the G203R mutant mice. The appendix
expands the analysis to other mouse models mentioned in Chapter 3. Chapter 5
summarizes and analyzes all the experimental data collected from Chapter 2 to Chapter
4. In addition, this chapter speculates on the future directions and the significance of this
project. This chapter also shows preliminary results of experiments that have not been
developed maturely in the appendix. These results may help the future efforts of
optimizing assays for further testing of GNAO1 mutations and for drug development or
repurposing.
36 !
APPENDIX
37 !
APPENDIX
SUPPLEMENTAL DATA
Table S1.1 A complete summary of clinical information regarding GNAO1 patients
No.
GNAO1
Mutations
Sex
Inheritance Age of onset
Seizures Hypotonia Chorea/athetosis Dystonia Myoclonus Ballismus
Dyskinesia
Stereotypies Developmental
Delay
Intellectual
Disability
Other
Reported involvement
F
F
M
M
F
M
F
F
M
F
F
F
F
F
F
de novo
de novo
de novo
de novo
birth
3 y
birth
2.5 mo
de novo
5 wk
de novo
15 hr
de novo
2 hr
de novo
infancy
de novo
NA
de novo,
somatic
mosaic
de novo
29 d
14 d
de novo
6 mo
de novo
de novo
3 mo
2 mo
de novo
2.5 mo
M
de novo
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
p.G40R
c.118G>A
p.G40R
c.118G>A
p.G40R
c.118G>A
p.G40R
c.118G>A
p.G40W
c.118G>T
p.G40E
c.119G>A
p.G40E
c.119G>A
p.G45E
c.134G>A
p.G45R
c. 133G>C
p. D174G
c.521A>G
p.T191_F197del
c.572_592del
p.R349_G352del
insQGCA
c.1046_1055del1
0ins10
p.L199P
c.596T>C
p.A227V
c.680C>T
p.Y231C
c.692 A>G
p.Y231C
c.692A>G
p.E246G
c.737A>G
p.N270H
c.808A>G
p.D273V
c.818A>T
p.D273V
c.818A>T
p.F275S
c.824T>C
p.I279N
c.836T>A
p.I279N
c.836T>A
p.I279N
c.836T>A
p.Y291N
c.871T>A
p.S47G
c.139A>G
p.I56T
c.167T>C
p.G203R
c.607G>A
p.G203R
c.607G>A
p.G203R
c.607G>A
p.G203R
c.607G>A
p.G203R
c.607G>A
p.G203R
c.607G>A
p.G203R
c.607G>A
p.G203R
c.607G>A
p.G203R
c.607G>A
p.G204R
c.610G>C
p.G42R
c.124G>C
p.S207Y
c.620C>A
F
F
F
F
F
M
F
M
F
M
F
F
F
F
M
F
F
F
F
M
M
F
M
5 d
6 mo
3 mo
2 d
2 d
3 d
9 d
4 d
1 h
2 mo
5 mo
4 y
7 mo
7 d
9 d
1 mo
3 mo
de novo
de novo
de novo
de novo
de novo
de novo
de novo
de novo
de novo
de novo
de novo
de novo
de novo
de novo
de novo
de novo
de novo
birth
de novo
de novo
de novo
birth
birth
12 d
de novo
24 mo
de novo
de novo
NA
infancy
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
38 !
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
microcephaly
ataxia
+
+
+
+
+
PEHO Syndrome;
Accompanying mutation
HESX1 (A9T)
cerebellar ataxia,
accompanying with
ATP2B3 (T113M)
Ohtahara syndrome
Ohtahara syndrome
+
oromotor apraxia
tetraparesis
acquired microcephaly
Ohtahara syndrome
lower limb spasticity
Ohtahara syndrome
bradycardia
+
+
+
+
+
+
+
+
+
+
+
tachycardia,
hyperthermia, sweating
frequent arching of the
back
Table S1.1 (cont’d)
No.
EEG Findings
Severe EEG
a disorganized background with frequent multi-focal high-
amplitude sharp and spike wave discharges, no definite
burst-suppression patterns
burst suppression at onset; slow background multifocal
spike waves
discontinous background, bilateral yemporal sharp waves
3 y: slow spike and wave, multifocal spikes
9 mo: right temporal seizures, focal spikes, focal slowing
2 mo: hypsar- rhythmia;
14 y: generalized onset of tonic seizures and epileptic
spasms, generalized slowing
9 d: multifocal spikes; 14 y: focal spikes and waves,
absence of normal awake and sleep features
NA
NA
burst suppression at 2 mo; hypsarrhythmia at 3 mo;
diffuse spike-and-slow-wave complex at 1yr 7 mo; sharp
waves at frontal lobe at 3 yr 9 mo
suppression-burst pattern at 2 week; hypsarrhythmia at 4
mo
activated posterior temporal and occipital spikes
7 y: sleep!
background slowing, multifocal high-voltaged sharp
waves and spike and slow-wave complexes
hypsarrhythmia and multifocal with ictal
modified burst-suppression at 3 mo; hypsarrhythmia in
awake burst-suppression pattern in sleep at 7 mo;
slowing of background activity with multifocal interictal
epileptiform discharge at 9 mo
Neonatal: multifocal epileptiform sharp waves;
20 mo: frequent bioccipital spikes
normal
hypsarrhythmia and slow background
Abnormal epileptiform activity
normal at first; later unkown
burst-suppression pattern, hypsarrhythmia, later slow
background with multifocal discharges
multifocal modified hypsarrhythmia, burst-suppression in
sleep, high-voltage midline central discharges during
spasms, generalized decrement with low voltage fast
activity
burst-suppression pattern at 4 d; multifocal sharp waves
at 1 yr, 4 mo; burst-suppression pattern ar 5 yr, 6 mo
Neonatal: multifocal sharp waves with high! amplitude
bursts (not burst suppression);
8 mo: modified hypsarrhythmia
1 y: multifocal spikes, focal seizures; 3 y: intermittent
posterior slowing
right frontotemporal spikes
left frontotemporal spikes
diffuse irregular spike-and-slow-wave complex at 5 yr
slow-wave bursts, migrating focal epileptiform discharges
delta and theta activity and rare multi-regional, bi-
hemispheric epileptic activity
multifocal and diffuse discharges, along with generalized-
onset seizures
background slowing
multifocal sharp waves, left temporal seizure pattern
hypsarrhythmia
multifocal paroxysmal activities in both temporal
hemispheres
NA
NA
NA
15 mo: slow posterior dominant rhythm
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
+
+
++
++
+
++
++
++
++
Brain MRI
normal
Severe MRI
mild ventricular enlargement; thin corpus
callosum
bilateral increased signal in frontal and
peritrigonal white matter in T1
4 mo: mildly prominent bifrontal subarachnoid
spaces
4 mo: bilateral mesial temporal sclerosis, diffuse
parenchymal atrophy, delayed myelination
2 y: status posttemporal lobectomy, left cerebral
atrophy
15 mo: nonspecific signal increase in globi
pallidi, normal myelination
cerebral and cerebellar atrophy
NA
delayed myelination and thin corpus callosum at
10 mo
normal at 3 mo
normal
delayed myelination and thin corpus callosum
progressive cerebral atrophy, thin corpus
callosum at 10 mo
delayed myelination, short and thin corpus
callosum and hippocampus
2 y: prominent subarachnoid spaces
mild loss of volume (atrophy) in generalized
distribution
minimal atrophy
normal
normal at first; later unknown
delayed myelination and thinning of white matter
moderate-severe progressive global atrophy
with delayed myelination, thin corpus callosum
normal at 1 mo; cerebral atrophy at 5 yr 6 mo
2.5 y: moderate to progressive atrophy with
delayed myelination
normal
ventricular enlargement; thin and dysmorphic
corpus callosum; mild hypoplasia of caudate
left frontal lesion (diffuse astrocytoma WHO
grade 2); low-lying cerebellar tonsils
delayed myelination at 1 yr, 3 mo; reduced
cerebral white matter, thin corpus callosum at 4
yr, 8 mo
progressive cerebral atrophy with delayed
myelination at 14 mo
mild atrophy
progressive diffuse cerebral atrophy and volume
loss in cerebellum
atrophy, thin corpus callosum (2 y)
mild atrophy (10 mo)
NA
thin corpus callosum
hypomyelination and atrophy
14 y: bilateral hyperintensities of the thalamus
on T2
NA
1 y: generalized thinning of corpus callosum,
relative paucity of deep white matter
39 !
+
+
+
++
++
++
++
++
++
++
++
+
+
+
++
++
++
++
++
++
++
++
+
++
+
+
NA
+
+
+
++
Table S1.1 (cont’d)
No.
Drug Trialed
Drug Positive Response
DBS
DBS
Positive
Response
(cid:7)(cid:11)(cid:12)(cid:8)(cid:13)
(cid:6)(cid:4) (cid:2)(cid:1)(cid:9)(cid:10)(cid:1)(cid:2)(cid:5)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
NA
multiple AEDs, ketogenic diet, vagus nerve
stimulation
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
pyridoxine, phenobarbital, levetiracetam,
topiramate, vigabatrin, ACTH, zonisamide,
clobazam
ACTH, levetiracetam,
clobazam, zonisamide
NA
NA
NA
ketogenic diet
NA
NA
NA
NA
prednisone, valproic acid, clonazepam
prednisone
phenobarbitone, vigabatrin, lamotrigine
NA
NA
NA
NA
N
NA
tetrabenazine
NA
NA
lamotrigine, valproic acid, trihexyphenidyl and
melatonin
seisures and dyskinetic
movements well-controlled
NA
valproate, ketogenic diet, pyridoxal-5-phosphate,
vigabatrin, levetiracetam, phenobarbitone,
prednisolone
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
tetrabenazine, well-controlled
on AED
well-controlled on AED
NA
phenobarbital
phenobarbital
lamotrigine, zonisamide
NA
Vit B6, sulthiame, levetiracetam, L-DOPA
Vit B6, phenobarbital, levetiracetam, topiramate,
valproic acid, vigabatrin, oxcarbazepine,
phenytoin, clobazam, lacosamide, ketogenic diet,
gabapentin, trihexyphenidyl, baclofen,
benzodiazepines
controlled epileptic activity but
not involuntary movements
NA
N
lamotrigine and zonisamide
controlled epileptic activity,
benzodiazepines reduced
paroxysmal dystonias
NA
topiramate, vigabatrin
phenobarbital, carbamazepine, benzodiazepines,
topiramate, clonazepam
topiramate, clonazepam
tetrabenazine, lorazepam, baclofen and
phenobarbital
phenobarbital
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Y
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
NA
NA
NA
tetrabenazine
Evaluated
NA
NA
N
N
40 !
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Law et al 2015
Danti et al 2017
Bruun et al 2017
Kelly et al 2019
Kelly et al 2019
Kelly et al 2019
Kelly et al 2019
Gawlinski et al 2016
26485252
28357411
28817111
30682224
30682224
30682224
30682224
27343026
Ueda et al 2015
DOI: 10.1055/s-0036-1597627
Nakamura et al 2013
Nakamura et al 2013
Kelly et al 2019
Marce-Grau A et al 2016
Saitsu et al 2015
23993195
23993195
30682224
27072799
25966631
Talvik et al 2015
DOI: 10.1177/2329048X15583717
NA
Kelly et al 2019
Danti et al 2017
EuroEPINOMICS-RES
Consortium 2014
Kelly et al 2019
Schirinzi et al 2018
EuroEPINOMICS-RES
Consortium 2014
Epi4k 2016
Nakamura et al 2013
Kelly et al 2019
Kelly et al 2019
Danti et al 2017
Danti et al 2017
Nakamura et al 2013
Saitsu et al 2015
Y
N
N
N
N
N
N
NA
NA
N
N
N
N
N
N
N
N
N
N
N
N
N
30682224
28357411
25262651
30682224
30642806
25262651
27476654
23993195
30682224
30682224
28357411
28357411
23993195
25966631
29429466
30642806
30642806
30103967
25590979
30682224
Dietel et al 2016
DOI: 10.1055/s-0036-1583625
Arya et al 2017
Schorling et al 2017
28202424
28628939
Schorling et al 2017
28628939
Xiong et al 2018
Schirinzi et al 2018
Schirinzi et al 2018
Koy et al 2018
Zhu et al 2014
NA
Kelly et al 2019
Table S1.1 (cont’d)
(cid:2)(cid:3)(cid:1)
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61#
62
63
64
65
66
67
68
69
70
71
72*
73*
74
75
76
77
78
79
80
81
GNAO1
Mutations
p.R209H
c.626G>A
p.R209H
c.626G>A
p.R209H
c.626G>A
p.R209H
c.626G>A
p.R209H
c.626G>A
p.R209H
c.626G>A
p.R209H
c.626G>A
p.R209L
c.626G>T
p.R209G
c.625C>G
p.R209C
c.625C>T
p.R209C
c.625C>T
p.R209C
c.625C>T
p.R209C
c.625C>T
p.R209C
c.625C>T
p.R209C
c.625C>T
p.R209C
c.625C>T
p.R209C
c.625C>T
p.R209C
c.625C>T
p. C215Y
c. 644G>A
p.A221D
c.662C>A
p.Q233P
c.698A>C
p.E237K
c.709G>A
p.E237K
c.709G>A
p.E237K
c.709G>A
p.E237K
c.709G>A
p.E237K
c.709G>A
p.E237K
c.709G>A
p.E246K
c.736G>A
p.E246K
c.736G>A
p.E246K
c.736G>A
p.E246K
c.736G>A
p.E246K
c.736G>A
p.E246K
c.736G>A
p.E246K
c.736G>A
p.E246K
c.736G>A
p.E246K
c.736G>A
p.E246K
c.736G>A
p.E246K
c.736G>A
p.L284S
c.851T>C
p.I344del
c.1030_1032delA
TT
c.723+1G>T
c.723+1G>A
Sex
Inheritance Age of onset
Seizures Hypotonia Chorea/athetosis Dystonia Myoclonus Ballismus
Dyskinesia
Stereotypies Developmental
Delay
Intellectual
Disability
Other
Reported involvement
M
M
M
M
M
F
M
M
F
F
M
M
F
F
F
F
F
M
M
F
F
M
M
F
F
M
M
F
M
F
F
M
M
F
F
F
F
F
F
F
F
F
de novo
de novo
de novo
de novo
de novo
de novo
de novo
de novo
1 y
18 mo
2 y
10 mo
3 y
6 mo
6 mo
birth
de novo
3 y 10 mo
de novo
11 mo
de novo
de novo
birth
7 mo
de novo
6 mo
de novo
de novo
de novo
6 mo
6 mo
birth
de novo
neonate
+
+
+
de novo
infant
+
de novo
de novo
12 yr
9 mo
de novo
13 mo
de novo
de novo
de novo
de novo
de novo
de novo
de novo
de novo
de novo
4 mo
3 mo
6 mo
6 mo
neonate
neonate
4 mo
4 y
4 y
de novo
6 mo
de novo
de novo
de novo
de novo
14 y
11 mo
3 mo
3 mo
de novo
13 mo
de novo
de novo
4 yr
30 mo
de novo
11 d
de novo
12 mo
de novo
3 y
de novo
4 mo
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
41 !
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
excessive movements
tarchycardia
ataxia
dysarthria
quadriplegia
excessive movements;
microcephaly
gait ataxia
peripheral spasticity
tachycardia,
hypertension
microcephaly
single seizure at 4 yr
tachycardia,
hyperthermia, sweating
microcephaly
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Table S1.1 (cont’d)
No.
EEG Findings
Severe EEG
Brain MRI
Severe MRI
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61#
62
63
64
65
66
67
68
69
70
71
72*
73*
74
75
76
77
78
79
80
81
normal
no irregularities other than diffuse slowing
no irregularities other than diffuse slowing
NA
NA
normal
NA
normal
NA
diffused low activities
low background activities
bilateral centrotemporal spikes
normal
diffuse slow activity
15 mo: slow posterior dominant rhythm
normal
NA
NA
NA
11 y: normal; 15 y: abnormal during sleep, frequent sharp
waves
NA
NA
NA
NA
NA
NA
NA
normal at 12 yr
NA
NA
NA
NA
normal
right-sided polyspike-wave formations
NA
NA
NA
NA
suppression-burst pattern
normal
NA
NA
+
+
+
+
+
++
++
42 !
normal
normal
normal
normal
global atrophy at 15 yr
13 mo: frontal lobe volume loss
normal
normal
normal at 13 mo
progressive cerebral and cerebellar atrophy,
brainstem atrophy, thin corpus callosum
ventricular enlargement; thin corpus callosum;
mild hypoplasia of caudate; hypoplasia of
inferior vermis
normal
normal
posterior left periventricular hypersignal
1 y: generalized thinning of corpus callosum,
relative paucity of deep white matte
temporal atrophy, ventricular enlargement and
mild temporal hypomyelination
Cortical (prominent frontally) subcortical atrophy.
Bilateral hypointense signals of globus pallidus
on SWI and T2* MRI sequences
Cortical (prominent frontally) subcortical atrophy.
Bilateral hypointense signals of globus pallidus
on SWI and T2* MRI
normal
normal
NA
NA
progressive global atrophy (8 y, 12 y)
NA
13 mo: mild hyperintensity in the occipital white
matter
Small medio- putaminal atrophy
normal
normal at 4 and 12 yr
normal at 12 mo
global atrophy at 5.5 yr
global atrophy and T2 hypointensity in globus
pallidi at 9 yr
T2 hypointensity in globus pallidi at 14 yr
normal
atrophy of right hippocampus
progressive global atrophy (1y, 5y, 8y)
NA
mild diffuse cortical atrophy
slight hyperintensity of the left pars triangularis
diffuse cerebral atrophy
normal
8 yr: cerebral atrophy
ventricular enlargement and dilated
subarachnoid spaces; moderate cortical atrophy;
dysmorphic corpus callosum; mild hypoplasia of
caudate muclei
++
++
++
++
+
++
++
++
++
++
+
+
++
++
+
+
++
+
++
++
++
Table S1.1 (cont’d)
No.
Drug Trialed
Drug Positive Response
DBS
NA
N
N
NA
Y
Y
tetrabenazine, trihexyphenidyl
Evaluated
Study
PMID or DOI
Menke et al 2016
Kulkarni et al 2015
Kulkarni et al 2015
27625011
26060304
26060304
Dhamija et al 2016
DOI: 10.1002/mdc3.12344
DBS
Positive
Response
NA
Y
Y
NA
N
N
N
NA
Menke et al 2016
Ananth et al 2016
Kelly et al 2019
Blumkin et al 2018
Ananth et al 2016
Saitsu et al 2016;
Sakamoto et al 2017
Danti 2017
Danti 2017
Waak et al 2018
Malaquias et al 2019
Kelly et al 2019
Schirinzi et al 2018
27068059
30682224
29801190
27625011
27068059
25966631; 27916449
28357411
28357411
28668776
31190250
30682224
30642806
Koy et al 2018
30103967
Koy et al 2018
Carecchio et al 2019
Kelly et al 2019
Yilmaz et al 2016
Feng at al. 2018
Waak et al 2018
Okumura et al 2018
Schirinzi et al 2018
Koy et al 2018
Koy et al 2018
Saitsu et al 2015
Ananth et al 2016
Ananth et al 2016
Ananth et al 2016
Ananth et al 2016
Schorling et al 2017
Schorling et al 2017
Waak et al 2018
Carecchio et al 2019
Carecchio et al 2019
Takezawa et al 2018
30103967
31216378
30682224
27278281
29758257
28668776
29935962
30642806
30103967
30103967
25966631
27068059
27068059
27068059
27068059
28628939
28628939
28668776
31076915
31076915
29761117
Gerald et al 2017
https://doi.org/10.1016/j.spen.2017.08.008
Kelly et al 2019
Koy et al 2018
Danti et al 2017
30682224
30103967
28357411
N
N
N
N
Y
N
N
N
Y
Y
N
N
Y
N
Y
N
N
Y
N
N
N
N
N
N
N
N
Y
Y
Y
N
N
N
Y
N
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61#
62
63
64
65
66
67
68
69
70
71
72*
73*
74
75
76
77
78
79
80
81
NA
clonazepam, valproic acid
clonidine, clonazepam
clonazepam, tetrabenazine, trihexyphenidyl
dexmedetomidine, opioids, benzodiazepam,
vecuronium, risperidone
NA
L-DOPA
NA
clonidine, valproic acid, clonazepam, bethanechol,
lorazepam, trihexyphenidyl, dexmedetomidine,
propofol, midazolam
diazepam, midazolam, clonazepam, phenobarbital,
bromazepam, haloperidol, tiapride, eperisone,
topiramate
NA
NA
carbamazepine, acetazolamide, oxcarbazepine
levodopa, clonazepam, trihexyphenidyl
NA
haloperidol, midazolam, propofol, phenytoin,
baclofen, clonazepam, levodopa, tetrabenazine,
clonidine, phenobarbital, lorazepam, curare
NA
NA
risperidone
NA
N
NA
N
topiramate
well-controlled on AED
well-controlled on AED
carbamazepine,
acetazolamide,
oxcarbazepine controlled
epileptic activity
N
NA
NA
N
tetrabenazine
trihexyphenidyl, clonazepam
trihexyphenidyl, clonazepam
NA
NA
midazolam, fentanyl, pimozide, clonazepam,
haploperidol, carbamazepine, acetazolamide,
diazepam, ketogenic diet
levetiracetam,clonazepam, tetrabenazine,
respiridone
baclofen, phenobarbitone, tetrabenazine
midazolam, fentanyl,
pimozide
levetiracetam, tetrabenazine
incomplete response to
tetrabenazine
NA
NA
NA
NA
NA
N
clonazepam, clonidine, trazodone, midazolam,
risperidone, tetrabenazine
clonazepam, clobazem, topiramate, levetiracetam,
valproic acid, clonidine, baclofen, tetrabenazine,
haploperido, diazepam, pentobarbital, propofol,
versed, fentanyl, dexmedetomidine
NA
NA
N
N
NA
N
tetrabenazine
baclofen, clobazem,
tetrabenazine, haploperido,
diazepam
oxcarbazepine, clonazepam, risperidone,
diazepam, tetrabenazine
diazepam, tetrabenazine
L-DOPA
levetiracetam
N
NA
haloperidol, baclofen, tetrabenazine, phenobarbital
tetrabenazine, phenobarbital
Trihexy-phenidyl, nitrazepam, clonazepam,
tetrabenazine, baclofen, L-DOPA, lev-etiracetam,
phenobarbital
Flunitrazepam, baclofen, trihexyphenidyl,
tetrabenazine, pimozide
NA
phenobarbital, clobazam, valproic acid, pyridoxine,
levetiracetam, fosphenytoin, valporate, clobazam,
zonisamide, rufinamide, high dose prednisone,
ACTH, IVIG, ketogenic diet
NA
NA
NA
NA
NA
NA
N
NA
N
well-controlled on AED
# Patient was presented to our clinic
43 !
N
N
N
NA
N
N
N
N
Y
N
N
N
Y
Y
N
N
Y
N
Y
N
N
Y
N
N
N
N
N
N
N
N
Y
Y
Y
N
N
N
Y
N
* Patients are siblings
Newly reported cases after Feng et al 2018 was publish
Figure S1.1 Correlation between seizure frequency and a severe EEG/MRI result
Clinical descriptions of EEG and MRI results were classified by one of the authors (C.S.)
as normal or not reported ( ), mild (+), or severe (++). The correlation of EEG and MRI
findings with either presence or absence of seizures or mutation status are illustrated
here and listed in Table S1.1. (A) Over 50% of patients with a seizure disorder (most
carrying LOF mutants) exhibit severe EEG. Also, patients carrying the GOF mutation
G203R frequently displayed seizure symptoms and severe EEG readings. (B) Patients
carrying the GOF mutations G203R and R209C showed occurrence of seizures, and
almost half also showed serious abnormalities in MRI. However, less than 50% of the
44 !
Figure S1.1 (cont’d) patients carrying GOF mutation E237K, E246K, or R209H showed
seizure activity, while almost 50% of them showed a severe MRI results. All values have
been shifted slightly (jitter with SD=5) to avoid overlap. Many patients with singleton LOF
mutations fell at 100% Seizures and 100% Severe MRI or EEG. The jitter was added to
better demonstrate how many different mutations result in patterns that fall in each
region of the graph.
45 !
REFERENCES
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Binds to the Extreme C Terminus of SNAP25 to Mediate the Action of
Gi/o-Coupled G Protein-Coupled Receptors. Mol Pharmacol, 89(1), 75-83.
doi:10.1124/mol.115.101600
Zwart, R., Reed, H., Clarke, S., & Sher, E.
(2016). A novel muscarinic
receptor-independent mechanism of KCNQ2/3 potassium channel blockade by
Oxotremorine-M.
221-228.
doi:10.1016/j.ejphar.2016.08.037
Pharmacol,
791,
Eur
J
65 !
CHAPTER 2: MOVEMENT DISORDER IN GNAO1 ENCEPHALOPATHY
ASSOCIATED WITH GAIN-OF-FUNCTION MUTATIONS
Modified from Feng, H., Sjögren, B., Karaj, B., Shaw, V., Gezer, A., & Neubig, R. R.
(2017). Movement disorder in GNAO1 encephalopathy associated with gain-of-function
mutations. Neurology, 89(8), 762-770.
DOI: https://doi.org/10.1212/WNL.0000000000004262
With permission from the American Academy of Neurology. All rights reserved.
Gezer, A. performed plasmid mutagenesis for the first set of GNAO1 mutations.
Wellhausen, N. did mutagenesis and cAMP assay in Figure S2.8.
Karaj, B. prepared six of the GNAO1 mutant plasmids used in Figure 2.2.
Vincent Shaw made Figure S2.5.
66!
2.1 Abstract
Objective: To define molecular mechanisms underlying the clinical spectrum of epilepsy
and movement disorder in individuals with de novo mutations in the GNAO1 gene.
Methods: We identified all GNAO1 mutations reported in individuals with epilepsy
(EIEE17) or movement disorders through April 2016; 15 de novo mutant alleles from 25
individuals were introduced into the Gαo subunit by site-directed mutagenesis in a
mammalian expression plasmid. We assessed protein expression and function in vitro in
HEK-293T cells by western blot and determined functional Gαo-dependent cyclic AMP
inhibition with a co-expressed α2A adrenergic receptor.
Results: Of the 15 clinical GNAO1 mutations studied, 9 show reduced expression and
loss of function (LOF, <90% maximal inhibition). Six other mutations show variable levels
of expression but exhibit normal or even gain-of-function (GOF) behavior, as
demonstrated by significantly lower EC50 values for α2A adrenergic receptor-mediated
inhibition of cAMP. The GNAO1 LOF mutations are associated with epileptic
encephalopathy while GOF mutants (such as G42R, G203R and E246K) or normally
functioning mutants (R209) were found in patients with movement disorders with or
without seizures.
Conclusions: Both LOF and GOF mutations in Gαo (encoded by GNAO1) are
associated with neurological pathophysiology. There appears to be a strong predictive
correlation between the in vitro biochemical phenotype and the clinical pattern of
67!
epilepsy vs. movement disorder.
2.2 Introduction
Epilepsy is one of the most common neurological disorders in the United States.
Severe early onset seizures can result in epileptic encephalopathy (Capovilla, Wolf,
Beccaria, & Avanzini, 2013). There are at least 52 different gene mutations that cause
early infantile epileptiform encephalopathy (EIEE) (McTague, Howell, Cross, Kurian, &
Scheffer, 2016). Mutations in the same genes also cause other neurodevelopmental
abnormalities (Berkovic et al., 2004; Sherr, 2003). A key challenge in genetic epilepsies
has been understanding the genotype/phenotype relations of causal genes; this may
require biochemical analysis (Noebels, 2015).
Mutations in the heterotrimeric G protein Gαo (GNAO1 gene) cause an autosomal
dominant epileptiform encephalopathy (EIEE17, OMIM: 615473) (Nakamura et al., 2013).
In this original paper, all 4 mutations were characterized as having loss-of-function (LOF).
More recently, an extended spectrum of “GNAO1 encephalopathies” was identified in
which individuals had movement disorders but minimal to no seizures (Ananth et al.,
2016; Marce-Grau et al., 2016). Here, we use “GNAO1 encephalopathy” to describe the
entire clinical spectrum of
individuals with pathological GNAO1 mutations. A
genotype-phenotype correlation was also recently noted (Menke et al., 2016); certain
mutations (e.g. E246K and several R209 alleles) were found specifically in children with
hypotonia, developmental delay, and chorea but no epilepsy. However, the mechanistic
68!
basis for this genotype-phenotype correlation remains unknown.
GNAO1 encodes the α subunit of Go, a heterotrimeric G protein (consisting of α and
βγ subunits) which is highly abundant in the central nervous system, comprising about 1%
of brain membrane protein (Sternweis & Robishaw, 1984). Gαo mediates signals from a
wide-range of inhibitory receptors including GABAB, α2A adrenergic, adenosine A1 and
dopamine D2 receptors. A canonical function of Gαi/o family proteins is inhibition of cAMP
(Ghahremani, Cheng, Lembo, & Albert, 1999). The identification of LOF mutations in
ADCY5 (which encodes adenylate cyclase 5, the enzyme that produces cAMP) in
dyskinesia and chorea patients directly links reduced cAMP to involuntary movement
disorders (Carapito et al., 2015; Chang et al., 2016; Mencacci et al., 2015; Morgan,
Kurek, Davis, & Sethi, 2016; Raskind et al., 2017). This is inconsistent with LOF behavior
of Gαo.
Here we assessed expression and function of human mutations in GNAO1 (Allen et
al., 2013; Ananth et al., 2016; Consortium, Project, & Consortium, 2014; Dhamija, Mink,
Shah, & Goodkin, 2016; Dietel, 2016; Epi KCEaekce, 2016; Kulkarni, Tang, Bhardwaj,
Bernes, & Grebe, 2016; Law et al., 2015; Marce-Grau et al., 2016; Nakamura et al., 2013;
Saitsu et al., 2016; Talvik, 2015; Zhu et al., 2015). Our biochemical analysis identified
both loss- and gain-of-function behaviors; the latter are associated with movement
disorders while the former are primarily found in individuals with epileptiform
encephalopathies. This mechanistic insight has important implications for therapies of
69!
GNAO1 encephalopathies.
Appendix B provides updated data on an additional list of 25 mutations analyzed
after the publication of Feng et al. 2017.
2.3 Materials and Methods
2.3.1 Materials
UK14,304 was from Sigma Aldrich (St Louis, MO). Forskolin was from Calbiochem
(San Diego, CA). Pertussis toxin (PTX) was from List Biological Laboratories (Campbell,
CA). Protease inhibitor cocktail was from Roche (Roche, Indianapolis, IN). If not
otherwise specified, all tissue culture reagents were from Thermo Fisher Scientific
(Waltham, MA) and all chemicals were from Sigma Aldrich.
2.3.2 DNA constructs and mutagenesis
Cloning of the porcine α2A AR in the pEGFP vector with an amino-terminal HA tag
has been previously reported (Brink, Wade, & Neubig, 2000). PTX-insensitive murine
Gαo (Gnao1; Gαo p.C351G PTXi) (Jeong & Ikeda, 2000) and RGS- and PTX-insensitive
murine Gαo (Gαo p.G184S/C351G; RGS/PTXi) (Jeong & Ikeda, 2000) in the pCI vector
were obtained from Dr. Stephen Ikeda (Guthrie Research Institute, Sayre, PA). Using the
PTXi murine Gαo C351G as a template, 15 point mutations or deletions were introduced
using
the Stratagene Quickchange
II Site-Directed Mutagenesis kit
(Agilent
Technologies, Santa Clara, CA). Primers for mutagenesis were designed using the
algorithm described at http://www.stratagene.com/sdmdesigner/default.aspx (Table
70!
S2.1). Mutations were verified by DNA sequencing at the RTSF Genomics Core at
Michigan State University and sequences were analyzed using Clone Manager 9 (Sci-Ed
Software, Denver, CO). The protein sequence of the human and murine Gαo are highly
similar (98% identical) and do not differ in sequence at any of the mutated positions
(Figure S2.1). As a positive control the RGS-insensitive GOF mutant Gαo p.G184S (Lan
et al., 1998) was used.
2.3.3 Cell culture and transfections
Human embryonic kidney (HEK-293T) cells were maintained in a humidified
incubator at 37°C with 5% CO2 and grown to 95% confluence in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml
penicillin and 100 µg/ml streptomycin. Cells were transfected using Lipofectamine 2000
according to the manufacturer’s recommended protocol. All transfections were
performed under serum-free conditions in Opti-MEM. Transfections were allowed to
proceed for 4-5 h before the media was changed back to DMEM with 10 % FBS.
Experiments were run 24 h after transfection.
For western blot, cells in 6-well plates were transfected using 2 µg of DNA and 8 µl of
Lipofectamine 2000 per well. For cAMP assays, cells were plated in 60-mm dishes. DNA
was kept constant at 4 µg (2 µg of Gαo or pcDNA and 2 µg of α2A AR) and 10 µl of
Lipofectamine2000 per plate was used. In the dominant-negative study, a total of 8 µg
DNA was added to cells in 60-mm dishes with 10 µl Lipofectamine2000 per plate. In all
71!
cases, empty vector (pcDNA3.1) was used to adjust the total amount of DNA.
2.3.4 SDS-PAGE and Western blot
Cells were harvested at 4oC in lysis buffer (20 mM Tris-HCl, pH7.4, 150 mM NaCl, 1
mM EDTA, 1 mM β-glycerophospate, 1% Triton X-100, 0.1% SDS, with protease
inhibitor) and then sonicated for 10 min at 4oC. Total protein concentrations in the cell
lysates were determined by BCA protein assay (Pierce; Rockford, IL) and adjusted with
an appropriate volume of Laemmli buffer (BioRad; Hercules, CA) with 5 %
2-mercaptoethanol (β-ME). Equal amounts of protein in each lane were resolved on a
12 % SDS-PAGE gel for 1 h at 160 V. Samples were transferred to an Immobilon-FL
PVDF membrane (Millipore, Billerica, MA) for 1 h at 100 V, 400 mA on ice and subjected
to Quantitive Infrared Western immunoblot analysis. The membrane was immersed in
Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 h with gentle shaking at
room temperature. The membrane was simultaneously incubated with anti-Gαo (rabbit;
1:1,000; sc-387; Santa Cruz biotechnologies, Santa Cruz, CA) and anti-actin (goat;
1:1,000; sc-1615; Santa Cruz) antibodies diluted in Odyssey blocking buffer with 0.1%
Tween-20 overnight at 4oC. Following four 5 min washes in phosphate-buffered saline,
0.1 % Tween-20 (PBS-T), the membrane was incubated for 1h at room temperature with
secondary antibodies (both 1:10,000; IRDye® 800CW Donkey anti-rabbit; IRDye®
680RD Donkey anti-goat; LI-COR Biosciences) diluted in Odyssey blocking buffer with
0.1 % Tween-20. The membrane was subjected to four 5 min washes in PBS-T and a
72!
final rinse in PBS for 5 min. The membrane was kept in the dark and the infrared signals
at 680 and 800 nm were detected with an Odyssey Fc image system (LI-COR
Biosciences). The Gαo polyclonal antibody recognizes an epitope located between
positions 90-140 Gαo (Santa Cruz, personal communication), which shouldn’t be
affected by any of the mutations studied.
2.3.5 cAMP measurements
LANCE Ultra cAMP assays (Perkin Elmer; Waltham, MA) were performed in
accordance with
the manufacturer’s
instructions. Briefly, HEK-293T cells were
transfected as indicated above. 100 ng/ml PTX was added the day before the assay to
inhibit endogenous Gi/o proteins. Cells were dissociated from dishes using Versene on
the day of experiment. Then cells (2,000 cells/well in 5 µl) were transferred to a white
384-well microplate (Perkin Elmer) and incubated with various concentrations of
UK14,304 and Forskolin (final 1µM; 5 µl/well) for 30 min at room temperature. A cAMP
standard curve was generated in triplicate according to the manual. Finally, europium
(Eu)-cAMP tracer (5µL) and ULight™-anti-cAMP (5µL) were added to each well and
incubated for 1h at room temperature. The plate was read on a TR-FRET microplate
reader (Synergy NEO; Biotek, Winooski, VT).
2.3.6 Data analysis and statistics
Quantification of infrared (IR) Western blot signals was performed using Image
Studio Lite
(LI-COR Biosciences).
Individual bands were normalized
73!
to
the
corresponding actin signals, and WT Gαo was set as control. All data was analyzed using
GraphPad Prism 6.0 (GraphPad; LaJolla, CA). Dose response curves were fit using
non-linear least squares regression. Expression levels and Normalized % inhibition were
analyzed with one-way ANOVA with Bonferroni’s post hoc tests for multiple comparisons.
Log EC50 values for the NF and GOF mutants were analyzed by paired t-test. Data are
presented as mean ± SEM. and a p-value less than 0.05 was considered significant.
2.4 Results
2.4.1 Most pathogenic GNAO1 mutations cause reduced Gαo protein expression
To evaluate 15 mutations in GNAO1 (Figure 2.1A and S2.1) that were previously
identified in patients with epilepsy or other neurodevelopmental disorders (Allen et al.,
2013; Ananth et al., 2016; Consortium et al., 2014; Dietel, 2016; Epi KCEaekce, 2016;
Kulkarni et al., 2016; Law et al., 2015; Marce-Grau et al., 2016; Nakamura et al., 2013;
Saitsu et al., 2016; Talvik, 2015; Zhu et al., 2015), we performed Western blots in
HEK-293T cells transiently transfected with each mutant. The majority of mutants (12)
showed significantly lower protein levels than wildtype (WT) Gαo, whereas three
separate Arg209 mutant alleles showed essentially normal expression as did the
previously described GOF mutant G184S (Fu et al., 2004) (Figure 2.1B-E).
74!
Figure 2.1 Location and protein expression levels of human GNAO1 mutations
related to epileptic encephalopathy.
(A) Location of 15 mutations (G40R, G42R, D174G, T191_F197del, L199P, G203R,
R209C, R209G, R209H, A227V, Y231C, E246K, N270H, F275S, and I279N) mapped on
the Gαo amino acid sequence. (B, C) Representative Western blots of Gαo protein
expression from HEK293T cells transiently transfected with each Gαo mutant. (D, E)
Quantification of relative protein levels of each Gαo mutant compared to WT Gαo. Graphs
are the result of 3 independent experiments and data are presented as mean ± SEM.
p<0.01**, p<0.0001**** using One-way ANOVA with Bonferroni’s post-hoc test for
pairwise comparison.
75!
2.4.2 Validation of an in vitro assay to assess function of GNAO1 mutations
We used inhibition of forskolin-stimulated cAMP levels as a functional readout to
allow efficient quantification of Gαo effects with
full concentration curves
for
agonist-mediated signaling. We co-transfected Gαo plasmids with α2A adrenergic
receptor (α2A AR) cDNA (Goldenstein et al., 2009). Robust inhibition of cAMP by the α2
adrenergic agonist UK14,304 depends on both the transfected receptor and the Gαo
protein (Figure S2.2A, S2.2B). A pertussis toxin (PTX)-insensitive Gαo (C351G) (Ikeda &
Jeong, 2004) was used to create all mutant constructs and WT, enabling inactivation of
endogenous Gi/o proteins using PTX.
When PTX eliminates the inhibitory signaling through Gi/o, α2A AR couples weakly to
Gs and stimulates adenylate cyclase (AC) (Wade et al., 1999), resulting in increased
cAMP levels after PTX treatment in the absence of a transfected Gαo (Figure S2.2B).
Consequently, the fractional inhibition of AC by Gαo mutants was assessed as the
decrease from the high control level of cAMP (PTX but no Gαo - 0%) to the low level with
WT Gαo (PTX and PTXi Gαo - 100%). This is termed Normalized % inhibition. PTX
treatment did not alter the ability of the PTX-insensitive Gαo to mediate α2A
AR-stimulated cAMP inhibition (Figure S2.2C). Hence, this is a good system to study
functional consequences of Gαo mutations.
76!
2.4.3 Nine GNAO1 mutations result in loss- (LOF) or partial loss-of-function
(PLOF)
Six mutants showed essentially complete loss-of-function (LOF) with normalized
inhibition below 40% (Figure 2.2A and Table 2.1). All of these mutants also showed low
expression levels (11-35% of control; Figure 2.1). Three mutants (A227V, Y231C, and
I279N; Figure 2.2A, 2.2E and Table 2.1) had intermediate effects and were classified as
partial loss-of-function (PLOF) mutants. The I279N mutation showed a modestly reduced
maximal inhibition of cAMP levels (Figure 2.2E and Table 2.1). This mutant also
produced a very low EC50 value (0.7 nM vs 25 nM for WT Gαo), which might explain the
discrepancy between the quite low expression levels (15% of WT) while maintaining
good maximal inhibition in the cAMP inhibition assay. Based on the maximum inhibition
below 90% of control, however, we classified this mutation as PLOF (Tables 2.1 and
2.3).
The dominant nature of the clinical picture in the GNAO1 encephalopathies raised
the question of whether the LOF mutations are actually dominant negative mutations that
interfere with the function of the remaining normal Gαo protein expressed in
heterozygous individuals. However, in co-expression studies of WT and mutant Gαo at
plasmid ratios of 1:1 or 1:2, there was no evidence of a dominant negative action (Figure
S2.3). This suggests that the effect of LOF mutations is through a haploinsufficiency
mechanism rather than a dominant negative one.
77!
Figure 2.2 Effect on α2A AR-mediated cAMP inhibition by GNAO1 mutants.
(A-C) Dose-response curves of representative GNAO1 mutants. (A) Dose-response
curves of LOF and PLOF mutants (G40R, L199P, N270H, F275S, A227V, Y231C)
showing changes in cAMP production in response to the AC activator forskolin and α2AR
agonist UK14,304, compared to the positive control (WT) and negative control (pcDNA).
(B) Dose-response curves of functioning Gαo mutants showing changes in cAMP
production in response to the α2AR agonist UK14,304. All dose-response curves are
shown in comparison with WT and G184S. (C) G42R displays a biphasic dose-response
78!
Figure 2.2 (cont’d) curve with cAMP inhibition at low concentrations (GOF), followed by
enhancement of cAMP levels at higher concentrations of UK14,304. (D) Quantification of
EC50 of functioning Gαo mutants. G42R, G203R and E246K exhibit significantly
increased potency for α2A AR-mediated cAMP inhibition similar to the known GOF
mutation G184S. p<0.05*, p<0.01**, p<0.001*** using paired t test between WT and
each mutant separately (Figure S2.4). E. Percentage of maximum inhibition (n=5) was
normalized to pcDNA (0%; resulting in activation of cAMP) and WT (100%).
p<0.0001**** using One-way ANOVA with Bonferroni’s post-hoc test for pairwise
comparison. Note that the maximum inhibition of G42R was calculated at UK14,304 of
15.8 nM.
Table 2.1 Functional data for loss-of-function (LOF) and partial loss-of-function
(PLOF) mutants.
Mutations
pcDNA (no Gαo)
WT
Expression
(% of WT)+
cAMP at 5µM
UK14,304
(% of un-stimulated)+
Normalized %
inhibition+
LogEC50+
EC50
(nM)
0
100
880 ± 30
23 ± 1
0
100
-6.48 ± 0.05
526
-7.71 ± 0.07
25
118G>A
Gly40Arg
G40R
27 ± 1 ****
900 ± 70
-2 ± 8 ****
-6.52 ± 0.13
355
521A>G
Asp174Gly
D174G
21 ± 7 ****
250 ± 20
-1 ± 7 ****
-6.41 ± 0.19
502
517_592del Thr191_Phe197del T191_F197del 11 ± 3 ****
290 ± 20
-16 ± 7 ****
-6.36 ± 0.20
525
596T>C
Leu199Pro
L199P
27 ± 2 ****
570 ± 30
36 ± 3 ****
-6.12 ± 0.07
784
680C>T
Ala227Val
A227V
24 ± 3 ****
320 ± 20
65 ± 3 ****
-6.32 ± 0.13
586
808A>C
Asp270His
N270H
30 ± 2 ****
860 ± 60
2 ± 7 ****
-6.42 ± 0.16
499
692A>G
Tyr231Cys
Y231C
35 ± 2 ****
340 ± 20
63 ± 2 ****
-6.34 ± 0.06
472
824T>C
Phe275Ser
F275S
22 ± 4 ****
720 ± 60
19 ± 7 ****
-6.46 ± 0.12
395
836T>A
Ile279Asp
I279N
15 ± 4 ****
100 ± 10
84 ± 1
-9.18 ± 0.15
0.7
+ Mean ± SEM; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, one-way ANOVA
79!
2.4.4 Six GNAO1 mutations result in gain-of-function (GOF) or normal function
(NF)
Unexpectedly, a significant number of pathological GNAO1 mutants showed
essentially normal or even GOF behavior (Figure 2.2B-E and Table 2.2). As a
benchmark for GOF behavior, we used our previously described RGS-insensitive G184S
mutant (Fu et al., 2004; Goldenstein et al., 2009; Kehrl et al., 2014; Lan et al., 1998),
which shows a mild seizure phenotype in mouse models (Kehrl et al., 2014). It produced
a small increase in the maximum inhibition of forskolin-stimulated cAMP levels in
response to UK14,304 (Table 2.2). More importantly, it had a significantly more potent
response to the α2AR agonist UK14,304 (Figure 2.2D & S2.4A). This represents a
2-3-fold increase in signal strength at low agonist concentrations. Three of the human
pathological GNAO1 mutants also showed GOF behavior by this criterion. The G203R,
E246K and G42R mutants produced robust inhibition of cAMP with significantly lower
EC50 values for the α2AR agonist UK14,304 (Figure 2.2D, S2.4 and Table 2.2). G203R
and E246K showed normal inhibition with modest decreases in EC50 (Table 2.2, Figure
2.2D, 2.2E and S.4). This is similar to the effect on EC50 seen for the bona fide GOF
mutant G184S. The G42R mutant showed the lowest EC50 of any of the mutants (Figure
S2.4), at least 50-fold lower than the WT protein (Figure 2.2C, 2.2D). However, the
inhibition mediated by G42R is followed by activation of cAMP with increasing
concentrations of UK14,304 (Figure 2.2C). The calculated Normalized % inhibition for
80!
the G42R mutant is essentially identical to that of WT Gαo which combined with its very
high potency for agonist-mediated inhibition suggests GOF behavior.
Three other patient-derived mutations (R209G, R209H, and R209C) showed almost
completely normal function and nearly normal expression levels and are designated as
normal function (NF) mutants (Figure 2.2B, 2.2D-E, S2.4, and Tables 2.2 & 2.3). The
EC50 values for R209G and R209H mutant were not significantly different from WT, while
the value for R209C was modestly but significantly higher (Table 2.2, Figure 2.2D &
S2.4).
81!
Table 2.2 Functional data for normal and gain-of-function (GOF) mutants.
Group
pcDNA (no Gαo)
WT
Expression
(% of WT)+
cAMP at 5µM
UK14,304
(% of unstimulated)+
Normalized %
inhibition+
LogEC50+
EC50
(nM)
-6.48 ± 0.05
525
-7.71 ± 0.07
0
100
0
100
880 ± 30
23 ± 1
15 ± 1
550G>A++ Gly184Ser
G184S
105 ± 5
108 ± 0.1
-8.06 ± 0.09 †
124G>C
Gly42Arg
G42R
70 ± 6 ****
170 ± 10
99 ± 0.1
-9.34 ± 0.09 †††
607G>A
Gly203Arg
G203R
72 ± 9 ****
736G>A Glu246Lys
E246K
74 ± 6 **
625C>G
Arg209Gly
R209G
77 ± 5
626G>A
Arg209His
R209H
109 ± 7
625C>T
Arg209Cys
R209C
96 ± 6
31 ± 3
39 ± 2
74 ± 4
21 ± 1
52 ± 2
100 ± 1.3
-8.06 ± 0.06 †††
98 ± 0.2
-8.00 ± 0.12 †
96 ± 0.2
-7.75 ± 0.09
100 ± 0.1
-7.52 ± 0.02
97 ± 0.2
-7.39 ± 0.08 ††
25
9.7
0.5
9.3
12
18
30
47
+ Mean ± SEM; ++ Not a human mutation; Underlined EC50 Values are significantly
different from that of WT; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, one-way ANOVA; †,
p<0.05; ††, p < 0.01; †††, p < 0.001 by paired t-test
82!
Table 2.3 Correlation between cAMP inhibition and clinical diagnosis.
#
Human GNAO1 Mutations
1
124G>
Gly42Arg
G42R
cAMP
Inh.
GOF
C
2
736G>A Glu246Lys
E246K
GOF
3
736G>A Glu246Lys
E246K
GOF
4
736G>A Glu246Lys
E246K
GOF
5
736G>A Glu246Lys
E246K
GOF
6
736G>A Glu246Lys
E246K
GOF
7
625C>
Arg209Gly
R209G
NF
G
8
626G>A Arg209His
R209H
NF
!
Epilepsy
Movement
Disorder
Age
Sex
Ref.
++
Unknown
F
(Zhu et
al.,
2015)
++
13 yrs
F
(Saitsu
et al.,
2016)
++
5.5 yrs
M
(Ananth
(twins)
et al.,
2016)
++
5.5 yrs
F
(Ananth
(twins)
et al.,
2016)
++
Decease
F
(Ananth
d at 10
yrs 3
months
et al.,
2016)
++
15 yrs
M
(Ananth
et al.,
2016)
++
Decease
F
(Ananth
d at 4 yrs
7 months
et al.,
2016)
++
16 yrs
M
(Ananth
et al.,
2016)
83!
Table 2.3 (cont’d)
9
626G>A Arg209His
R209H
NF
10 626G>A Arg209His
R209H
NF
11 626G>A Arg209His
R209H
NF
++
8 yrs
M
(Kulkarn
i et al.,
2016)
++
6yrs
M
(Kulkarn
i et al.,
2016)
++
5 yrs
M
(Radhik
a
Dhamija
, 2016)
12 625C>T Arg209Cys R209C
NF
+
++
18 yrs
F
(Saitsu
et al.,
2016)
13 607G>A Gly203Arg G203R
GOF
++
++
8 yrs
F
(Nakam
ura et
al.,
2013)
14 607G>A Gly203Arg G203R
GOF
+
++
14
F
(Saitsu
15 607G>A Gly203Arg G203R
GOF
++
++
3 yrs
F
(Dietel,
months
et al.,
2016)
2016)
4 yrs
F
(Talvik,
9 months
2015)
20
F
(Saitsu
months
et al.,
2016)
16 692A>G Tyr231Cys
Y231C
PLOF
++
17 680C>T Ala227Val
!
A227V
PLOF
++
84!
Table 2.3 (cont’d)
18 836T>A
Ile279Asp
I279N
PLOF
++
19 836T>A
Ile279Asp
I279N
PLOF
++
20 118G>A Gly40Arg
G40R
LOF
++
21 521A>G Asp174Gly D174G
LOF
++
13 yrs
F
(Nakam
ura et
al.,
2013)
2 yrs
M
(Epi
KCEaek
ce,
2016)
10
F
(Law et
months
al.,
2015)
4 yrs
F
(Nakam
1 month
ura et
al.,
2013)
22
517_59
Thr191_
T191_
LOF
++
+
Decease
F
(Nakam
2del
Phe197del
F197del
d at 11
months
ura et
al.,
2013)
23 596T>C
Leu199Pro
L199P
LOF
++
+
20
F
(Marce-
24 824T>C Phe275Ser
F275S
LOF
++
25 808A>C Asp270His
N270H
LOF
++
!
months
Grau et
al.,
2016)
9 yrs
F
(Allen et
al.,
2013)
3 yrs
F
(Consort
ium et
al.,
2014)
GOF: gain of function; NF: normal function; PLOF: partial loss of function; LOF: loss of
function. ++: major symptoms, +: minor symptoms
85!
2.4.5 Clinical correlation with biochemical behavior of mutant GNAO1 alleles
To address genotype/phenotype correlations for GNAO1 encephalopathy, we
reviewed the case reports of all 25 individuals who had GNAO1 mutations that had been
reported by April, 2016. They have a range of clinical patterns, which extend from early
severe epileptic encephalopathy with prominent tonic seizure activity to individuals with a
dominant choreo-athetotic movement disorder with virtually no evidence of seizures.
There are also individuals (Dietel, 2016; Saitsu et al., 2016), including one of the original
4 cases (Nakamura et al., 2013) (patient #13 – G203R; Table 2.3), who had multiple
seizures but also showed prominent choreo-athetosis.
In 2016, a clinical report (Ananth et al., 2016) described a unique series of 6 patients
with GNAO1 mutations and a pronounced movement disorder, virtually without seizures.
They had global developmental delay and hypotonia from infancy and all developed
chorea by ages 4-11. In the majority of cases it was intractable, leading to death in two
cases. Four patients carried the E246K allele, which we have found to be a GOF
mutation. The other two mutations found in this group (R209G and R209C) exhibited
essentially normal function in our cAMP inhibition measurements. There are several
other reports (Allen et al., 2013; Ananth et al., 2016; Consortium et al., 2014; Epi
KCEaekce, 2016; Kulkarni et al., 2016; Law et al., 2015; Marce-Grau et al., 2016; Saitsu
et al., 2016; Talvik, 2015; Zhu et al., 2015) of GNAO1 mutations in individuals with a
predominant movement disorder with or without seizures. This distinction of clinical
86!
patterns based on certain mutant alleles in GNAO1 encephalopathy patients was noted
very recently (Menke et al., 2016) but without information about biochemical
mechanisms. Table 2.3 summarizes the Gαo biochemical function from the present
report and its relation to seizure disorder or movement disorder in literature reports for
these mutations. GOF and NF mutations are nearly always found when movement
disorder is the predominant feature of the clinical pattern. Mutations that have pure LOF
or PLOF biochemical phenotypes are seen in individuals with epileptic encephalopathy
without pronounced choreoathetosis. A number of patients exhibit both seizures and
movement disorder. We have indicated in Table 2.3 with + or ++ which of these features
is predominant or less so. Further studies will be needed based on new cases and/or
additional mutations, but there does appear to be a clear pattern emerging about a
genotype-phenotype correlation that is driven by a GOF/LOF difference in mutant
GNAO1 alleles.
2.4.6 Location of mutations linked to GNAO1 encephalopathies in the Gαo protein
To investigate the structural basis for the effects of mutations in Gαo, the mutations
were modelled onto the published crystal structure of Gαo in complex with RGS16 (PDB:
3C7K; Figure S2.5). The locations of mutations within the Gαo structure segregated
according to their function. The GOF mutations are all near G184S and close to the
ribose and phosphate moieties of the bound GDP. The LOF mutants are more broadly
scattered throughout the GTPase domain and may destabilize protein folding or stability
87!
consistent with their markedly reduced expression levels. The PLOF mutations are
clustered in the GTPase domain but away from the bound GDP. This striking
structure-function correlation may facilitate prediction of the function of new mutations
but ultimately a rigorous biochemical analysis will provide definitive understanding of
function.
2.5 Discussion
The concept of a “GNAO1 encephalopathy” has developed based on the
identification of at least 15 different mutations in the GNAO1 gene (Allen et al., 2013;
Ananth et al., 2016; Consortium et al., 2014; Epi KCEaekce, 2016; Kulkarni et al., 2016;
Law et al., 2015; Marce-Grau et al., 2016; Nakamura et al., 2013; Saitsu et al., 2016;
Talvik, 2015; Zhu et al., 2015) associated with various combinations of epilepsy,
developmental delay, hypotonia, and choreo-athetotic movement disorders. Our study
demonstrates GOF as well as LOF mutations in GNAO1 and describes a clear
correlation between biochemical and clinical characteristics. The existence of these
unexpected GOF mutations has important therapeutic implications. Specifically, one
might expect that different approaches to therapy would be needed for different
mutations (i.e. agonists for LOF and antagonists for GOF mutants).
We chose inhibition of cAMP production as the functional readout to assess the Gαo
mutants because of
the
robust measurements permitting complete agonist
concentration-response studies. This was critical to our findings since the GOF mutants
88!
were detected primarily through their ability to increase signals at low agonist
concentrations (Figure 2.2 and Table 2.2). Our previously studied GOF mutant (G184S)
which is insensitive to the inhibitory influence of RGS proteins shows such a “left-shift” of
agonist concentration response curves in vitro (Clark, Harrison, Zhong, Neubig, &
Traynor, 2003; Fu et al., 2004) and in vivo (Goldenstein et al., 2009; Lamberts et al.,
2013), and also has a mild seizure phenotype in a mouse model (Kehrl et al., 2014). One
might argue that cAMP is not the best choice of functional measures for epilepsy since
N-type Ca++ channels or the synaptic release mechanism proteins are critical for
regulation of neurotransmitter release. However, the apparent correlation of clinical
patterns with the biochemical behavior in our cAMP assay does suggest that function
assessed in this way is relevant to functionality in humans. The clear pathological effect
of the R209 mutations (with at least 3 individuals carrying distinct alleles), however, does
raise the question of why a protein with normal expression and function would cause
pathology. It is possible that the R209 mutations have a selective loss of one of the other
functional outputs while retaining a normal ability to inhibit AC. Alternatively; there may
be selective alterations in expression or localization in neurons that are not accurately
reflected in our HEK-293T cell studies of cAMP regulation. A full understanding of the
causal mechanisms in GNAO1 encephalopathies requires additional studies of these
mutant Gαo proteins in neurons and with different functional readouts.
The locations of mutations in the protein structure may partially explain their
89!
functional influences. All functioning mutants (NF and GOF) are located around the RGS
binding domain, while most of the LOF or PLOF mutants are near the GDP binding
region. Two exceptions are D174G and T191_F197del. D174 forms a salt bridge with
R162 and mutations in this position may disrupt this interaction. T191_F197del truncates
two beta sheets as well as their linking region, which would be expected to decrease
protein stability of Gαo.
A dominant genetic effect from GOF mutants is not unusual but the fact that the LOF
mutations result in a severe autosomal dominant disorder is a bit surprising. We have
ruled out a biochemical dominant negative mechanism of these mutations, at least for
cAMP regulation, suggesting a haploinsufficiency mechanism. In mice, homozygous Gαo
knockouts exhibit seizures as well as hyperactive turning behavior (Jiang et al., 1998).
We did not, however, observe spontaneous seizures or an increased sensitivity to
pentylenetetrazol (PTZ) kindling in heterozygous Gnao1+/- knockouts (Kehrl et al., 2014).
This suggests that humans are more susceptible to haploinsufficiency of Gαo than are
mice. In contrast, we observed enhanced kindling sensitivity and reduced survival in our
Gnao1+/G184S knock-in mouse model possibly due to seizures (Kehrl et al., 2014).
Furthermore, these mice display early neonatal lethality (Kehrl et al., 2014) of unclear
mechanism which may be similar to the hypotonia seen in human patients carrying GOF
mutations. We do not know whether the abnormalities in these mice are due to brain
developmental abnormalities or acute signaling effects. Further studies are needed to
90!
better understand this and to determine whether our Gnao1+/G184S mutant mouse might
represent a useful pre-clinical model for individuals with GNAO1 GOF mutations.
To date, all characterized GNAO1 mutants have been reported as LOF mutations.
The G203R mutant in the original paper (Nakamura et al., 2013) was reported as a LOF
mutant for regulation of N-type Ca++ channels. Similarly, a G42R mutation in Gαi1 was
reported as a LOF mutant based on biochemical studies (Bosch et al., 2012). The unique
approach that we have taken with detailed cAMP dose-response studies in a mammalian
cell model permitted our recognition of the GOF mechanisms (e.g. the Gαo G42R
mutation). The patient with the G203R mutation, which we found to have GOF for cAMP
inhibition, had a very different clinical pattern than the other 3 patients in the original
study. She had a much later onset of disease (7 months) as well as developmental delay
and severe chorea with only localized seizures (Nakamura et al., 2013). A similar clinical
pattern was observed in two more, recently described, patients with this same mutation
(Dietel, 2016; Saitsu et al., 2016). All patients carrying the GOF mutations identified here
appear distinct from the strict EIEE pattern (see Table 3). In comparison, patients with
LOF or PLOF mutations were diagnosed with either Ohtahara syndrome (Y231C, I279N,
D174G, T191_F197del) or early-infantile epileptic encephalopathy (A227V, L199P,
N270H, F275S). Thus GOF and LOF mutations in Gαo appear to result in different
disease mechanisms likely requiring different therapeutic approaches.
It has remained challenging to convert knowledge about genetic epilepsy mutations
91!
into therapies. The GNAO1 encephalopathies may be different because of the eminently
targettable nature of the receptors that drive Gαo signaling pathways. A critical question
then becomes which receptors might be involved. Interestingly, activation of many
Gi/o-coupled receptors is associated with suppression of seizures. Adenosine A1
receptors may play a role in the efficacy of the ketogenic diet (Masino et al., 2011) and
agonists at group II metabotropic glutamate receptors are anticonvulsant in various
models (Dalby & Thomsen, 1996). The opposite situation is also seen; GABABR agonists
exacerbate absence seizures while GABABR antagonists suppress them (Han HA, 2012).
Identifying which receptors or downstream signaling effectors of Gαo contribute to
mechanisms of encephalopathy from LOF or GOF GNAO1 mutations could therefore
reveal potential targets for novel anti-convulsant drug development.
We have identified distinct biochemical mechanisms of pathogenic human GNAO1
mutations that may improve the understanding of the heterogeneous clinical spectrum of
GNAO1-associated epilepsy and movement disorders. Furthermore, these results also
carry significant implications for personalized therapeutics in GNAO1 encephalopathies.
92!
APPENDICES
93!
APPENDIX A
SUPPLEMENTAL DATA
94!
Table S2.1 Primer sequences for mutagenesis to create GNAO1 mutants.
Mutation
Protein
R209H
(Arg209His)
F: 5'-GTGGATCCACTTCTTGTGTTCAGATCGCTGGCC-3'
R: 5'-GGCCAGCGATCTGAACACAAGAAGTGGATCCAC-3'
Primer Sequences
DNA
626
G>A
G203R
(Gly203Arg)
E246K
(Glu246Lys)
G42R
(Gly42Arg)
R209C
(Arg209Cys)
I279N
(Ile279Asp)
T191_F197del
(Thr191_Phe1
97del)
R209G
(Arg209Gly)
A227V
(Ala227Val)
F275S
(Phe275Ser)
N270H
(Asp270His)
G40R
(Gly40Arg)
607
G>A
736
G>A
F: 5’-AGATCGCTGGCCTCTGACGTCAAACAGCC-3’,
R: 5’-GGCTCTTTGACGTCAGAGGCCAGCGATCT-3’
F: 5'-AAGAGCATGAGAGACTTGTGCATGCGGTTCGTG-3'
R: 5'-CACGAACCGCATGCACAAGTCTCTCATGCTCTT-3'
124
G>C
F: 5'-TTTTTCCTGATTCTCGAGCCCCCAGCAGGAG-3'
R: 5'-CTCCTGCTGGGGGCTCGAGAATCAGGAAAAA-3'
625
C>T
836T
>A
572_
592d
el
F: 5'-GATCCACTTCTTGCATTCAGATCGCTGGCCCC-3'
R: 5'-GGGGCCAGCGATCTGAATGCAAGAAGTGGATC-3'
F: 5’-GTCAAAGGTGACTTCTTGTTCTTCTCGCCAAAGAGA-3’
R: 5’-ATCTCTTTGGCGAGAAGAACAAGAAGTCACCTTTGAC-3’
F: 5’-GCATCGTAGAAACCCACTTCAGGCTGTTTGACGTC-3’,
R:5’-GACGTCAAACAGCCTGAAGTGGGTTTCTACGATGC-3’
625
C>G
Forward 5'-ATCCACTTCTTGCCTTCAGATCGCTGGCCC-3'
Reverse 5'-GGGCCAGCGATCTGAAGGCAAGAAGTGGAT-3'
680
C>T
F: 5'-GGTCATAGCCGCTGAGTACGACACAGAAGATGATG-3',
R: 5'-CATCATCTTCTGTGTCGTACTCAGCGGCTATGACC-3'
824T
>C
808A
>C
118
G>A
F: 5'-ATCTTCTCGCCAGAGAGGTCTTTCTTGTTGAGGAAG-3',
R:5'-CATTCCTCAACAAGAAAGACCTCTCTGGCGAGAAGAT-3'
F:5'-CAAAGAGGTCTTTCTTGTGGAGGAAGAGGATGATGGA-3
R:5'-TCCATCATCCTCTTCCTCCACAAGAAAGACCTCTTTG-3'
F: 5'-CCTGATTCTCCAGCCCTCAGCAGGAGTAATTTC-3'
R: 5'- GAAATTACTCCTGCTGAGGGCTGGAGAATCAGG -3'
95!
Table S2.1 (cont’d)
D174G
(Asp174Gly)
L199P
(Leu199Pro)
Y231C
(Tyr231Cys)
692A>G
521A>G
596T>C
F: 5’-GGTTCGGAGGATGCCCTGCTCGGTGGG-3’,
R: 5’-CCCACCGCGCAGGGCATCCTCCGAACC-3’
F: 5'-CCCCGACGTCAAACGGCCTGAAGTGGAGG-3'
R: 5'-CCTCCACTTCAGGCCGTTTGACGTCGGGG-3'
F: 5’-AGCACCTGGTCACAGCCGCTGAGTGCG-3’
R: 5’-CGCACTCAGCGGCTGTGACCAGGTGCT-3’
96!
Figure S2.1 Alignment of the human and mouse Gαo protein sequences.
Human Gαo (NCBI accession number NP_066268.1) and mouse Gαo (NCBI accession
number NP_034438.1) were aligned using Clustal Omega(Sievers et al., 2011). The
Sequences are 98% identical at the protein level. All the mutations in the current study
are highlighted in blue and the inter-species homology is 100% in all those positions.
The known gain-of-function mutation G184S is highlighted in green.
97!
Figure S2.2 Validation of the Lance Ultra cAMP assay with transient transfection
of α2A adrenergic receptor (α2A AR) and Pertussis toxin (PTX)-insensitive Gαo. (A)
Co-expression of both α2A AR and Gαo C351G (PTX insensitive) results in strong
inhibition of forskolin-stimulated cAMP levels. In the absence of Gαo the α2A AR produces
a very modest inhibitory effect. (n=3). (B) α2A AR activation results in cAMP activation in
the presence of PTX (n=3). (C) Dose-response curves of co-expression of α2A AR and
Gαo C351G exhibit similar EC50 in the absence and presence of PTX (with PTX
EC50=9.4nM; without PTX EC50=9.9nM, n=3).
98!
Figure S2.3 Assessment of dominant-negative effect of complete LOF mutants
G40R, N270H, D174G, T191_F197del, L199P, and F275S.
(A) Dose response curves of changes in cAMP production of HEK293T cells
co-transfected with different combinations of WT/D174G and α2A AR. D174G did not
show an increase of inhibition when co-transfected with WT in different concentrations
(n=3). (B) Dose response curves of changes in cAMP production of HEK293T cells
co-transfected with different combination of WT/T191_F197del and α2A AR. Although a
slight upward shift was observed with WT/T191_F197del 2µg/4µg, the trend did not
continue with WT/T191_F197del 2µg/4µg (n=3). (C) Dose response curves of changes
99!
Figure S2.3 (cont’d) in cAMP production of HEK293T cells co-transfected with different
combination of WT/L199P and α2A AR (n=2). (D) Dose response curves of changes in
cAMP production of HEK293T cells co-transfected with different combination of
WT/F275S and α2A AR (n=2). (E) Dose response curves of changes in cAMP production
of HEK293T cells co-transfected with different combination of WT/G40R and α2A AR
(n=2). (F) Dose response curves of changes in cAMP production of HEK293T cells
co-transfected with different combinations of WT/N270H and α2A AR (n=2).
100!
Figure S2.4 Paired t-test analysis of LogEC50 values for normal and GOF mutants.
(A-D) Apart from the known gain of function mutant G184S, G42R, G203R and E246K
show a significant decrease of EC50 (n=5). (E) R209C shows an increase of EC50 (n=5).
(F, G) R209G and R209H do not display any significant change in EC50 (n=3).
101!
Figure S2.5 Mapping of mutations on the structure of Gαo-GDP bound to RGS16.
(A) Gαo`GDP ` RGS16 complex with the nucleotide-binding domain in cyan, α-helical
domain in grey, switch regions in blue and RGS16 in orange (PDB: 3C7K). (B-D)
Localization of mutations on the Gαo`GDP complex. LOF mutants are in red, PLOF
mutants are in yellow, GOF mutants are in green and NF mutants are in grey. Each
mutant presented in the structure has been changed to its mutated amino acid. (B)
R209H serves as a representative to all three mutants at R209. (D) T191_F197del has
only been labeled out in red. Protein structure adapted from Slep et al.(Slep et al., 2008)
using Pymol (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC)
102!
APPENDIX B
VALIDATION OF THE GENOTYPE-PHENOTYPE CORRELATION OF
GNAO1-ASSOCIATED NEUROLOGICAL DISORDERS
103!
Since the first fifteen GNAO1 mutations covered by our previous study (Feng et al.,
2017), many more GNAO1 mutations have been reported. To validate our
genotype-phenotype correlation model, we have tested twenty-five newly reported
mutations’ functional changes using our cAMP assay. Previously, we used HEK293T
cells transiently transfected with α2AR and Gαo proteins for all of our assays. However, in
HEK293T cells, α2AR couples to Gαs when the system lacks Gαi or Gαo protein (Figure
S2.6C) (Wade et al., 1999). This complicates the interpretation of the results. When
HEK293T cells are transfected with LOF mutations or only pcDNA, stimulation of the
α2AR results in an increase in cAMP concentration (Figure S2.6C). Here we used another
HEK cell line without the Gαs protein (kindly provided by Dr. Kirill Martemyanov from the
Scripps Institute; GNAS KO cell line; Figure S2.6) (Masuho et al., 2018), and tested the
functions of newly reported GNAO1 mutations. The maximum inhibition of cAMP is less
prominent in the GNAS KO cell line (Figure S2.6A & S2.6B). However, in this new
system, non-functioning mutations transfected cells, including cells only transfected with
pcDNA and α2AR, show no effects on cAMP production (Figure S2.6C, S2.6D & S2.7F,
S2.8D). The functioning mutations inhibited the cAMP production when α2AR was
activated by UK14,304 (Figure S2.6B, D, F & S2.7E & S2.8C). One interesting aspect
using the GNAS KO cell line is that there is a clear rightward shift in the
concentration-response curve when the system contains more Gαo protein (Figure S2.6B:
without PTX, EC50 for α2AR vs EC50 for α2AR + Gαo: 1.2 nM vs 10.6 nM); S2.6F: EC50 for
104!
α2AR + Gαo - PTX vs EC50 for α2AR + Gαo + PTX: 10.6 nM vs 42.6 nM). This rightward
shift can be explained by the Gβγ-mediated cAMP inhibition.
All adenylate cyclase (AC) isoforms can be stimulated by forskolin, however,
stimulated AC activities can be further regulated in a subtype specific manner (Ammer &
Christ, 2002). For example, type I AC not only can be inhibited by Gαo, but also by Gβγ
directly (Bayewitch et al., 1998a; Taussig, Quarmby, & Gilman, 1993), while type V AC is
not affected by Gαo but can be inhibited by Gαi subunits and Gβγ (Bayewitch et al.,
1998b; Taussig & Gilman, 1995). Gβγ subunits exhibit a surprising stimulatory effect on
type II and type IV ACs, although this effect is highly conditional and only detectable with
the presence of GαS (Bayewitch et al., 1998a; Gao & Gilman, 1991; Tang & Gilman,
1991; Taussig & Gilman, 1995). In particular, stimulation of type II AC by Gβγ requires a
significantly higher concentration of Gβγ than GαS (Taussig & Gilman, 1995). Human
kidney, from which HEK293 cells are developed, mainly expresses Type VI AC (Defer,
Best-Belpomme, & Hanoune, 2000), which can be inhibited by Gβγ dimers (Bayewitch et
al., 1998b). It is possible that in this system Gαo functions as a restraint for Gβγ’s
inhibition of AC; therefore with more Gαo present, the EC50 increases significantly.
Similar to our previously described trend, functioning Gαo mutants showed a
relatively normal protein expression pattern comparing to WT Gαo (Figure S2.7A, C &
S2.8A, B) with the exception of Q223P (Figure S2.7; 0.066 ± 0.019). Non-functioning
GNAO1 mutations all exhibited significantly reduced protein expression pattern (Figure
105!
S2.7B, D & S2.8A, B). Some of the mutant alleles identified were obtained from genomic
database or personal communication from families so clinical information is limited in
these cases.
However, it is still clear that functioning GNAO1 mutations are associated with
movement disorder patients, while non-functioning GNAO1 mutations are mainly related
to the onset of epilepsy (Table S2.4). The genotype-phenotype between the mutation
functions and the onset of epilepsy or movement disorders still stand with the new HEK
cell line (Table S2.4).
106!
Figure S2.6 Comparison of validation of the Lance Ultra cAMP assay between
HEK293T and GNAS KO HEK293 cells with transient transfection of α2A
adrenergic receptor (α2AR) and Pertussis toxin (PTX)-insensitive Gαo. Figure S2.6A,
S2.6C, & S2.6E are taken from Figure S2.2. (A, B) Co-expression of both α2A AR and
Gαo C351G (PTX insensitive) results in strong inhibition of forskolin-stimulated cAMP
levels in both cell lines. In the absence of Gαo the α2A AR produces a very modest
inhibitory effect in HEK 293T cells but a strong inhibition effect in GNAS KO cells (n=3).
(C, D) α2A AR activation results in cAMP activation in the presence of PTX in HEK293T
but no effect in GNAS KO cells due to the lack of Gαs (n=3). (E, F) Dose-response
curves of co-expression of α2A AR and Gαo C351G exhibit similar EC50 in the absence
and presence of PTX in both cell line (HEK 293T: with PTX EC50=9.4 nM; without PTX
EC50=9.9 nM, n=3. GNAS KO: with PTX EC50=42.6 nM; without PTX EC50=10.6 nM,
n=3).
107!
Figure S2.7 GNAO1 mutations’ functionalities correlate to their protein expression
patterns. (A-D) Representative and quantification of relative protein levels of each Gαo
mutant compared to wild-type Gαo grouped by functioning mutants (A, C) and
non-functioning mutants (B, D). (E, F) Dose-response curves for α2 agonist-mediated
inhibition of AC with different GNAO1 mutants. In the GNAS KO HEK cell line,
functioning GNAO1 mutants inhibit cAMP production (E) while non-functioning GNAO1
mutants do not show any inhibition of cAMP production (F). Graphs are the results of 3
independent experiments and data are presented as mean ± SEM. *p<0.05, **p<0.01,
****p<0.001 using one-way ANOVA analysis of variance with Bonferroni post-hoc test for
pairwise comparison. PTX = pertussis toxin.
108!
Figure S2.8 GNAO1 mutations’ functionalities correlate to their protein expression
patterns (assays done by Nils Wellhausen with a different group of mutations
from those in Figure S2.7). (A) Representative mutations of and (B) quantification of
relative protein levels of each Gαo mutant compared to wild-type Gαo. (C, D)
Dose-response curves for α2AR regulation of AC with GNAO1 mutants. In GNAS KO
HEK cell line, functioning GNAO1 mutants inhibit cAMP production (C) while
non-functioning GNAO1 mutants do not show any inhibition of cAMP production (D).
Graphs are the results of 3 independent experiments and data are presented as mean ±
SEM. *p<0.05, **p<0.01, ****p<0.001 using one-way ANOVA analysis of variance with
Bonferroni post-hoc test for pairwise comparison. PTX = pertussis toxin.
109!
Table S2.2 All non-functioning GNAO1 mutations tested with GNAS KO cells
Expression
(% of WT)
0
100
99 ± 3
7 ± 2
4
7
0
5 ± 2
10
12 ± 1
6 ± 3
3 ± 2
-2 ± 5
4 ± 1
-
6 ± 4
cAMP at 5 µM
UK14, 304 (% of
unstimulated)
116 ± 12
64 ± 4
60 ± 7
110 ± 7
103 ± 4
90 ± 4
102 ± 4
107 ± 6
101 ± 7
120 ± 10
123 ± 13
105 ± 2
108 ± 7
98 ± 5
110 ± 5
86 ± 3
Normalized
% Inhibition
0
100
129 ± 14
13 ± 14
29 ± 9
60 ± 9
32 ± 8
21 ± 12
35 ± 14
-10 ± 21
-18 ± 27
-1 ± 4
-10 ± 17
15 ± 12
-31 ± 16
43 ± 7
LogEC50
-5.70 ± 1.30
-7.14 ± 0.67
-7.43 ± 0.07
-
-
-
-
-
-
-
-
-
-
-
-
-6.66 ± 0.17
EC50 (nM)
2014
73
38
-
-
-
-
-
-
-
-
-
-
-
-
218
Group
pcDNA
WT
G184S Gly184Ser
Gly45Gln
G45E
G45R
Gly45Arg
Asp273Val
D273V
Tyr291Asn
Y291N
Leu157Pro
L157P
G40W
Gly40Trp
N270Y
Asn270Tyr
E246G Glu246Gly
L284S
Leu284Ser
S229R Ser229Arg
Leu39Pro
L39P
Gln52Pro
Q52P
G40E
Gly40Glu
550G>A
134G>A
133G>C
818A>T
871T>A
470T>C
118G>T
808A>T
737A>G
851T>C
687C>A
116T>C
155A>C
119G>A
Table S2.3 All functioning GNAO1 mutations tested with GNAS KO cells
550G>A
698A > C
626G>T
844T>A
167T>C
139A>G
709G>A
812A>G
863T>C
725A>C
448T>C
649G>A
1030_1032
delATT
Group
pcDNA
WT
G184S Gly184Ser
Q233P Gln233Pro
R209L
Arg209Leu
Ser282Thr
S282T
Ile56Thr
I56T
Ser47Gly
S47G
E237K
Glu237Lys
Lys271Arg
K271R
Phe288Ser
F288S
Asn242Thr
N242T
I163T
Ile163Thr
Glu217Lys
E217K
Expression
(% of WT)
0
100
106 ± 11
7 ± 2
102 ± 36
63 ± 15
39 ± 6
27 ± 8
24 ± 2
5 ± 1
4 ± 1
-
-
65 ± 14
cAMP at 5µM
UK14, 304 (% of
unstimulated)
116 ± 12
64 ± 4
60 ± 7
73 ± 5
70 ± 4
63 ± 3
79 ± 6
70 ± 6
66 ± 5
97 ± 8
73 ± 4
73 ± 4
80 ± 4
85 ± 5
Normalized
% Inhibition
0
100
129 ± 14
100 ± 10
107 ± 7
123 ± 6
87 ± 12
106 ± 12
115 ± 10
17 ± 19
74 ± 10
93 ± 10
37 ± 11
46 ± 12
LogEC50
-5.70 ± 1.30
-7.14 ± 0.67
-7.43 ± 0.07
-7.60 ± 0.32
-6.93 ± 0.19
-7.85 ± 5.08
-7.77 ± 0.31
-7.72 ± 0.29
-7.37 ± 0.17
-8.00 ± 0.88
-7.62 ± 0.31
-7.50 ± 0.24
-7.17 ± 1.12
-6.71 ± 0.64
EC50 (nM)
2014
73
38
25
116
14
17
19
43
10
24
31
67
195
I344del
Ile344del
49 ± 15
95 ± 4
22 ± 9
-7.42 ± 0.22
38
110!
Table S2.4 Genotype-phenotype correlation of the newly reported GNAO1
mutations
GNAO1
Mutations
Movement
Disorder
cAMP Inhibition
Epilepsy
Gender
Age of
onset
birth
4 y
13 mo
4 mo
3 mo
NA
5 mo
12 mo
infancy
NA
6 mo
NA
birth
2.5 mo
NA
NA
15 hr
2 hr
11 d
M
F
F
M
M
F
M
F
F
M
F
F
F
F
M
F
M
F
F
R209L
I56T
Q233P
E237K
E237K
S282T*
S47G
I344del
G45E
G45R
E246G
N270Y*
G40W*
Y291N*
L157P*
D273V*
G40E
G40E
L284S
Functioning
Functioning
Functioning
Functioning
Functioning
Functioning
Functioning
Functioning
Non-Functioning
Non-Functioning
Non-Functioning
Non-Functioning
Non-Functioning
Non-Functioning
Non-Functioning
Non-Functioning
Non-Functioning
Non-Functioning
Non-Functioning
+
+
+
++
++
++
+
++
+
++
+
+
++
+
++
++
++
+
+
+
++
+
+
+
+
+
+
* Personal communication from GNAO1 patient support group
NA: not available
++ Severe symptoms; + Mild symtoms
111!
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112!
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CHAPTER 3: BEHAVIORAL ASSESSMENT OF MOUSE MODELS WITH
GNAO1-ASSOCIATED MOVEMENT DISORDER AND EPILEPSY
Modified from Feng, H., Larrivee, C. L., Demireva, E. Y., Xie, H., Leipprandt, J. R., &
Neubig, R. R. (2019). Mouse models of GNAO1-associated movement disorder:
Allele-and sex-specific differences in phenotypes. PloS one, 14(1), e0211066.
DOI:10.1371/journal.pone.0211066
With permission from PLOS ONE. All rights reserved.
&
From Larrivee, C. L., Feng, H., Leipprandt, J. R., Demireva, E. Y., Xie, H., & Neubig, R.
R. (2019). Mice with GNAO1 R209H Movement Disorder Variant Display
Hyperlocomotion Alleviated by Risperidone. bioRxiv, 662031.
DOI: https://doi.org/10.1101/662031
Demireva, E. Y. and Xie, H. did the design and production of the G203R and R209H
mutant mice.
Larrivee C. L. performed Rotarod and Open Field studies for the G203R and R209H
mutant mice.
Leipprandt, J. R. did the breeding and genotyping for the mutant mice.
119!
3.1 Abstract
Infants and children with dominant de novo mutations in GNAO1 exhibit movement
disorders, epilepsy, or both. Children with loss-of-function (LOF) mutations and
partial-loss-of-function (PLOF) mutations exhibit Epileptiform Encephalopathy 17
(EIEE17). Gain-of-function (GOF) or those with normal functioning (NF) mutations in an
in vitro assay are found in patients with Neurodevelopmental Disorder with Involuntary
Movements (NEDIM). There is no animal model with a human mutant GNAO1 allele.
Here we assess the behavioral patterns in several mouse models. Mouse models
with Gnao1 knock-in mutation G203R (GOF), R209H (NF) or ΔT191F197 (LOF) were
created by CRISPR/Cas9 methods to determine whether the clinical features of patients
with a particular GNAO1 mutation which could include epilepsy and/or movement
disorder would be evident in the mouse model. These three newly developed models are
compared with previously developed Gnao1 mouse model, Gnao1+/G184S and Gnao1+/-.
Gnao1+/G203R mutant mice were viable and gained weight comparably to controls.
Homozygotes were not non-viable. Grip strength was decreased in both males and
females. Male Gnao1+/G203R mice were strongly affected in movement assays (RotaRod
and DigiGait) while females were not. Male Gnao1+/G203R mice also showed enhanced
seizure propensity in the pentylenetetrazole kindling test. Mice with a G184S GOF
knock-in also showed movement-related behavioral phenotypes but females were more
strongly affected than males. In contrast, the Gnao1+/R209H mouse model exhibited
120!
hyperkinetic movements, which have not been seen in any other Gnao1 mutant mouse
line. Gnao1+/R209H mice also did not show a strong sex difference in our behavioral
battery test. Mice carrying the strong epilepsy allele, Gnao1+/ΔT191F197 did not gain weight
like their WT siblings and the mutant mice developed seizures around P7 and all mutant
mice died before P16. In contrast, Gnao1+/- mice survived and never developed
spontaneous seizures.
Gnao1+/G203R, Gnao1+/R209H and Gnao1+/ΔT191F197 mice all shared similar phenotypes
regarding to the onset of epilepsy and/or movement disorders as children with the same
heterozygous GNAO1 mutations. Although Gnao1+/ΔT191F197 mice did not survive to
breeding age and the line was lost, both Gnao1+/G203R and Gnao1+/R209H mouse models
should be useful tools in mechanistic and preclinical studies of GNAO1-related
movement disorders and epilepsy.
3.2 Introduction
Neurodevelopmental Disorder with Involuntary Movements (NEDIM) is a newly
defined neurological disorder associated with mutations in GNAO1. It is characterized by
“hypotonia, delayed psychomotor development, and infantile or childhood onset of
hyperkinetic involuntary movements” (OMIM 617493). NEDIM is monogenetic and
associated with GOF mutations and NF mutations in GNAO1 (Feng et al., 2017). The
GNAO1 gene has also been associated with early infantile epileptic encephalopathy 17
(EIEE17; OMIM 615473). However, 36% of patients showed both epilepsy and
121!
movement disorder phenotypes. This includes many different GNAO1 alleles such as:
G40R, G45R, S47G, I56T, T191_F197del, L199P, G203R, R209C, A227V, Y231C and
E246G (Feng, Khalil, Neubig, & Sidiropoulos, 2018).
GNAO1 encodes Gαo, the most abundant membrane protein in the mammalian
central nervous system (Jiang & Bajpayee, 2009). Gαo is the α-subunit of the Go protein,
a member of the Gi/o family of heterotrimeric G proteins. Gi/o proteins couple to many
important G protein-coupled-receptors (GPCRs) involved in movement control like
GABAB, dopamine D2, adenosine A1 and adrenergic α2A receptors (Franek et al., 1999;
Gazi, Nickolls, & Strange, 2003; Lorenzen, Lang, & Schwabe, 1998; Tian, Duzic, Lanier,
& Deth, 1994). Upon activation, Gαo and Gβγ separate from each other and modulate
separate downstream signaling pathways. Gαo mediates inhibition of cyclic AMP (cAMP),
and Gβγ mediates inhibition of AC and N-type calcium channels while activating
G-protein activated inward rectifying potassium channels (GIRK channels) (Zhang,
Pacheco, & Doupnik, 2002). Go is expressed mainly in the central nervous system and it
regulates neurotransmitter release by modulating intracellular calcium concentrations in
pre-synaptic cells (Li et al., 2004). It has also been suggested that Go plays a role in
neurodevelopmental processes like neurite outgrowth and axon guidance (Bromberg,
Iyengar, & He, 2008; Strittmatter, Fishman, & Zhu, 1994). Consequently, Go is an
important modulator of neurological functions.
Previously, we defined a functional genotype-phenotype correlation for GNAO1
122!
disorders (Feng et al., 2017); GOF and NF mutations are found in patients with
movement disorders, while loss-of-function (LOF) and partial-loss-of-function (PLOF)
mutations are associated with epilepsy (Feng et al., 2017). I recently published a
mechanistic review of this genotype-phenotype correlation (Feng et al., 2018). The
experimental study of human GNAO1 mutant alleles, however, was done in HEK293T
cells, which lack complex physiological context of the brain. Therefore, it was important
to see whether mouse models with GNAO1 mutations would share clinical
characteristics of the human patients. Such a result would verify the previously - reported
genotype-phenotype correlation and would provide a system for more detailed
mechanistic studies and preclinical testing models for possible new therapeutics.
Previously, we reported that Gnao1+/G184S mutant mice carrying a human-engineered
GOF mutation (G184S) showed heightened sensitization to pentylenetetrazol (PTZ)
kindling and had an elevated frequency of interictal epileptiform discharges on EEG
(Kehrl et al., 2014). Here, we tested whether the Gnao1+/G184S mice also exhibit
movement disorders although G184S has not been found in human (Feng et al., 2019).
A Gnao1+/- mouse model was also described previously. They are hyperalgesic and
display severe motor control impairment (Jiang et al., 1998). Gnao1-/- mice are
hyperactive and also exhibit an abnormal turning behavior (Jiang et al., 1998). In our
hands, they are poorly viable.
The G203R is a GOF GNAO1 mutation in the cAMP assay (Feng et al., 2017). It is
123!
one of the most common GNAO1 mutations found clinically (Table 3.1) (Arya, Spaeth,
Gilbert, Leach, & Holland, 2017; Feng et al., 2018; Nakamura et al., 2013; Saitsu et al.,
2016; Schorling et al., 2017; Xiong et al., 2018). Most patients with this mutation exhibit
both seizures and movement disorders (Arya et al., 2017; Feng et al., 2018; Nakamura
et al., 2013; Saitsu et al., 2016; Schorling et al., 2017; Xiong et al., 2018).
The R209 is a mutation hotspot in the Gαo protein, which has been reported in over
ten patients. Previously, the R209H mutation was in more than seven patients (Ananth et
al., 2016; R. Dhamija, Mink, Shah, & Goodkin, 2016; Kelly et al., 2019; Kulkarni, Tang,
Bhardwaj, Bernes, & Grebe, 2016; Marecos, Duarte, Alonso, Calado, & Moreira, 2018),
all of who develop severe chorea/athetosis, dystonia, hypotonia and developmental
delay (Table 3.1). All three previously reported R209 mutations (R209H, R209C, and
R209G) were NF GNAO1 mutations in our cAMP assay (Feng et al., 2017). However, it
remained a question why GNAO1 mutations that showed normal function in the cellular
assay were pathogenic.
The ΔT191F197 in-frame deletion mutation was the most severe LOF pathogenic
GNAO1 mutation reported in patients (Feng et al., 2017; Nakamura et al., 2013) (Table
3.1). It is the most representative LOF mutation with the lowest protein expression level
and lowest % inhibition (Feng et al., 2017); therefore it was a good candidate for
generating a LOF mouse model that would possibly develop spontaneous neurological
disorders. However, since all mutant ΔT191F197 mice died before P16, we were not
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able to generate a line carrying this mutation. In this chapter, I only provide limited data
on survival and growth of this mouse model.
Table 3.1 The status of Gnao1 mutant mice
Allele
cAMP
Clinical
Mouse Status
Inhibition
GOF
LOF
GOF
G184S
KO
G203R
N/A
N/A
EIEE17
& MD
Rare adult lethality; breeds well (Kehrl et al., 2014)
Homozygous early lethality (Jiang et al., 1998)
Rare adult lethality; breeds well (Feng et al., 2019)
R209H
ΔT191F197
NF
LOF
MD
Hyperactivity; gait phenotypes (Larrivee et al., 2019)
EIEE17 Heterozygous early lethality (P7-P14); loss of strains
(Figure 3.4)
We intended to develop mouse models with the representative human GOF (G203R),
NF (R209H), and LOF (ΔT191F197) mutations to see if they replicated the clinical
phenotype of GNAO1 mutation-associated neurological disorders. If so, they would be
valuable tools to understand neural mechanisms underlying the complex phenotypic
spectrum of patients with GNAO1 mutations. In this chapter, I show the behavioral
assessment of two mouse lines carrying Gαo GOF mutation Gnao1+/G203R and NF
mutation Gnao1+/R209H. They are compared with two previously described mouse models:
one with a known GOF function mutation G184S (Gnao1+/G184S) and the other the Gnao1
KO model (Gnao1+/-). These two mouse models (Gnao1+/G203R and Gnao1+/R209H) present
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the possibility of studying GNAO1-associated neurological defects in animal models.
3.3 Materials and Methods
3.3.1 Animals
Animal studies were performed in accordance with the Guide for the Care and Use of
Laboratory Animals established by the National Institutes of Health. All experimental
protocols and personnel were approved by the Michigan State University Institutional
Animal Care and Use Committee (IACUC). Mice were housed on a 12-h light/dark cycle
and had free access to food and water. They were studied between 8-12 weeks old.
3.3.2 Generation of Gnao1 mutant mice
3.3.2.1 Generation of Gnao1+/G203R mouse model
Gnao1+/G184S (Feng et al., 2017; Fu et al., 2004; Goldenstein et al., 2009; Kehrl et al.,
2014) and Gnao1+/- mice (Kehrl et al., 2014) were generated as previously described
and used as N10 or greater backcross on the C57BL/6J background.
Gnao1G203R mutant mice were generated using CRISPR/Cas9 genome editing on
the C57BL/6NCrl strain. gRNA
targets within exon 6 of
the Gnao1
locus
(ENSMUSG00000031748) were used to generate the G203R mutation (Figure 3.1A).
Synthetic single-stranded DNA oligonucleotides (ssODN) were used as repair templates
carrying the desired mutation and short homology arms (Table 3.2). CRISPR reagents
were delivered as ribonucleoprotein (RNP) complexes. RNPs were assembled in vitro
using wild-type S.p. Cas9 Nuclease 3NLS protein, and synthetic tracrRNA and crRNA
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(Integrated DNA Technologies, Inc.). TracrRNA and crRNA were denatured at 95°C for 5
min and cooled to room temperature in order to form RNA hybrids, which were incubated
with Cas9 protein for 5 min at 37°C. RNPs and ssODN templates were electroporated
into C57BL/6NCrl zygotes as described previously(Qin et al., 2015), using a Genome
Editor electroporator (GEB15, BEX CO, LTD). C57BL/6NCrl embryos were implanted
into pseudo-pregnant foster dams. Founders were genotyped by PCR (Table 3.2)
followed by T7 Endonuclease I assay (M0302, New England BioLabs) and validated by
Sanger sequencing.
Figure 3.1 Development of Gnao1+/G203R mouse model. (A) Targeting of the Gnao1
locus. The location of the gRNA target protospacer and the PAM, and double stranded
breaks following Cas9 cleavage are indicated on the WT allele. Deleted or modified
sequences are highlighted in blue. The resulting edited allele sequence and translation
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Figure 3.1 (cont’d) are presented along with the sequences used as references for
ssODN synthesis. (B) Heterozygous Gnao1+/G203R mutant mice are largely normal in size
and behavior. Photo comparing mutant mouse with its littermate control is shown. (C)
Gnao1+/G203R mice have a relatively normal survival; while homozygous Gnao1G203R/G203R
mice die perinatally (P0-P1). (D) Gnao1+/G203R mice develop normally and gain weight
similarly to their WT littermate controls.
Table 1. Location, sequence and genotyping of gRNA targets in Gnao1
locus.
Table 3.2 Location, sequence and genotyping of gRNA targets in the Gnao1 locus.
DSB
location
gRNA
target
ssODN
Gnao1 G203R
chr 8: 93,950,314
5’ TGCAGGCTGTTTGACGTCGG GGG 3’ (+)
5’ ATGGCCGTGACATCCTCAAAGCAGTGGATCCAC
TTCTTGCGTTCAGATCGCTGGCCGCGGACGTCAAA
CAGTTTGCAGGGAGTCAGGGAAAGCTGT 3’
PCR
primers
Genotyping
Fwd: 5’ GACAGGTGTCACAGGGGATG 3’
Rev: 5’ TCCTAGCCAAGACCCCAACT 3’
PCR product = 462bp
SacII site created by G203R mutation
gRNA target – 20bp protospacer and PAM sequences are listed, strand
orientation indicated by (+) or (-). Sequence of ssODN used as repair template
is listed. For G203R, mutated codon is highlighted in bold. DSB – double
gRNA target – 20bp protospacer and PAM sequences are listed, strand orientation
indicated by (+) or (-). The sequence of the ssODN used as a repair template is listed.
For G203R, the mutated codon is highlighted in bold. DSB – double stranded break.
PAM – protospacer adjacent motif.
stranded break. PAM – protospacer adjacent motif.!
The likelihood of an off-target site being edited is very low. Based on the number and
position of mismatches several predictive algorithms were used to assign guide
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specificity scores from 0 to 100 (100 is the best) to rank gRNAs by specificity with
respect to off-targets occurring (Doench et al., 2016; Haeussler M, 2016; Hsu et al.,
2013). The gRNA target used for this experiment has a specificity score of 94, which is
the highest seen by the MSU Transgenic and Gene Editing Facility in over 40 similar
targeting experiments (E. Demivera personal communication). This greatly reduces the
probability of an off-target edits occurring. After examining the off-target lists (Table
S3.9), we did not identify any off-target loci with less than 3 mismatches or with an
off-target binding score > 0.5 which we deem as thresholds for further validation. We
also did not identify any off-target loci with significant scores that were on the same
chromosome and would be less likely to be removed from the genome after breeding of
several generations. Furthermore, the RNP (ribonucleoprotein) approach of delivering
CRISPR reagents to mouse embryos we employed further lowers the risk of off-target
events (Iyer et al., 2018).
Nevertheless, we directly validated several predicted off-target loci within coding
regions for the G203 gRNA target TGCAGGCTGTTTGACGTCGG GGG. One off-target
site with 4 mismatches and a score of 0.52 was validated
for
locus
ENSMUSG00000041390. We also analyzed two other off-target sites with 4 mismatches
and scores of 0.15 and 0.069 respectively, predicted to occur on the same chromosome
(chr 8) ENSMUSG00000086805 and ENSMUSG00000097637. To test these 3 off-target
sites, DNA from WT and founder animals from which the line was expanded were
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analyzed by PCR and sequencing and we found that no off-target effects had occurred
for all 3 off-target loci analyzed (see Supplemental Materials).
3.3.2.2 Generation of Gnao1+/R209H mouse model
Mutant Gnao1+/R209H mice were generated via CRISPR/Cas9 genome editing on a
C57BL/6J genomic background. CRISPR gRNA selection and locus analysis were
performed using the Benchling platform (Benchling, Inc. San Francisco, CA.). A gRNA
targeting exon 6 of the Gnao1 locus (ENSMUSG00000031748) was chosen to cause a
double strand break (DSB) 3bp downstream of codon R209. A single-stranded
oligodeoxynucleotide (ssODN) carrying the R209H mutation CGC > CAC with short
homology arms was used as a repair template (Figure 3.2 and Table 3.3).
Ribonucleoprotein (RNP) complexes consisting of a synthetic crRNA/tracrRNA hybrid
and Alt-R® S.p. Cas9 Nuclease V3 protein (Integrated DNA Technologies, Inc. Coralville,
IA), were used to deliver CRISPR components along with the ssODN to mouse zygotes
via electroporation as previously described (Feng et al., 2019). Edited embryos were
implanted into pseudo-pregnant dams using standard techniques. Resulting litters were
screened by PCR (Phire Green HSII PCR Mastermix, F126L, Thermo Fisher, Waltham,
MA.), T7 Endonuclease I assay (M0302, New England Biolabs Inc.) and Sanger
sequencing (GENEWIZ, Inc. Plainfield, NJ) for edits of the target site.
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Table 3.3 Location and sequence of gRNA and ssODN template for CRISPR-Cas
targeting Gnao1 locus; primers and genotyping method for Gnao1+/R209H mice
Location
gRNA target
5’ N20-PAM -3’
Gnao1 R209H
Chr 8: 93,950,334
5’ AGCGATCTGAACGCAAGAAG TGG 3’
ssODN template
(reverse complement)
GTTTCGTCCTCGTGGAGCACCTGGTCATAGCCGCT
GAGTGCGACACAGAAGATGATGGCCGTGACATCCTCAAA
GCAGTGGATCCACTTCTTGtGTTCAGATCGCTGGCCCCCG
ACGTCAAACAGCCTGCAGGGAGTCAGGGAAAGCTGTGA
GGGCGGGGACGCCTA
PCR primers
Genotyping
O586 FWD: 5' GGACAGGTGTCACAGGGGAT 3’
O587 REV: 5' ACTGGCCTCCCTTGGCAATA 3'
By Sanger Sequencing
Figure 3.2 Targeting of the mouse Gnao1 locus. (A) Mouse Gnao1 genomic locus
(exon size not to scale), red outline is magnified in (B) showing exon 6 and relative
location of codon 209, and PCR primers O586 and O587. (C) Location and exact
sequence of gRNA target within exon 6, dotted red line denotes DSB, PAM is highlighted
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Figure 3.2 (cont’d) and sequence corresponding to gRNA protospacer is underlined
(also in E). (D) Raw gel electrophoresis images showing PCR of the target region and T7
Endonuclease I (T7 Endo I) digestion analysis of founders 1324 – 1335 (n=12), with WT,
H2O (-) and T7Endo I (+) controls. Founder 1324 (red number) was positive for the
mutation on one allele and WT on the other, note that the single bp mismatch was not
reliably detected by T7 Endo I assay. (E) Exact sequence of edited founder 1324 as
aligned to WT reference genome, two peaks (G and A) are detected on the sequence
chromatogram, indicating the presence of both WT and edited R209H allele.
3.3.3 Genotyping and Breeding
3.3.3.1 Genotyping Gnao1+/G203R mice
Heterozygous Gnao1+/G203R mutant founder mice were crossed against C57BL/6J
mice to generate Gnao1+/G203R heterozygotes (N1 backcross). Further breeding was
done to produce N2 backcross heterozygotes while male and female N1 heterozygotes
were crossed to produce homozygous Gnao1G203R/G203R mutants. Studies were done on
N1 or N2 G203R heterozygotes with comparisons to littermate controls.
All mice had ears clipped before weaning. DNA was extracted from earclips by an
alkaline lysis method (Truett et al., 2000). The G203R allele of Gαo was identified by Sac
II digests (WT 462 Bp and G203R 320 & 140Bp) of genomic PCR products generated
with
primers
(Fwd
5'
GACAGGTGTCACAGGGGATG
3';
Rev
5'
TCCTAGCCAAGACCCCAACT 3'). Reaction conditions were: 0.8 µl template, 4 µl 5x
Promega PCR buffer, 0.4 µl 10 mM dNTPs, 1 µl 10 µM Forward Primer, 1 µl 10 µM
Reverse Primer, 0.2 µl Promega GoTaq and 12.6 µl DNase free water (Promega catalog
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# M3005, Madison WI). Samples were denatured for 4 minutes at 95 oC then underwent
32 cycles of PCR (95 oC for 30 seconds, 60 oC for 30 seconds, and 72 oC for 30 seconds)
followed by a final extension (7 minutes at 72 oC). After PCR, samples were incubated
with Sac II restriction enzyme for 2 hrs.
3.3.3.2 Genotyping Gnao1+/R209H mice
Studies were done on N1 R209H heterozygotes with comparisons to littermate
controls. To generate Gnao1+/R209H heterozygotes
(N1 backcross), 2
founder
Gnao1+/R209H mice, 1 male and 1 female, were crossed with C57BL/6J mice.
DNA was extracted by an alkaline method (Hirata, Takahashi, Shimoda, & Koide,
2016) from ear clips done before weaning. PCR products were generated with primers
flanking
the mutation site
(Fwd 5' GGACAGGTGTCACAGGGGAT 3’; 5'
ACTGGCCTCCCTTGGCAATA 3'). Reaction conditions were: 0.8 µl template, 4 µl 5x
Promega PCR buffer, 0.4 µl 10mM dNTPs, 1 µl 10 µM Forward Primer, 1 µl 10 µ M
Reverse Primer, 0.2 µl Promega GoTaq and 12.6 µl DNase free water (Promega catalog
# M3005, Madison WI). Samples were denatured for 4 minutes at 95 ̊ C then underwent
32 cycles of PCR (95 ̊ C for 30 seconds, 63 ̊C for 30 seconds, and 72 ̊C for 30 seconds)
followed by a 7-minute final extension at 72 ̊C. Ethanol precipitation was done on the
PCR products and then samples were sent for Sanger sequencing (GENEWIZ, Inc.
Plainfield, NJ).
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3.3.4 Behavioral Studies
Researchers conducting behavioral experiments were blinded until the data analysis
was completed. Before each experiment, mice were acclimated in the testing room for at
least 10 min. The timeline of behavioral protocols is described in Figure 3.3. Female
experimenters conducted all behavioral studies.
Figure 3.3 The timeline for utilizing animals in this study. Open field, Rotarod and
Grip strength tests were performed on the same group of 8-week-old animals in this as
showed above. DigiGait tests were done on naïve 8-week-old animals. Animals finishing
the motor behavior studies were used for the PTZ kindling study for 3 weeks.
3.3.4.1 Open Field
The Open Field test was conducted in Fusion VersaMax 42 cm x 42 cm x 30 cm
arenas (Omnitech Electronics, Inc., Columbus, OH). Mice and their littermate controls
were placed in the arena for 30 minutes to observe spontaneous activities. Using the
Fusion Software, distance traveled (cm) was evaluated for novel (first 10 minutes),
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sustained (10-30 minutes), and total (0-30 minutes) activity. Center Time was also
measured. Center Time was defined as the time spent in the center portion (20.32cm x
20.32cm) of the Open Field cage.
3.3.4.2 RotaRod
Motor skills were assessed using an Economex accelerating RotaRod (Columbus
Instruments, Columbus, OH). The entire training and testing protocol took two days. On
day 1, mice were trained for three 2-minute sessions, with a 10-minute rest between
each training period. During the first two sessions, the RotaRod was maintained at a
constant speed of 5 rpm. In the third training session, the rod was started at 5 rpm and
accelerated at 0.1 rpm/sec for 2 minutes. On day 2, mice were trained with two more
accelerating sessions for 2 minutes each with a 10-minute break in between. The final
test session was 5 minutes long, starting at 5 rpm then accelerating to 35 rpm (0.1
rpm/sec). For all training and test trials, the time to fall off the rod was recorded. RotaRod
learning curves were done on a separate group of mice with 10 tests in one day with a
5-min rest between each test. The learning rate of each group of animals was calculated
as described (Hirata et al., 2016).
3.3.4.3 Grip Strength
Mouse grip strength data was collected following a protocol adapted from Deacon et
al (Deacon, 2013) using seven home-made weights (10, 18, 26, 34, 42, 49, 57 grams).
Briefly, the mouse was held by the middle/base of the tail and lowered to grasp a weight.
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A total of three seconds was allowed for the mouse to hold the weight with its forepaws
and to lift the weight until it was clear of the bench. Three trials were done starting with
the 10g weight to permit the mice to lift the weights with a 10-second rest between each
trial. If the mouse successfully held a weight for 3 seconds, the next heavier weight was
given; otherwise the maximum time/weight achieved was recorded. A final total score
was calculated based on the heaviest weight the mouse was able to lift up and the time
that it held it (Deacon, 2013). The final score was normalized to the body weight of each
mouse, which was measured before the trial.
3.3.4.4 DigiGait
Mouse gait data were collected using a DigiGait Imaging System (Mouse Specifics,
Inc., Framingham, MA) (Hansen & Pulst, 2013). The test is used for assessment of
locomotion as well as
the
integrity of
the
cerebellum and muscle
tone/equilibrium(Franco-Pons, Torrente, Colomina, & Vilella, 2007). Briefly, after
acclimation, mice were allowed to walk on a motorized transparent treadmill belt. A
high-speed video camera was mounted below to capture the paw prints on the belt. Each
paw image was treated as a paw area and its position recorded relative to the belt.
Seven speeds (18, 20, 22, 25, 28, 32 and 36 cm/s) were tested per animal with a
5-minute rest between each speed. An average of 4-6 s of video was saved for each
mouse, which is sufficient for the analysis of gait behaviors in mice (Franco-Pons et al.,
2007). For each speed, left & right paws were averaged for each animal while fore and
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hind paws were evaluated separately. Stride length was normalized to animal body
length.
3.3.5 PTZ Kindling Susceptibility
A PTZ kindling protocol was performed as described before (Kehrl et al., 2014) to
assess epileptogenesis. Briefly, PTZ (40 mg/kg, i.p. in 5 mg/ml) was administered every
other day starting at 8 weeks of age. Mice were monitored and scored for 30 minutes for
behavioral signs of seizures as described(Grecksch et al., 2004; Kehrl et al., 2014;
Wilczynski et al., 2008). Kindling is defined as death or the onset of a tonic-clonic seizure
on two consecutive treatment days. The number of injections for each mouse to reach a
sensitization was reported in survival curves. This experiment lasted up to 4 weeks with
a maximum of 12 doses. Each animal in the study was checked every day for health and
seizure development.
Animals were humanely euthanized with CO2 immediately after kindling or after 12
PTZ injections and observation. In total, 40 animals were used for this study, among
which 27 died of tonic-clonic seizures and 13 were euthanized after 12 doses of PTZ
injections.
3.3.6 Data Analysis
All data was analyzed using GraphPad Prism 7.0 (GraphPad; La Jolla, CA). Data are
presented as mean ± SEM and a p value less than 0.05 was considered significant. All
statistical tests are detailed in Figure Legends. Multiple comparison correction of the
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dataset from DigiGait was performed via a false discovery rate (FDR) correction at a
threshold value of 0.01 in an R environment using the psych package.
3.4 Results
3.4.1 The growth patterns of the three newly developed Gnao1 mutant mouse
models (G203R, R209H and ΔT191F197)
3.4.1.1 Gnao1+/G203R mice showed normal viability and growth.
Genotypes of offspring of Gnao1+/G203R x WT crosses (N1 - C57BL/6NCrl x
C57BL/6J) were observed at the expected frequency (29 WT and 27 heterozygous). All
three homozygous mice from Gnao1+/G203R x Gnao1+/G203R crosses died by P1. The small
numbers of offspring observed from these crosses so far, however, were not significantly
different from expected frequencies (4 wt, 14 het, and 3 homozygous). Heterozygous
Gnao1+/G203R mice did not show any growth abnormalities compared to Gnao1+/+ mice
(Figure 3.1B & 3.1D) and they had relatively normal survival. There were two
spontaneous deaths (~5-7 weeks) seen for Gnao1+/G203R mice out of 33 (Figure 3.1C).
This is reminiscent of the spontaneous deaths seen previously with the Gnao1+/G184S
GOF mutant mice (Kehrl et al., 2014). Gnao1+/G203R mice did not exhibit any obvious
spontaneous seizures or abnormal movements.
3.4.1.2 Gnao1+/R209H mice have expected frequency and normal viability
Two founder Gnao1+/R209H mice, 1 male and 1 female, were crossed with C57BL/6J
mice. Out of 98 offspring of a cross of Gnao1+/R209H with WT mice, 51 heterozygotes and
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47 WT were observed. Gnao1+/R209H mice exhibit no overt postural or movement
abnormalities or seizures at basal conditions. Adult mice showed no statistically
significant differences in weight between WT and Gnao1+/R209H genotypes of either sex
(data not shown).
3.4.1.3 Gnao1+/ΔT191F197 mice developed spontaneous seizures at P7 and died
before P16.
Gnao1ΔT191F197 mutant mice were generated using CRISPR/Cas9 genome editing on
the C57BL/6NCrl strain. gRNA
targets within exon 6 of
the Gnao1
locus
(ENSMUSG00000031748) were used to generate the ΔT191F197 mutation (Figure
3.4A). Only one viable founder (male) was obtained. Genotypes of offsprings of this male
founder Gnao1+/ΔT191F197 x WT crosses (C57BL/6NCrl x C57BL/6J) were observed.
Heterozygous Gnao1+/ΔT191F197 mice were very rare and all died perinatally within P16
(Figure 3.4C &3.2D). Gnao1+/ΔT191F197 mice also developed spontaneous seizure at P7
(Figure 3.4B). Previously, we described that Gnao1 G184S heterozygous mutant mice
on a 129 background (N6 129S1/SvImJ) lived a relatively normal life (Kehrl et al., 2014).
To investigate whether a 129 background is also protective towards the ΔT191F197
mutant mice, we crossed our Gnao1+/ΔT191F197 with WT mice on the 129 background and
assessed their offsprings. Unfortunately, 129 alleles did not appear to provide a
dominant protective effect against the spontaneous death observed in the heterozygous
ΔT191F197 mutant mice (Figure 3.4C).
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Figure 3.4 Gnao1+/ΔT191F197 mice developed spontaneous seizures at P7 and died
before P16. (A) Targeting of the Gnao1 locus. The location of the gRNA target
protospacer and the PAM, and double stranded breaks following Cas9 cleavage are
indicated on the WT allele. Deleted or modified sequences are highlighted in blue. The
resulting edited allele sequence and translation are presented along with the sequences
used as references for ssODN synthesis. (B) Video snapshot of one heterozygous
Gnao1+/ΔT191F197 mutant mouse developed spontaneous seizure at P7. Photo comparing
mutant mouse with its littermate control is shown. (C) Gnao1+/ΔT191F197 on both C57BL/6J
and B6/129 backgrounds died prematurely. (D) Gnao1+/ΔT191F197 mice did not develop or
gain weight normally comparing to WT littermate controls.
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3.4.2 Behavioral assessment of mutant Gnao1 mouse models (G184S, G203R,
R209H and KO) for movement patterns
3.4.2.1 Female Gnao1+/G184S and male Gnao1+/G203R mice show similar movement
abnormalities and gait disturbances
Since GOF alleles of GNAO1 in children result primarily in movement disorder, we
tested motor coordination in two mouse lines with GOF mutations. One carried an
engineered GOF mutant G184S, designed to block RGS protein binding (DiBello et al.,
1998; Fu et al., 2004; Lan et al., 1998). The other is the G203R GOF mutant, which has
been seen in at least 9 children (Chapter 1) (Feng et al., 2018; Feng et al., 2017). First,
we used a two-day training and testing procedure on the RotaRod (Figure 3.5A & 3.5B).
Gnao1+/G184S and Gnao1+/G203R mice were compared to their same-sex littermate controls.
Female Gnao1+/G184S mice exhibited a reduced retention time on the accelerating
RotaRod (unpaired t-test, p<0.001, Figure 3.5A) while male mice remained unaffected.
In contrast, male Gnao1+/G203R mice exhibited reduced time to stay on the rotating rod
(unpaired t-test, p<0.05, Figure 3.5B) while female Gnao1+/G203R mice did not show any
abnormalities. Results from all the RotaRod training and testing sessions are shown in
Figure S1. Neither Gnao1+/G184S nor Gnao1+/G203R mice showed a significant difference in
learning rate on RotaRod (Figure S3.3), suggesting that the differences we observed in
the RotaRod study was due to movement deficits rather than learning difficulties.
Grip strength was assessed as described (Deacon, 2013). This test is widely done in
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combination with the RotaRod motor coordination test. This may be relevant to the
hypotonia, seen in many GNAO1 patients (Ananth et al., 2016; Bruun et al., 2018; Danti
et al., 2017; Euro, Epilepsy Phenome/Genome, & Epi, 2014; Gawlinski et al., 2016;
Honey et al., 2018; Kulkarni et al., 2016; Law et al., 2015; Marce-Grau et al., 2016;
Saitsu et al., 2016; Schorling et al., 2017; Waak et al., 2018; Yilmaz et al., 2016; Zhu et
al., 2015). Similar to the RotaRod results, female Gnao1+/G184S mice also showed
reduced forepaw grip strength compared to their littermate controls (unpaired student
t-test, p<0.05, Figure 3.5C) while males did not exhibit a significant difference (Figure
3.5C). In contrast, both male and female Gnao1+/G203R mice displayed reduced forepaw
grip strength (unpaired t-test, p<0.05, Figure 3.5D).
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Figure 3.5 Female Gnao1+/G184S mice and male Gnao1+/G203R mice show reduced
time on RotaRod and reduced grip strength. (A&B) Quantification of RotaRod studies.
(A) Female Gnao1+/G184S mice lose the ability to stay on a RotaRod (unpaired t-test;
***p<0.001), while male Gnao1+/G184S mice appeared unaffected. (B) Male Gnao1+/G203R
also showed reduced motor coordination on RotaRod (unpaired t-test, *p<0.01). (C&D)
Quantification of grip strength results. Scores for each mouse were normalized to the
body weight of the animal measured. (C) Female Gnao1+/G184S mice are less capable of
lifting weights compared to their Gnao1+/+ siblings (unpaired t-test, *p<0.05). (D) Both
male and female Gnao1+/G203R mice showed reduced ability to hold weights (unpaired
t-test, *p<0.05). Data are shown as mean ± SEM.
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The open field test provides simultaneous measurements of locomotion, exploration
and surrogates of anxiety. It is a useful tool to assess locomotive impairment in rodents
(Tatem et al., 2014), however, environmental salience may reduce the impact of the
motor impairment on behaviors (Parr & Friston, 2017). Therefore, we divided the 30-min
open field measurements into two periods, with the first 10 min assessing activity in a
novel environment and the 10-30 minute period designated as sustained activity (Figure
3.6C & 3.6D). The novelty measurement showed a significant difference between
Gnao1+/G184S mice and their littermate controls for both male and female mice (2-way
ANOVA, p<0.01 for female, p<0.05 for male, Figure 3.6C). Female, but not male,
Gnao1+/G184S mice showed reduced activity in the sustained phase of open field testing
(2-way ANOVA, *p<0.05, **p<0.01, ****p<0.0001). Both male and female Gnao1+/G184S
mice also showed reduced total activity (2-way ANOVA, p<0.001, Figure 3.6A & 3.6C).
Neither male nor female Gnao1+/G203R mice performed differently in the open field arena
compared to their littermate controls (Figure 3.6B & 3.6D). No significant difference was
observed in the time mice spent in the center of the arena (Figure S3.2).
144!
Figure 3.6 G184S mutant mice showed reduced activities in Open Field Test but
G203R mutants do not. (A&C) Female and male Gnao1+/G184S mice showed decreased
activity in the open field test. A total of 30 min activity was recorded which was divided
into Novelty (0-10 min) and Sustained (10-30 min) period. (A) Representative heat map
of overall activity comparison between Gnao1+/+ and Gnao1+/G184S mice in both sexes. (C)
Quantitatively, both male and female Gnao1+/G184S travelled less in the open field arena
(2-way ANOVA; ****p< 0.0001, **p<0.01, *p<0.05). (B & D) Neither male nor female
Gnao1+/G203R mice showed abnormalities in the open field arena. (B) Sample heat map
tracing both female and male mouse movement in open field. (D) Quantification showed
no difference between Gnao1+/+ and Gnao1+/G203R mice in distance traveled (cm) in the
open field arena (2-way ANOVA; n.s.). Data are shown as mean ± SEM. Numbers of
animals are indicated on bars.
145!
In addition to the above behavioral tests, we also performed gait assessment on
Gnao1+/G184S and Gnao1+/G203R mice of both sexes. Gait is frequently perturbed in rodent
models of human movement disorders even when the actual movement behavior seen in
the animals does not precisely phenocopy the clinical movement pattern (Song, Fan,
Exeter, Hess, & Jinnah, 2012; Stroobants, Gantois, Pooters, & D'Hooge, 2013). The
multiple parameters assessed in DigiGait allow it to pick up subtle neuromotor defects
and makes it more informative than the RotaRod test.
The gait analysis largely confirmed the sex differences between the two strains in
RotaRod tests. Thirty-seven parameters were measured for both front and hind limbs.
Given the large number of measurements, we used false discovery rate (FDR) analysis
with a Q of 1% as described in Methods to reduce the probability of Type I errors (Figure
S3.4 & S3.5, Table S3.1-S3.4). Gnao1+/G184S female mice showed 22 significant
differences (Q<0.01) and males showed 8 (Figure S3.4, Table S3.3 & S3.4). For
Gnao1+/G203R mice, the opposite sex pattern was seen with 27 parameters in females
and 8 parameters in males showing significant differences from WT (Figure S3.5, Table
S3.1 & S3.2). Two of the most highly significant parameters and ones that had face
validity in terms of clinical observations (stride length and paw angle variability) were
chosen for further analysis.
Across the range of treadmill speeds, female Gnao1+/G184S mice showed significantly
reduced stride length (2-way ANOVA, p<0.01, Figure 3.7A) and increased paw angle
146!
variability (2-way ANOVA, p<0.0001, Figure 3.7E) compared to WT littermates. Male
Gnao1+/G184S mice only had a difference in paw angle variability (2-way ANOVA,
p<0.0001), not in stride length (Figure 3.7C & 3.7G). These results are consistent with
the results of RotaRod and grip strength measurements in that female Gnao1+/G184S mice
showed a stronger phenotype than males. In contrast to the Gnao1+/G184S mice, male
Gnao1+/G203R mice appeared to be more severely affected in gait compared to female
Gnao1+/G203R mice. Male Gnao1+/G203R mice had highly significantly reduced stride length
(2-way ANOVA, p<0.0001, Figure 3.7D) and increased paw angle variability (2-way
ANOVA, p<0.05, Figure 3.7H). In contrast, female Gnao1+/G203R mice did not show any
significant differences in stride length or paw angle variability (Figure 3.7B & 3.7F). In
addition to these quantitative gait abnormalities a qualitative defect was seen. A
significant number of Gnao1+/G203R mice of both sexes failed to run when the belt speed
exceeded 22 cm/s (Mann-Whitney test, female and male p<0.05, Figure 3.7J). For
reasons that are not clear such a difference was not seen for Gnao1+/G184S mice (Figure
3.7I).
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Figure 3.7 DigiGait Imaging System reveals sex-specific gait abnormalities in
Gnao1+/G184S mice and Gnao1+/G203R mice. (A-D) Female Gnao1+/G184S mice showed
significant gait abnormalities, while female Gnao1+/G203R mice remain normal. (A & B)
Female Gnao1+/G184S mice showed reduced stride length (2-way ANOVA with Bonferroni
multiple comparison post-test) while female Gnao1+/G203R mice were unchanged from
control (2-way ANOVA; n.s.). (C) Female Gnao1+/G184S mice also showed increased paw
angle variability (2-way ANOVA, p<0.0001) while female Gnao1+/G203R mice showed
normal paw angle variability. (E-H) Male Gnao1+/G203R and Gnao1+/G184S mutant mice
showed distinct gait abnormalities. (E & G) Male Gnao1+/G184S mice showed significantly
148!
Figure 3.7 (cont’d) increased paw angle variability (2-way ANOVA p <0.0001 overall
with significant Bonferroni multiple comparison tests; **p<0.01 and *p<0.05). There was
no effect on stride length. (F & H) In contrast, male Gnao1+/G203R mice showed markedly
reduced stride length (2-way ANOVA p<0.0001 with Bonferroni multiple comparison
post-test; ***p<0.001, **p<0.01, and *p<0.05) and modestly elevated paw angle
variability (overall p<0.05). (I) Gnao1+/G184S mice did not show significant differences in
the highest treadmill speed successfully achieved. (J) Both male and female
Gnao1+/G203R mice showed reduced capabilities to run on a treadmill at speeds greater
than 25 cm/s (Mann-Whitney test; *p<0.05).
3.4.2.2 Gnao1+/R209H mouse model exhibits unique hyperactive behavior in the
open field arena but no abnormal motor coordination in other behavior tests and
minor disturbance in gait analysis
All patients with R209H mutation were diagnosed with hyperkinetic movements
including chorea/athetosis and dystonia (Ananth et al., 2016; R. Dhamija et al., 2016;
Kelly et al., 2019; Kulkarni et al., 2016; Marecos et al., 2018). To assess whether
Gnao1+/R209H mice phenocopy the patients’ symptoms, we repeated the above behavior
tests with the newly developed heterozygous R209H mice.
Unlike Gnao1+/G184S and Gnao1+/G203R mice, both sexes of Gnao1+/R209H mice were
hyperactive in the open field arena (Figure 3.8A& 3.8B) but completely normal in Rotarod
(Figure 3.8C) and grip strength assessment (Figure 3.8D). Male and
female
Gnao1+/R209H mice also showed reduced time spent in the center of the arena (Figure
3.8A & 3.8B), which is an indication for possible anxiety-linked behavior (Wilmshurst,
149!
Byrne, & Webb-Peploe, 1989).
Figure 3.8 Gnao1 +/R209H mice show significant hyperactivity and reduced time in
center in the open field arena. (A) Representative heat maps of Gnao1+/R209H mice and
Gnao1+/+ mice in the open field arena (B) Time spent in the open field arena was
separated into 0-10 minutes (novelty) and 10-30 minutes (sustained). Gnao1+/R209H male
and female mice exhibit increased locomotion in the novelty period. Hyperactivity was
maintained throughout the sustained period as mice continued to show significant
150!
Figure 3.8 (cont’d) increase in distance traveled (2-way ANOVA; ****p < 0.0001, ***p <
0.001, * p < 0.05). Gnao1+/R209H mice of both sexes spend less time in center areas of the
open field arena compared to WT littermates. (C) Neither male nor female Gnao1+/R209H
mice show significant differences on the Rotarod. (D) There is no significant difference
between grip strength between WT and Gnao1+/R209H mice. Data are shown as mean ±
SEM.
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Figure 3.9 Male and female Gnao1+/R209H mice shows gait abnormalities in different
tests on the DigiGait imaging system. (A & B) Male Gnao1+/R209H mice showed
reduced stride length compared to wildtype littermates (2-way ANOVA with Bonferroni
multiple comparison post-test), while female Gnao1+/R209H mice show a normal stride
151!
Figure 3.9 (cont’d) length. (C & D) Neither male nor female Gnao1+/R209H exhibited
significant differences in paw angle variability compared to WT littermates. (E) At speeds
greater than 25 cm/s female Gnao1+/R209H shows reduced ability to run on a treadmill.
Gait analysis was done with a Digigait video system. Similar to Gnao1+/G203R mouse
model, male Gnao1+/R209H mice showed a highly significant genotype effect with reduced
stride length compared to wildtype littermates (**p<0.01, 2-way ANOVA). Females
showed no difference from WT. No difference was seen in paw angle variability of
Gnao1+/R209H of either sex. However, both male and female Gnao1+/R209H mice failed to
consistently run at higher speeds (>20 cm/s; Figure 3.9E, **p<0.01, Student’s t-test). The
difference observed was not due to a reduced body length (WT: 9.54 cm vs R209H:
10.17 cm) or weight. Comparisons of other parameters assessed by the Digigait system
were shown in the appendix (Figure S3.6 & Table S3.5-S3.6). Compared to the number
of parameters with significant difference detected for Gnao1+/G203R and Gnao1+/G184S
mice respectively, Gnao1+/R209H mice only did not display as many significantly different
gait abnormalities (Figure S3.6).
3.4.2.3 Previously described Gnao1+/- mouse model did not show any
abnormalities in the behavioral test battery
A Gnao1+/- KO mouse model was previously described for the study of the
mechanisms of Go protein (Jiang et al., 1998; Valenzuela et al., 1997). In these reports,
homozygous Gnao1-/- mice lived but exhibited a reduced lifespan and developed severe
152!
motor control impairment (Jiang et al., 1998). Gnao1-/- mice were also reported to be
hyperactive and had an abnormal turning behavior (Jiang et al., 1998). Heterozygous
Gnao1+/- mice did not have any spontaneous abnormal behavior (Jiang et al., 1998). We
have been unable to generate homozygous (Gnao1-/-) KO mice. In our behavior battery,
heterozygous Gnao1+/- mice did not show any abnormalities in open field (Figure
3.10A-D), Rotarod (Figure 3.10E), or grip strength (Figure 3.10F). However, male
Gnao1+/- mice did exhibit significantly reduced stride length (Figure 3.11B, p<0.001,
2-way ANOVA). Furthermore, male Gnao1+/- mice also showed several DigiGait
parameters that are significantly different compared to the WT mice (Figure S3.7 & Table
S3.7-S3.8). To our surprise, Gnao1+/- mice also did not develop any spontaneous
seizure activity in contrast to the LOF mutant mice Gnao1+/ΔT191F197, which were severely
impaired by spontaneous seizures perinatally and died prematurely (Figure 3.4).
153!
Figure 3.10 Male and female Gnao1+/- mice do not show any abnormalities in the
behavioral tests including open field, Rotarod, and grip strength. (A-D) Neither sex
of Gnao1+/- mice has normal activity in the open field arena in any time period. The
activity pattern of Gnao1+/- mice (A) is also similar to Gnao1+/+ (B). The overall activity is
also comparable between male (D) and female (C) WT and Gnao1+/- mice. (E) Gnao1+/-
mice do not show any reduced motor coordination capability in Rotarod. (F) Grip strength
test shows that neither sex of Gnao1+/- mice decreases their capability of lifting heavy
weight.
154!
Figure 3.11 DigiGait Imaging System reveals the decreased stride length in male
Gnao1+/- mice. Female Gnao1+/- mice do not show any difference comparing to female
Gnao1+/- mice in either stride length (A) or paw angle variability (C). (B) However, male
Gnao1+/- mice exhibit a significantly decreased stride length (p<0.0001, 2-way ANOVA)
but not (D) paw angle variability.
3.4.3 PTZ kindling study of G203R and R209H mice.
Kindling studies for Gnao1+/G184S and Gnao1+/- mice have been reported previously
by Kehrl et al (Kehrl et al., 2014).
3.4.3.1 Male Gnao1+/G203R mice are sensitized to PTZ kindling.
Epilepsy has been observed in 100% of patients with GNAO1 G203R mutations
(Arya et al., 2017; Feng et al., 2018; Nakamura et al., 2013; Saitsu et al., 2016; Xiong et
al., 2018). Also in the Gnao1+/G184S GOF mutant mice, we previously reported
spontaneous lethality as well as increased susceptibility to kindling by the chemical
155!
anticonvulsant PTZ for both males and females (Kehrl et al., 2014). Kindling is a
phenomenon where a sub-convulsive stimulus, when applied repetitively and
intermittently, leads to the generation of full-blown convulsions. To determine if the
G203R GOF mutant mice mimicked the G184S mutants and phenocopied the human
epilepsy pattern of children with the G203R mutation, we assessed PTZ-induced kindling
in Gnao1+/G203R mutant mice. As expected for C57BL/6 mice, females were more prone
to kindling than male mice, half kindled at 4 and 8-10 injections, respectively (Figure
3.12A & 3.12B). Despite the increased sensitivity of females in general, female
Gnao1+/G203R mice did not show significantly higher sensitivity to PTZ compared to their
littermate controls (Figure 3.12A). On the contrary, male Gnao1+/G203R mice were more
sensitive to PTZ kindling than controls (Figure 3.12B, Mantel-Cox Test, p<0.05). Also,
three spontaneous deaths were seen (two male and one female) among the 33 G203R
mice observed for at least 100 days, similar to the early lethality seen in G184S mutant
mice. We cannot, however, attribute those deaths to seizures at this point.
156!
Figure 3.12 Gnao1+/G203R male mice have an enhanced Pentylenetetrazol
(PTZ)-Kindling response. (A) Female Gnao1+/G203R did not show heightened sensitivity
to PTZ injection. (B) Male Gnao1+/G203R mice developed seizures earlier after repeated
PTZ injections (Mantel-Cox Test; p<0.05).
3.4.3.2 R209H mutant mice are not hypersensitive to PTZ kindling
Repeated application of a sub-threshold convulsive stimulus, leads to the generation of
full-blown convulsions (Dhir, 2012). GNAO1 variants differ in their ability to cause
epileptic seizures in patients. Children carrying the R209H mutant allele do not exhibit a
seizure disorder. In accordance with the patients’ pattern, Gnao1+/R209H mice did not
show increased susceptibility to kindling-induced seizures (Figure 3.13A & 3.13B). This
contrasts with the increased kindling sensitivity in male G203R mutant mice (Figure 3.13)
(Feng et al., 2019; Larrivee et al., 2019).
157!
Figure 3.13 Gnao1+/R209H mice do not have an enhanced pentylenetetrazol (PTZ)
kindling response. (A&B) Neither male nor female Gnao1+/R209H mice showed
significant differences in sensitivity to PTZ injection compared to WT littermates (n.s.;
Mantel-Cox test).
3.5 Discussion
In this chapter, I describe three newly developed Gnao1 mutant mouse models
(ΔT191F197, G203R, and R209H) and compare them with two previously published
mouse models (G184S and KO). These data verify the genotype-phenotype correlation
that I describe in chapter 2. Also, among the three different newly developed mouse
models, only two mouse models (G203R and R209H) produced viable strains (Table
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3.3). Through the established behavioral pattern of the mouse models, we intend to
explore mechanisms of Gnao1-associated movement disorders in the next chapter.
Heterozygous male mice carrying the G203R mutation (GOF) in Gnao1 exhibit both
a mild increase in seizure propensity and evidence of abnormal movements. This fits
precisely with the variable seizure pattern of the children who carry this mutation as well
as their severe choreo-athetotic movements (Arya et al., 2017; Dietel, 2016; Feng et al.,
2018; Nakamura et al., 2013; Saitsu et al., 2016; Schirinzi et al., 2019; Schorling et al.,
2017; Xiong et al., 2018). Heterozygous mice carrying the R209H mutation (NF) only
develop hyperkinetic movements without loss of motor coordination on RotaRod or loss
of capability of lifting heavy weights (Figure 3.8). This mimics patients with R209H
mutations (Ananth et al., 2016; Blumkin et al., 2018; R. M. Dhamija, J, W.; Shah, B, B.;
Goodkin, H, P., 2016; Kelly et al., 2019; Kulkarni et al., 2016; Marecos et al., 2018;
Menke et al., 2016). In comparison, mice model with the LOF mutation ΔT191F197
developed spontaneous seizures at an early age and died prematurely before P16. This
fits the clinical description of the patient carrying the same mutation (Nakamura et al.,
2013) who died at 11 months (Figure 3.4). A summary of phenotypes of viable mutant
mice is shown in Table 3.4. For comparison purposes, we have also tested the
previously reported GOF mutant mouse model (G184S) and KO mouse model. The
G184S mouse model exhibits a sex-dependent movement disorder while KO mice did
not show any severe motor disability.
159!
Table 3.4 Phenotypes of Gnao1 mutant mice
Open Field
Balance
(RotaRod)
Grip Strength
Gait Analysis
Seizure
Susceptibility
G203R
Normal
R209H
G184S
Hyperactivity
Hypoactivity
Reduced
Normal
Reduced
KO
Normal
Normal
Reduced
Normal
Reduced
Normal
↓↓ stride length
↓stride length
↑ variability
Increased
Normal
Increased
↓ stride length
↑↑ variability
↓ stride length
Normal
(Kehrl et al., 2014)
(Kehrl et al., 2014)
In mouse models of movement disorders, the mouse phenotype is usually not as
striking or as easily observed as the clinical abnormalities in the patients (Oleas, Yokoi,
DeAndrade, Pisani, & Li, 2013; Wilson & Hess, 2013), however they are often
informative about mechanism and therapeutics. The male Gnao1+/G203R mutant mouse
carrying patient-derived mutation very closely replicates the mild seizure phenotype of
female Gnao1+/G184S mice (Kehrl et al., 2014). I now show that the female Gnao1+/G184S
mice also exhibit gait and motor abnormalities. Both the GNAO1 G203R and the G184S
mutations show a definite but modest GOF phenotype in biochemical measurements of
cAMP regulation (Feng et al., 2017). In each case, the maximum percent inhibition of
cAMP is not greatly increased but the potency of the a2A adrenergic agonist, used in
those studies to reduce cAMP levels, was increased about 2-fold (Feng et al., 2017).
This effectively doubles signaling through these two mutant G proteins at low
160!
neurotransmitter concentrations (i.e. those generally produced during physiological
signaling). This, however, does not prove that cAMP is the primary signal mechanism
involved in pathogenesis of the disease. The heterotrimeric G protein, Go, is the defining
subunit to many different effectors (Feng et al., 2018; Strittmatter et al., 1994;
Wettschureck & Offermanns, 2005). We recently reviewed the mutations associated with
genetic movement disorders and identified both cAMP regulation and control of
neurotransmitter release as two mechanisms that seem highly likely to account for the
pathophysiology of GNAO1 mutants (Chapter 1) (Feng et al., 2018). Since many Go
signaling effectors (including cAMP and neurotransmitter release) can be mediated by
the Gbg subunit released from the Go heterotrimer, other effectors could also be involved
in the disease mechanisms. A recent hypothesis has also been raised that intracellular
signaling by Gao may be involved (Solis & Katanaev, 2018). The observation that one of
the most common movement disorder-associated alleles (R209H and other mutations in
Arg209) does not markedly alter cAMP signaling in in vitro models, does suggest that the
mechanism is more complex than a simple GOF vs LOF distinction at cAMP regulation.
The R209H mutation was only tested for regulation of cAMP levels. It remains an
unanswered question why a NF mutation still would lead to movement disorder in human
patients and hyperactivity in our mouse models. This is a potential drawback of our in
vitro assessment of cAMP only in an engineered HEK293T cell system. Since Gao
regulates at least six different pathways (Jiang & Bajpayee, 2009), cAMP may not be the
161!
affected downstream target of the R209H mutation. Therefore, the R209H animal model
should be more valuable foe mechanistic studies since it will provide a more relevant
physiological environment for studying the regulation of Gao in isolated neurons.
Another interesting observation lies in the comparison between the KO mouse model
and the LOF mouse model ΔT191F197. Although ΔT191F197 proved to be an
epileptogenic and lethal mutation in both mouse and human, the actual KO mouse model
did not develop any obvious seizure phenotype. Previously, homozygous Gnao1 KO
mice were reported with a mild seizure phenotype (Jiang et al., 1998), however,
heterozygous KO mice were relatively normal. One explanation could be the
compensation effect of Gai protein, which takes over the mechanistic pathways that were
once regulated by Gao protein. More likely, ΔT191F197 has some unknown mechanism,
which would lead to the abnormal fetal development and infantile lethality.
We also observed a striking sex difference in the phenotypes of our mouse models.
Female Gnao1+/G184S mice and male Gnao1+/G203R mice showed much more prominent
movement abnormalities than male G184S and female G203R mutants. However, the
patterns of changes in the behavioral tests did not exactly overlap. G184S mutants
showed significant changes in open field tests while G203R mutants did not. Conversely,
G203R mutants showed a striking reduction in ability to walk/run at higher treadmill
speeds while G184S mutants did not. For both mutant alleles, the seizure phenotype
was also worse in the sex with more prominent movement disorder. For the NF mutant
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line, Gnao1+/R209H mice did not show any sex difference in their hyperkinetic
movemements in the open field arena (Figure 3.8), but they did have male dominated
gait abnormalities, shown as decreased stride length (Figure 3.9).
GNAO1 encephalopathies are slightly more prevalent (60:40) in female than male
patients (Feng et al., 2018). It is not uncommon to have sex differences in epilepsy or
movement disorder disease progression. One possible explanation is that estrogen
prevents dopaminergic neuron depletion (Smith & Dahodwala, 2014). The Gi/o coupled
estrogen receptor, GPR30, contributes to estrogen physiology and pathophysiology
(Revankar, Cimino, Sklar, Arterburn, & Prossnitz, 2005). Also, PD is more common in
male than female human patients (Wooten, Currie, Bovbjerg, Lee, & Patrie, 2004),
therefore, the pro-dopaminergic properties of estrogen may exacerbate conditions
mediated by hyper-dopaminergic symptoms like chorea in Hungtington’s disease (HD)
(Smith & Dahodwala, 2014). Chorea/athetosis is the most prevalent movement pattern
seen in GNAO1-associated movement disorders (Feng et al., 2018) so the female
predominance correlates with that in HD. Clearly mechanisms of sex differences are
complex including differences in synaptic patterns, neuronal densities and hormone
secretion (Gillies, Murray, Dexter, & McArthur, 2004; Kompoliti, 1999; Smith &
Dahodwala, 2014), but it is beyond the scope of this chapter to explain how the
molecular differences contribute to the distinct behavioral patterns. A more detailed
analysis on sex difference is provided in Chapter 5.
163!
Since GNAO1 encephalopathy is often associated with developmental delay and
cognitive impairment (Feng et al., 2018), it would be interesting to see whether the
movement phenotype we have seen in female Gnao1+/G184S, male Gnao1+/G203R or
Gnao1+/R209H mice is due to a neurodevelopmental malfunction or to ongoing active
signaling alterations. Go-coupled GPCRs play an important role in hippocampal memory
formation (Madalan, Yang, Ferris, Zhang, & Roman, 2012; Schutsky, Ouyang, & Thomas,
2011). Additional behavioral tests will be valuable to assess the learning and memory
ability of the Gnao1 mutant mice.
With the increasing recognition of GNAO1-associated neurological disorders, it is
important to learn about the role of Go in the regulation of central nervous system. The
novel Gnao1 G203R and R209H mutant mouse models reported here, and further
models under development, should facilitate our understanding of GNAO1 mechanisms
in the in vivo physiological background rather simply in in vitro cell studies. The animal
models can also be used for preclinical drug testing and may permit a true allele-specific
personalized medicine approach in drug repurposing for the associated movement
disorders or epilepsy.
164!
APPENDIX
165!
APPENDIX
SUPPLEMENTAL DATA
Figure S3.1 RotaRod test was conducted with 5 training sessions and 1 test
session over two consecutive days. (A) Female Gnao1+/G184S mice showed
significantly motor abnormalities in test trial at day 2 (unpaired t-test; ***p<0.001). (B)
Male Gnao1+/G184S mice did not show any significance in any training or test session. (C)
Female Gnao1+/G203R mice did not exhibit any motor abnormalities in any RotaRod trial or
test session. (D) Male Gnao1+/G203R mice showed significantly decreased capability in
motor balance (unpaired t-test; *p<0.05).
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A
B
F Gnao1+/+
F Gnao1+/G184S
M Gnao1+/+
M Gnao1+/G184S
50
40
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10
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15
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0
F Gnao1+/+
F Gnao1+/G184S
M Gnao1+/+
M Gnao1+/G184S
50
40
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20
10
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%
0
F Gnao1+/+
F Gnao1+/G203R
M Gnao1+/+
M Gnao1+/G203R
16
10
10
11
F Gnao1+/+
F Gnao1+/G203R
M Gnao1+/+
M Gnao1+/G203R
Figure S3.2 Time spent at the center in the Open Field Test. (A) No significant
differences were observed between Gnao1+/G184S mice and their littermate controls. (B)
No significant differences were observed between Gnao1+/G203R mice and their littermate
controls.
167!
Figure S3.3 RotaRod learning curve was collected in 10 consecutive tests with a
5-min break between each test. (A, C & E) Short-term learning curve comparison
between Gnao1+/+ and Gnao1+/G203R in both sexes. (A & C) Both male and female
Gnao1+/G203R mice showed reduced capability of keeping balance on RotaRod. (E) No
significant difference in either sex between Gnao1+/+ and Gnao1+/G203R mice was
observed comparing the rate of learning. (B, D & F) Short-term learning curve
comparison between Gnao1+/+ and Gnao1+/G184S in both sexes. (B & D) Both male and
female Gnao1+/G184S mice showed reduced capability of keeping balance on RotaRod. (F)
No significant difference in either sexes between Gnao1+/+ and Gnao1+/G184S mice was
observed comparing the rate of learning.
168!
A
Fore Limb
F Gnao1+/+ vs Gnao1+/G184S
10
l
e
u
a
v
r
e
t
e
m
a
r
a
P
1
Hind Limb
F Gnao1+/+ vs Gnao1+/G184S
B
100
10
1
l
e
u
a
v
r
e
t
e
m
a
r
a
P
0.1
0.1
StrideLength
MAX.dA.dT
Swing
Paw.Area.at.Peak.Stance.in.sq..cm
Axis.Distance
Overlap.Distance
Stride.Frequency
Stride
Paw.Angle.Variability
Fore Limb
C
M Gnao1+/+ vs Gnao1+/G184S
10
e
u
l
a
v
r
e
t
e
m
a
r
a
P
1
Paw.Drag
Propel
Stance.Swing
StrideLength
MAX.dA.dT
Swing
Paw.Area.at.Peak.Stance.in.sq..cm
Overlap.Distance
Stance
Stride.Frequency
Stride
Midline.Distance
Paw.Angle.Variability
Hind Limb
0.01
D
M Gnao1+/+ vs Gnao1+/G184S
100
10
1
l
e
u
a
v
r
e
t
e
m
a
r
a
P
0.1
MAX.dA.dT
0.1
Paw.Area.at.Peak.Stance.in.sq..cm
Paw.Angle.Variability
Paw.Drag
MAX.dA.dT
Tau...Propulsion
0.01
Swing
Paw.Area.at.Peak.Stance.in.sq..cm
Figure S3.4 False discovery rate (FDR) calculation probed of significantly different
parameters from the DigiGait data in Gnao1+/G184S mice. All parameters that showed
significance at belt speed 25 cm/s are plotted. (A&B) Female Gnao1+/G184S and their
littermate controls showed parameters with significance detected by the FDR analysis.
(C&D) Male Gnao1+/G184S and their littermates controls showed parameters with
significance detected by the FDR analysis. FDR is calculated by a two-stage step-up
method of Benjamini, Krieger and Yekutiel. Significant values are defined as q < 0.01.
169!
A
Hind Limb
1000
F Gnao1+/+ vs Gnao1+/G203R
100
10
l
e
u
a
v
r
e
t
e
m
a
r
a
P
1
0.1
0.01
Stance.Swing
StrideLength
Swing
Overlap.Distance
Tau..Propulsion
X.StanceStride
X..Shared.Stance
Midline.Distance
X.SwingStride
M Gnao1+/+ vs Gnao1+/G203R
Hind Limb
C
1000
100
l
e
u
a
v
r
e
t
e
m
a
r
a
P
10
1
B
Fore Limb
M Gnao1+/+ vs Gnao1+/G203R
10
1
l
e
u
a
v
r
e
t
e
m
a
r
a
P
0.1
0.1
0.01
Swing
StrideLength
Stride.Frequency
Stride
Absolute.PawAngle
Overlap.Distance
Stride.Length.CV
X.Steps
0.01
Propel
Stance.Swing
StrideLength
X.BrakeStance
MAX.dA.dT
Paw.Area.at.Peak.Stance.in.sq..cm
Swing
Brake
X.BrakeStride
Overlap.Distance
X.StanceStride
X..Shared.Stance
Stride.Length.CV
X.PropelStance
X.Steps
Swing.Duration.CV
Midline.Distance
Paw.Angle.Variability
X.SwingStride
Figure S3.5 False discovery rate (FDR) calculation probed of significantly different
parameters from the DigiGait data in Gnao1+/G203R mice. All parameters that showed
significance are plotted here. (A) Female Gnao1+/G203R and their littermate controls
showed 9 parameters with significance only in hind limb data detected by the FDR
analysis. (B&C) Male Gnao1+/G203R and their littermates controls exhibited 27 parameters
with significance detected by the FDR analysis in fore and hind limb data combined. FDR
is calculated by a two-stage step-up method of Benjamini, Krieger and Yekutiel.
Significant values are defined as q < 0.01.
170!
A
Fore Limb
F Gnao1+/+ vs Gnao1+/R209H
F Gnao1+/+ vs Gnao1+/R209H
Hind Limb
B
1000
100
10
1
l
e
u
a
v
r
e
t
e
m
a
r
a
P
0.1
0.01
C
10
1
l
e
u
a
v
r
e
t
e
m
a
r
a
P
0.1
0.01
Overlap.Distance
Fore Limb
M Gnao1+/+ vs Gnao1+/R209H
D
Propel
X.PropelStride
X.BrakeStance
X.PropelStance
Swing
Swing.Duration.CV
Stride.Frequency
Midline.Distance
Hind Limb
M Gnao1+/+ vs Gnao1+/R209H
e
u
l
a
v
r
e
t
e
m
a
r
a
P
10
1
0.1
0.01
Paw.Drag
Overlap.Distance
1000
100
e
u
l
a
v
r
e
t
e
m
a
r
a
P
10
1
Overlap.Distance
0.1
Paw.Drag
MAX.dA.dT
Paw.Area.at.Peak.Stance.in.sq..cm
Overlap.Distance
Figure S3.6 False discovery rate (FDR) calculation probed of significantly different
parameters from the DigiGait data in Gnao1+/R209H mice. All parameters that showed
significance are plotted here. (A&B) Female Gnao1+/R209H and their littermate controls
showed 9 parameters with significance detected by the FDR analysis. (C&D) Male
Gnao1+/R209H and their littermates controls exhibited fewer parameters with significance
comparing to female detected by the FDR analysis in fore and hind limb data combined.
FDR is calculated by a two-stage step-up method of Benjamini, Krieger and Yekutiel.
Significant values are defined as q < 0.01.
171!
M Gnao1+/+ vs Gnao1+/KO
Fore Limb M
A
100
l
e
u
a
v
r
e
t
e
m
a
r
a
P
10
1
0.1
0.01
Swing
X.BrakeStride
StrideLength
SLVar
B
1000
100
l
e
u
a
v
r
e
t
e
m
a
r
a
P
10
1
0.1
0.01
Hind Limb M
M Gnao1+/+ vs Gnao1+/KO
Swing
X.SwingStride
X.StanceStride
Stance.Swing
StrideLength
Absolute.PawAngle
Paw.Drag
SLVar
Paw.Area.Variability.at.Peak.Stan
Swing.Duration.CV
Midline.Distance
Figure S3.7 False discovery rate (FDR) calculation probed of significantly different
parameters from the DigiGait data in Gnao1+/- mice. All parameters that showed
significance are plotted here. (A&B) Female Gnao1+/- and their littermate controls did not
show any significant difference in any parameters given. Male Gnao1+/- and their
littermates controls exhibited several parameters with significance comparing to female
detected by the FDR analysis in fore and hind limb data combined. FDR is calculated by
a two-stage step-up method of Benjamini, Krieger and Yekutiel. Significant values are
defined as q < 0.01.
172!
Table S3.1 Gait analysis parameters of male Gnao1 G203R mutant mice
Feng et al Table S1 Gait analysis parameters Male Gnao1 G203R mutants
Measured Parameter
Fore Limb
p
X.SwingStride
Swing
Brake
X.BrakeStride
Propel
X.PropelStride
Stance
X.StanceStride
Stride
X.BrakeStance
X.PropelStance
Stance.Swing
StrideLength
Stride.Frequency
PawAngle
Absolute.PawAngle
Paw.Angle.Variability
StanceWidth
StepAngle
SLVar
SWVar
StepAngleVar
X.Steps
Stride.Length.CV
Stance.Width.CV
Step.Angle.CV
Swing.Duration.CV
Paw.Area.at.Peak.Sta
nce.in.sq..cm
Paw.Area.Variability.
at.Peak.Stan
Hind.Limb.Shared.St
ance.Time
X..Shared.Stance
StanceFactor
Gait.Symmetry
MAX.dA.dT
MIN.dA.dT
Tau..Propulsion
Overlap.Distance
PawPlacementPositi
oning.PPP.
Ataxia.Coefficient
Midline.Distance
Axis.Distance
Paw.Drag
0.000086
0.560859
0.653649
0.060086
0.003957
0.105634
0.020015
0.560859
0.001621
0.073312
0.073312
0.496204
<0.000001
0.002659
0.255886
0.000619
FDR
Yes
No
No
No
No
No
No
No
Yes
No
No
No
Yes
Yes
No
Yes
0.477312
0.50287
0.530415
0.19286
0.753361
0.566174
0.000053
0.000167
0.957513
0.205403
0.004977
0.111782
0.534568
0.656453
0.034845
0.926219
0.31531
0.000567
0.009576
0.065892
0.000511
0.813623
No
No
No
No
No
No
Yes
Yes
No
No
No
No
No
No
No
No
No
Yes
No
No
Yes
No
Hind Limb
p
<0.000001
0.000966
0.000399
<0.000001
0.006698
0.088909
0.413195
0.000966
0.010476
0.000004
0.000004
0.000155
<0.000001
0.024506
0.576762
0.7785
0.000239
0.718181
0.990996
0.158581
0.324024
0.422419
0.000013
0.001176
0.256205
0.276243
0.001453
0.004248
0.074908
0.286417
0.000002
0.927493
0.034845
0.001727
0.646552
0.455592
0.000567
0.009576
0.031149
0.000002
0.774606
0.013423
FDR
Yes
Yes
Yes
Yes
Yes
No
No
Yes
No
Yes
Yes
Yes
Yes
No
No
No
Yes
No
No
No
No
No
Yes
Yes
No
No
Yes
Yes
No
No
Yes
No
No
Yes
No
No
Yes
No
No
Yes
No
No
Fore Limb
M Gnao1+/G203R
0.08760833
37.85083333
0.06865
29.43125
0.076825
32.71791667
0.1454875
62.14916667
0.23310833
47.23583333
52.76416667
1.67083333
5.60791667
4.49708333
-1.50833333
5.14166667
8.31791667
4.51666667
86.1125
1.28591667
17.77083333
92.2875
21.81875
23.578375
83.45
96.21666667
29.98779167
M Gnao1+/+
0.09211667
38.07777778
0.06939444
28.40222222
0.08235556
33.52
0.15173889
61.92222222
0.24383333
45.82444444
54.17555556
1.65222222
6.09777778
4.30055556
-2.13944444
3.90722222
8.12333333
4.77777778
92
1.24272222
18.37222222
85.73888889
24.00833333
20.82244444
82.95
81.05555556
27.594
SD
0.0111967
3.54836583
0.016458
4.89821488
0.01995907
4.68472378
0.02844761
3.54836583
0.03618381
7.20043157
7.20043157
0.25378818
0.77998392
0.63908954
4.87883924
3.61305873
2.56029197
4.11368551
97.32018227
0.32258874
19.78855562
114.0590048
4.7819712
6.52396481
94.95293012
111.6869989
7.89706951
0.30805556
0.04937709
0.02894444
0.01608279
0.2975
0.07788791
0.02995833
0.01688003
1
1
1
0
0
0
11.30555556
1.01388889
16.896
-5.16177778
11.54350393
0.04501259
3.37636885
1.45335565
11.8375
1.02416667
16.8605
-5.31579167
12.53391424
0.05217581
4.22700441
1.6247547
1.4025
0.4440782
1.55470833
0.44461341
0.47255556
0.899
-2.22233333
0.01011111
1
0.209826
0.30885281
0.34125483
0.83342464
0
0.53175
0.95725
-2.37679167
-0.00895833
0.24509639
0.328734
0.51249994
0.80948622
0
1
1
1
1
n
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
SD
0.01176783
4.2332119
0.01707632
5.96865439
0.01889352
5.25089869
0.02613979
4.2332119
0.03278618
8.50521667
8.50521667
0.29341328
0.74607749
0.67417378
6.12588422
3.64193574
2.92469857
3.82128663
93.39500767
0.34549218
19.10096449
116.870725
5.87860657
7.92635878
95.25775208
127.9222556
9.08822921
0
0
0
n
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
M Gnao1+/+
0.09002222
36.68666667
0.03542222
14.345
0.12215
48.96611111
0.15759444
63.31333333
0.24762222
22.61888889
77.38111111
1.75555556
6.17777778
4.25111111
0.39222222
16.34777778
4.79555556
9.53888889
46.90555556
0.89744444
8.52777778
83.06111111
23.67777778
14.70616667
128.2111111
99.85555556
21.31672222
SD
0.01187409
3.98361729
0.0096027
3.47436381
0.0268225
4.21758972
0.03072707
3.98361729
0.03807229
5.02721964
5.02721964
0.3053018
0.72670955
0.64785757
17.03757417
4.65733584
1.82575682
8.65887596
62.27966276
0.27275979
12.16526356
110.7694855
4.70635557
4.90287218
154.5150275
121.4064747
6.46662759
0.64966667
0.12237364
0.05305556
0.033092
22
118.0833333
13.59444444
1.01388889
46.49827778
-8.88127778
178.9333333
1.4025
0.47255556
0.63983333
1.76005556
0.02372222
218.8277778
26.92406039
67.80483618
13.27605364
0.04501259
9.49935633
1.91057189
101.5146521
0.4440782
0.209826
0.24794907
0.28026224
1.34792041
112.0513066
n
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
Hind Limb
M Gnao1+/G203R
0.08343333
35.24083333
0.0396125
16.53625
0.115625
48.225
0.15522917
64.75916667
0.23864167
25.36166667
74.63875
1.88625
5.73458333
4.39541667
1.335
16.2
5.57333333
9.85416667
46.975
0.95095833
9.69583333
92.04583333
21.32291667
16.811125
111.1208333
113.7708333
24.35395833
SD
0.01239566
4.70652526
0.01337189
4.86819351
0.0221926
4.54530426
0.02815843
4.70652526
0.03329752
6.5864662
6.58555209
0.375531
0.71873598
0.64873536
17.17832949
5.77465245
2.32933748
8.99741847
62.43865106
0.45003245
11.87042946
115.4575489
5.88566941
7.52811028
150.8803385
135.1570046
11.41084435
0.61033333
0.14986149
0.05908333
0.03507305
24.97916667
152.3458333
13.475
1.02416667
43.518625
-8.980875
186.7458333
1.55470833
0.53175
0.7065
1.57204167
-0.01433333
190.6458333
29.29998471
74.75690858
13.32500873
0.05217581
9.64248797
2.39585688
109.3986127
0.44461341
0.24509639
0.35345432
0.46433242
1.34621132
117.356213
n
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
240
173!
Table S3.2 Gait analysis parameters of female Gnao1 G203R mutant mice
Gait analysis parameters Female Gnao1 G203R mutants
Feng et al Table S2
n
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
F Gnao1+/+
0.08701905
37.98428571
0.03703333
15.94428571
0.10746667
46.06619048
0.14451429
62.01571429
0.23155238
25.63047619
74.36952381
1.66857143
5.80761905
4.54809524
0.40666667
17.21047619
5.24428571
8.97619048
46.68095238
0.82042857
7.22380952
93.55714286
23.34761905
14.45657143
127.6666667
96.90952381
20.35714286
SD
0.0103341
4.4849659
0.0117622
4.3353439
0.0239663
4.7852095
0.029293
4.4849659
0.0346545
6.3902157
6.3902157
0.3092867
0.7083501
0.6820467
18.071952
5.3985099
2.5795276
8.1636612
62.270502
0.3400398
9.302076
122.72527
5.9556269
6.7501268
155.40339
123.48735
10.045441
0.624
0.138264
0.06057143
0.0355498
19.06190476
116.7095238
13.52380952
1.0192381
43.48771429
-9.69980952
164.8380952
1.42585714
0.44066667
0.62028571
1.8667619
-0.004
212.1142857
23.988055
70.404616
13.510298
0.0513119
9.941629
2.6687581
101.35854
0.3488621
0.1970416
0.3216493
0.3241286
1.2942361
132.61357
n
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
Hind Limb
F Gnao1+/G203R
0.08259735
36.3
0.03834513
16.8199115
0.10812832
46.88185841
0.14648673
63.70044248
0.22903982
26.23938053
73.76061947
1.79070796
5.5659292
4.59734513
1.02699115
15.92699115
5.44778761
8.96902655
53.61946903
0.86349558
8.37610619
89.84955752
24.08628319
15.80809735
123.6283186
105.039823
22.57513274
SD
0.011866
3.9776487
0.0114719
4.766196
0.0226036
4.0616338
0.0268555
3.9787156
0.0339969
6.5617814
6.5617814
0.3146108
0.6710776
0.6955682
16.942806
5.7724885
3.1063833
8.1553754
67.358321
0.299672
10.810084
112.67252
5.4387774
6.1679082
157.26751
125.67239
8.4641854
0.60482301
0.1192093
0.05575221
0.029599
21.98230088
144.300885
13.12389381
1.01123894
42.92628319
-8.8219469
206.1415929
1.30376106
0.47349558
0.69393805
1.61292035
-0.00207965
215.9734513
25.48742
66.869808
12.926894
0.0292767
8.6321179
2.4798691
106.91923
0.3572146
0.2414856
0.3180957
0.3222468
1.2662788
114.3211
n
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
226
11.6952381 12.247545
1.0192381
0.0513119
16.9396667 4.1600988
-5.1652381 1.8030427
12.27876106
1.01123894
16.29477876
-4.63566372
12.170536
0.0292767
3.8423131
1.6700964
1.42585714 0.3488621
1.30376106
0.3572146
1
1
1
0
0
0
1
1
1
1
0
0
0
0
Fore Limb
F Gnao1+/G203R
0.08465044
37.58362832
0.07067699
31.14867257
0.07115487
31.26681416
0.14184071
62.41637168
0.22645575
49.8840708
50.1159292
1.68141593
5.50530973
4.64778761
-1.51504425
4.13274336
7.53495575
4.28318584
83.71238938
1.13561947
15.69026549
88.42920354
24.34070796
21.13402655
82.69469027
100.6283186
27.70469027
SD
0.011645
3.1433241
0.0163127
5.0584954
0.0169689
4.8963937
0.0252212
3.1433241
0.0342293
7.5687729
7.5687729
0.2274687
0.6725528
0.7174148
4.944406
3.0979403
2.560203
3.588795
92.843532
0.3330556
16.365318
114.15516
5.3805268
7.3727667
91.571949
129.68952
8.038455
0.30084071
0.0747244
0.03048673
0.0186722
SD
31.35
F Gnao1+/+
0.0862619
0.0112876
38.2376191 3.4885409
0.06979524
0.019761
30.4114286 5.6522649
0.07122857 0.0153668
4.8383758
0.14104286 0.0265394
61.762381
3.4885409
0.22737619 0.0347068
8.051334
49.1166667
50.8833333
8.051334
1.63809524 0.2335009
5.70190476 0.7409397
4.63333333 0.7236493
-2.0414286 5.1145489
4.27380952 3.4631111
7.08
2.9170313
4.65238095 3.9041285
87.4904762 95.888554
1.11104762 0.3637969
14.8666667
16.15035
94.5571429 123.21106
23.7642857 5.9102466
20.0334286 7.9432229
71.4761905
83.50877
92.3857143 125.23065
25.4634286 9.9336491
0.31547619 0.0784235
0.03247619 0.0177047
0.44066667 0.1970416
0.85080952
0.36314
-1.9551905 0.4090709
-0.0137143
0.794259
n
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
210
Fore Limb
Hind Limb
Measured Parameter
X.SwingStride
Swing
Brake
X.BrakeStride
Propel
X.PropelStride
Stance
X.StanceStride
Stride
X.BrakeStance
X.PropelStance
Stance.Swing
StrideLength
Stride.Frequency
PawAngle
Absolute.PawAngle
p
0.143574
0.040102
0.610655
0.151434
0.962205
0.858596
0.747729
0.040102
0.780626
0.30554
0.30554
0.050429
0.003872
0.83429
0.275233
0.653751
FDR
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
p
0.000042
0.000039
0.239211
0.045932
0.766869
0.055104
0.463647
0.000039
0.445327
0.327424
0.327424
0.000053
0.000285
0.456267
0.711622
0.017128
FDR
Yes
Yes
No
No
No
No
No
Yes
No
No
No
Yes
Yes
No
No
No
SLVar
SWVar
StanceWidth
StepAngle
Paw.Angle.Variability 0.083677
0.304131
0.676222
0.46197
0.597496
0.590118
0.287034
0.134218
0.183108
0.500567
0.009699
Stride.Length.CV
Stance.Width.CV
Step.Angle.CV
StepAngleVar
Swing.Duration.CV
Paw.Area.at.Peak.Sta
X.Steps
nce.in.sq..cm
Paw.Area.Variability.
at.Peak.Stan
Hind.Limb.Shared.St
ance.Time
X..Shared.Stance
StanceFactor
Gait.Symmetry
MAX.dA.dT
MIN.dA.dT
Tau..Propulsion
Overlap.Distance
PawPlacementPositi
oning.PPP.
Ataxia.Coefficient
Midline.Distance
Axis.Distance
Paw.Drag
0.046636
0.25505
0.618239
0.044325
0.093152
0.001558
0.000346
0.122248
0.125326
0.844898
0.882318
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
0.459013
0.992695
0.265709
0.160659
0.235126
0.742411
0.176573
0.029457
0.787718
0.496458
0.012817
0.120876
0.123726
0.219455
0.000033
0.752284
0.044325
0.528498
0.000411
0.000043
0.000346
0.122248
0.01669
<0.000001
0.987517
0.744503
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
No
No
No
Yes
Yes
Yes
No
No
Yes
No
No
0.47349558
0.90159292
-1.96256637
-0.00261062
1
0.2414856
0.3272514
0.3777178
0.7706896
0
174!
Table S3.3 Gait analysis parameters of male Gnao1 G184S mutant mice
Gait analysis parameters Male Gnao1 G184S mutants
Feng et al Table S3
Fore Limb
M Gnao1+/G184S
0.08857143
37.6702381
0.05385714
22.75119048
0.09477381
39.57857143
0.14857143
62.3297619
0.23721429
36.49047619
63.50952381
1.67142857
5.93333333
4.44761905
0.91547619
4.55119048
8.06666667
3.79761905
46.78571429
1.32916667
14.6547619
39.98809524
22.25595238
22.82166667
35.82142857
40.55952381
26.30416667
SD
0.011722
3.3344055
0.0134703
4.3988359
0.023563
4.5858365
0.0298184
3.3344055
0.0389603
6.6003216
6.6003216
0.2341719
0.693226
0.7554582
5.4220321
3.046922
3.1549286
3.0528666
50.288342
0.4364264
15.444026
52.336606
5.1316958
8.3634236
40.621426
53.357704
9.8202879
0.26928571
0.0702111
0.02559524
0.0187148
n
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
n
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
M Gnao1+/+
0.09
36.51428571
0.02879592
11.57857143
0.13060204
51.91632653
0.15944898
63.48571429
0.24947959
18.21020408
81.78979592
1.79183673
6.26122449
4.22346939
-0.05204082
17.08877551
4.2877551
7.12244898
25.30612245
0.91683673
6.80612245
42.21428571
21.0255102
14.93540816
57.7244898
48.95918367
21.32826531
SD
0.0127724
5.1117008
0.0075135
2.4412372
0.029081
4.6134205
0.0336318
5.1117008
0.0391545
3.3839064
3.3839064
0.3701974
0.9164738
0.6697901
17.660751
4.1070692
1.9761081
6.2363506
32.434997
0.309591
7.6934276
51.158184
4.4032395
5.4905719
69.576003
59.29655
7.197867
0.70244898
0.1316122
0.04530612
0.030127
14.24489796
74.91836735
9.25510204
1.01755102
51.415
-8.24183673
103.755102
1.40122449
0.43704082
0.61469388
1.40795918
0.01989796
107.2653061
17.100709
45.56058
9.6744016
0.0511529
10.373366
2.1764496
45.984547
0.3999259
0.1825829
0.248655
0.2798857
1.3185305
45.749779
n
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
98
Hind Limb
M Gnao1+/G184S
0.08357143
35.3
0.02907143
12.08333333
0.12857143
52.62738095
0.15763095
64.7
0.24122619
18.70238095
81.29880952
1.89642857
6.01071429
4.40238095
0.675
16.66309524
5.24047619
7.78571429
22.44047619
0.9572619
7.66666667
36.42857143
21.95238095
16.00988095
59.47619048
58.16666667
23.49071429
SD
0.0102043
5.2765451
0.0081415
2.8270992
0.0333879
5.3388179
0.0377269
5.2765451
0.0423552
4.2032969
4.2045728
0.4203975
0.634551
0.7735034
17.87932
6.2551719
2.7850772
7.0198882
30.489718
0.4562322
9.442704
44.194064
5.1569459
7.6712154
70.164948
63.81578
11.914931
0.60595238
0.1286643
0.05357143
0.0337833
15.35714286
87.82142857
9.55952381
1.01452381
44.32047619
-9.5402381
72.35714286
1.61261905
0.51178571
0.6727381
1.51952381
0.00738095
72.89285714
17.924637
42.745862
9.5328869
0.0503579
10.399373
2.7031744
49.842316
0.5000846
0.1905993
0.3411474
0.4244029
1.4239324
52.00487
n
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
84
Fore Limb
Hind Limb
Measured Parameter
X.SwingStride
X.BrakeStride
X.PropelStride
X.StanceStride
Swing
Brake
Propel
Stance
Stride
X.BrakeStance
X.PropelStance
Stance.Swing
StrideLength
Stride.Frequency
PawAngle
Absolute.PawAngle
p
0.043466
0.726843
0.826349
0.180502
0.148248
0.318489
0.27969
0.726843
0.140428
0.197014
0.197014
0.810208
0.047635
0.153098
0.223604
0.062068
FDR
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
p
0.000278
0.117241
0.812719
0.197797
0.66152
0.336379
0.731504
0.117241
0.173925
0.382853
0.384084
0.075995
0.036347
0.096182
0.783411
0.583264
FDR
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
SLVar
SWVar
StanceWidth
StepAngle
Paw.Angle.Variability 0.000004
0.931208
0.781449
0.018345
0.268206
0.51712
0.201763
0.009988
0.245913
0.386308
0.495053
Stride.Length.CV
Stance.Width.CV
Step.Angle.CV
StepAngleVar
Swing.Duration.CV
Paw.Area.at.Peak.Sta
X.Steps
nce.in.sq..cm
Paw.Area.Variability.
at.Peak.Stan
Hind.Limb.Shared.St
ance.Time
X..Shared.Stance
StanceFactor
Gait.Symmetry
MAX.dA.dT
MIN.dA.dT
Tau..Propulsion
Overlap.Distance
PawPlacementPositi
oning.PPP.
Ataxia.Coefficient
Midline.Distance
Axis.Distance
Paw.Drag
<0.000001
0.002006
0.396136
0.688994
<0.000001
0.891498
0.001809
0.00764
0.055735
0.998012
0.836286
Yes
No
No
No
No
No
No
No
No
No
No
Yes
No
No
No
Yes
No
No
No
No
No
No
0.007857
0.50059
0.542101
0.480115
0.49907
0.419338
0.192532
0.274044
0.866251
0.314724
0.13408
0.000001
0.082786
0.669308
0.051594
0.831523
0.688994
0.000008
0.000429
0.000017
0.001809
0.00764
0.187315
0.035382
0.951005
0.000004
No
No
No
No
No
No
No
No
No
No
No
Yes
No
No
No
No
No
Yes
Yes
Yes
Yes
No
No
No
No
Yes
M Gnao1+/+
0.09253061
37.83979592
0.05344898
21.91020408
0.09986735
40.25714286
0.15326531
62.16020408
0.24587755
35.25306122
64.74693878
1.66326531
6.17959184
4.29387755
-0.00102041
3.73163265
6.31938776
3.83673469
48.89795918
1.18122449
12.31632653
45.3877551
21.35714286
19.70622449
29.3877551
33.97959184
25.39285714
SD
0.0141627
3.1936386
0.0115937
4.0355505
0.0236179
4.5416969
0.028498
3.1936386
0.0396769
6.2745798
6.2745798
0.2230934
0.9320147
0.6895115
4.7029821
2.8371467
1.7027969
3.0348283
51.835284
0.4015749
12.961161
58.864099
4.3328046
7.7656016
33.936154
48.807634
8.1613281
0.32091837
0.0605148
0.01877551
0.0098719
0.43704082
0.81897959
-2.76357143
0.03887755
1
0.1825829
0.3422138
0.3019703
0.8386733
0
1
1
1
0
0
0
8.70408163
1.01755102
19.58153061
-4.68867347
9.4859736
0.0511529
3.7431478
0.9273804
7.57142857
1.01452381
16.61440476
-4.71535714
8.2932484
0.0503579
3.9087463
1.6546185
1.40122449
0.3999259
1.61261905
0.5000846
1
1
1
0
0
0
0.51178571
0.91821429
-2.76369048
0.01333333
1
0.1905993
0.3516473
0.3417171
0.8200578
0
175!
Table S3.4 Gait analysis parameters of female Gnao1 G184S mutant mice
Gait analysis parameters Female Gnao1 G184S mutants
Feng et al Table S4
Fore Limb
Hind Limb
Measured Parameter
X.SwingStride
X.BrakeStride
X.PropelStride
X.StanceStride
Swing
Brake
Propel
Stance
Stride
X.BrakeStance
X.PropelStance
Stance.Swing
StrideLength
Stride.Frequency
PawAngle
Absolute.PawAngle
p
0.000002
0.352054
0.018172
0.605581
0.073513
0.303687
0.004809
0.352054
0.000183
0.490318
0.489918
0.335195
0.0007
0.000035
0.167612
0.167612
FDR
Yes
No
No
No
No
No
No
No
Yes
No
No
No
Yes
Yes
No
No
p
0.00019
0.571929
0.041827
0.827244
0.003264
0.75231
0.001181
0.571929
0.000242
0.875453
0.875453
0.536831
0.000352
0.000096
0.464223
0.464223
FDR
Yes
No
No
No
Yes
No
Yes
No
Yes
No
No
No
Yes
Yes
No
No
F Gnao1+/+
0.08906667
38.21333
0.062156
26.46
0.083178
35.33444
0.145322
61.78667
0.234389
42.76444
57.23556
1.64
5.707778
4.468889
4.784444
4.784444
6.265556
1.684444
64.84444
1.152444
0.349556
13.42622
18.32778
20.47044
21.188
21.42844
25.59067
SD
0.012567
3.504418
0.017608
5.698922
0.019603
5.498685
0.026527
3.504418
0.035201
8.767346
8.767346
0.240692
0.807719
0.682797
2.854089
2.854089
2.252647
0.330259
7.514911
0.381574
0.098164
4.439098
5.488579
6.723017
6.590364
8.660338
8.521292
0.398111
0.152248
0.031556
0.019309
1.009333
1.012889
25.22511
6.588111
0.075179
0.039413
9.069926
2.658642
1.749778
0.686131
0.443222
0.794889
2.572
0.838222
1
0.221435
0.295526
0.38345
0.176753
0
n
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
SLVar
SWVar
StanceWidth
StepAngle
Paw.Angle.Variability 0.000023
0.022922
0.998777
0.164572
0.200099
0.882892
0.00456
0.568182
0.007756
0.734363
0.412289
Stride.Length.CV
Stance.Width.CV
Step.Angle.CV
StepAngleVar
Swing.Duration.CV
Paw.Area.at.Peak.Sta
X.Steps
nce.in.sq..cm
Paw.Area.Variability.a
t.Peak.Stan
Hind.Limb.Shared.Sta
nce.Time
X..Shared.Stance
StanceFactor
Gait.Symmetry
MAX.dA.dT
MIN.dA.dT
Tau..Propulsion
Overlap.Distance
PawPlacementPositio
ning.PPP.
Ataxia.Coefficient
Midline.Distance
Axis.Distance
Paw.Drag
0.000002
0.291841
0.167306
0.25904
<0.000001
0.014025
0.001549
0.979063
0.203884
0.039565
0.000293
0.055735
0.998012
0.836286
Yes
No
No
No
No
No
No
No
No
No
No
Yes
No
No
No
Yes
No
Yes
No
No
No
Yes
No
No
No
0.003025
0.068763
0.466437
0.379246
0.091884
0.454172
0.006296
0.504883
0.02606
0.682633
0.494238
0.000015
0.294115
0.112442
0.28745
0.357268
0.25904
0.000014
0.119243
0.253295
0.001549
0.979063
0.38151
0.000029
0.015682
0.00008
Yes
No
No
No
No
No
No
No
No
No
No
Yes
No
No
No
No
No
Yes
No
No
Yes
No
No
Yes
No
Yes
Hind Limb
F Gnao1+/G184S
0.082333
37.79697
0.026402
12.075
0.110394
50.11818
0.136818
62.20303
0.219182
19.34394
80.65606
1.667424
5.420455
4.823485
16.82121
16.82121
5.02197
2.410606
54.86515
0.757652
0.259242
12.06667
19.98864
14.31485
10.9447
22.30394
18.80689
SD
0.012478
3.394359
0.00874
3.589752
0.023384
3.746567
0.026794
3.394359
0.03649
5.293627
5.293627
0.242599
0.691799
0.795393
5.22535
5.22535
2.460662
0.412918
8.435848
0.273303
0.129024
3.238623
4.73676
5.874303
5.480517
6.351191
6.401079
0.660227
0.230086
0.059773
0.045725
0.051152
36.32273
1.019545
1.018333
51.24644
10.95538
0.138298
1.463712
0.442424
0.579848
1.190758
1.220076
5.45697
0.019511
8.859942
0.061129
0.032011
18.95255
5.069048
0.06125
0.629242
0.222697
0.279277
0.564684
0.219397
2.551102
n
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
n
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
132
F Gnao1+/+
0.088633
37.53889
0.029
12.18
0.119811
50.28222
0.148811
62.46111
0.237511
19.45556
80.54444
1.687778
5.781111
4.415556
16.32778
16.32778
4.153333
2.566667
56.06222
0.793778
0.220667
12.55133
18.08889
13.79056
8.748444
22.82822
18.19433
SD
0.011621
3.24618
0.01003
3.40298
0.022828
3.871491
0.026549
3.24618
0.035089
5.068056
5.068056
0.237888
0.775306
0.680369
4.441025
4.441025
1.479538
0.475299
8.526091
0.335337
0.097547
3.478382
5.451305
5.541985
4.294501
6.986445
6.749598
0.818667
0.301436
0.067
0.056299
0.057378
37.59111
1.009556
1.012889
64.31333
12.05456
0.150978
1.749778
0.443222
0.546444
1.539222
1.299111
7.0439
0.021
8.462962
0.047095
0.039413
24.77673
5.245177
0.103392
0.686131
0.221435
0.277777
0.641965
0.261708
3.321218
n
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
90
Fore Limb
F Gnao1+/G184S
0.080841
37.76212
0.056508
26.05303
0.078295
36.1803
0.134818
62.23788
0.215674
41.91894
58.08182
1.672727
5.342424
4.908333
4.209848
4.209848
7.730303
1.536364
64.84697
1.092273
0.375
13.54348
20.31818
20.97265
25.38455
22.01364
24.72242
SD
0.012049
3.562839
0.017191
5.79599
0.02004
6.320866
0.027276
3.562839
0.03649
9.074334
9.073969
0.252647
0.755956
0.810338
3.153645
3.153645
2.613352
0.333114
9.138359
0.261539
0.104679
3.867871
4.78319
6.21845
8.830206
9.055266
7.146609
0.311288
0.11312
0.029091
0.015356
0.988788
1.018333
19.5603
5.764015
0.077291
0.032011
6.647057
2.269378
1.463712
0.629242
0.442424
0.845379
2.450985
0.752045
1
0.222697
0.285901
0.455068
0.167454
0
176!
Table S3.5 Gait analysis parameters of male Gnao1 R209H mutant mice
N
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
M WT Mean
0.08889286
36.2732143
0.03811607
15.1651786
0.12082143
48.5633929
0.15891964
63.7267857
0.24777679
23.6625
76.3375
1.80178571
6.23035714
4.21964286
1.14821429
17.0785714
4.3625
7.05357143
31.9732143
0.83419643
5.17857143
46.75
24.3080357
13.5108036
65.6696429
52.7767857
19.8541964
SD
0.009719
4.576024
0.011977
3.560158
0.021481
3.628342
0.029695
4.576024
0.033051
4.618239
4.618239
0.371018
0.794104
0.559736
17.7401
4.6622
1.635481
6.172942
43.08831
0.271292
5.429672
60.6561
4.825909
4.479219
74.19895
64.92819
5.777298
0.57464286
0.063401
0.044375
0.019162
16.2678571
82.4196429
10.7142857
1.00232143
40.355625
-7.7479464
90.5625
1.03803571
0.41776786
0.58044643
1.52928571
0.01223214
108.973214
19.84035
46.81026
10.60293
0.026913
4.795198
1.135325
46.16808
0.333044
0.151887
0.24559
0.19725
1.234583
56.80795
N
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
Hind Limb
M R209H Mean
0.08860417
35.76979167
0.03653125
14.56770833
0.12441667
49.66354167
0.1609375
64.23020833
0.2495625
22.54375
77.45729167
1.83645833
6.13854167
4.20104167
0.85520833
15.265625
4.95104167
7.17708333
28.34375
0.89395833
6.15625
48.04166667
24.65104167
14.95729167
76.25
54.9375
21.98125
SD
0.012193
4.245595
0.012519
4.416536
0.023144
4.175722
0.028273
4.245595
0.033977
5.984204
5.983552
0.379784
0.76066
0.578291
16.02805
4.705849
2.276886
6.412151
38.24184
0.326185
6.875323
58.69079
5.644336
6.409749
82.21333
64.68544
8.948453
0.53677083
0.068032
0.04375
0.019317
17.125
88.27083333
10.6875
0.9975
37.9240625
-7.51552083
104.5729167
1.251875
0.4609375
0.65375
1.506875
-0.01677083
96.90625
19.99961
45.32908
10.76477
0.044745
5.440301
1.551981
64.28223
0.401419
0.193277
0.31857
0.277653
1.264412
61.0565
N
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
96
Fore Limb
Hind Limb
P value
0.742713
0.186611
0.07883
0.046618
0.476735
0.224488
0.459252
0.186611
0.606739
0.144106
0.143904
0.254797
0.514326
0.599868
0.409773
0.056783
0.708628
0.384371
0.543298
0.435215
0.920715
0.24211
0.8498
0.286712
0.642162
0.453618
0.065786
FDR
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
P value
0.849568
0.414505
0.352604
0.281478
0.246967
0.043317
0.617997
0.414505
0.701773
0.130071
0.129692
0.507041
0.397668
0.814209
0.901338
0.005873
0.031802
0.887767
0.524421
0.15068
0.253516
0.876653
0.63707
0.057982
0.33056
0.810819
0.040295
FDR
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
0.269777
0.906173
0.904653
0.339967
0.337523
0.043386
0.00004
0.072965
0.131163
0.193089
0.734326
No
No
No
No
No
No
Yes
No
No
No
No
0.000048
0.815503
0.757282
0.36289
0.985628
0.339967
0.000739
0.215019
0.069796
0.00004
0.072965
0.062689
0.49868
0.867511
0.141646
Yes
No
No
No
No
No
Yes
No
No
Yes
No
No
No
No
No
Measured Parameters
X.SwingStride
X.BrakeStride
X.PropelStride
X.StanceStride
Swing
Brake
Propel
Stance
Stride
X.BrakeStance
X.PropelStance
Stance.Swing
StrideLength
Stride.Frequency
PawAngle
StanceWidth
StepAngle
SLVar
SWVar
StepAngleVar
X.Steps
Stride.Length.CV
Stance.Width.CV
Step.Angle.CV
Absolute.PawAngle
Paw.Angle.Variability
Swing.Duration.CV
Paw.Area.at.Peak.Stanc
e.in.sq..cm
Paw.Area.Variability.at.P
eak.Stan
Hind.Limb.Shared.Stan
ce.Time
X..Shared.Stance
StanceFactor
Gait.Symmetry
MAX.dA.dT
MIN.dA.dT
Tau..Propulsion
Overlap.Distance
PawPlacementPositioni
ng.PPP.
Ataxia.Coefficient
Midline.Distance
Axis.Distance
Paw.Drag
M WT Mean
0.09575
39.058929
0.0748482
30.158036
0.0765714
30.778571
0.1515089
60.941071
0.2472232
49.605357
50.395536
1.5875
6.2089286
4.2294643
-1.589286
3.075
6.4607143
3.4107143
48.8125
1.0917857
12.625
45.919643
24.424107
17.998393
31.107143
49.205357
24.166161
SD
0.010045
3.751313
0.018216
5.195713
0.02064
5.958209
0.02807
3.751313
0.03416
8.646812
8.64638
0.262395
0.784024
0.586429
3.516355
2.31787
2.427261
2.559397
54.21986
0.27152
12.67695
60.59437
4.986563
5.348469
36.02711
63.63878
6.335163
0.2744643
0.031902
0.0205357
0.010209
1
1
1
9.5178571
1.0023214
14.877321
-3.363571
9.432532
0.026913
2.009157
0.813505
0
0
0
0.4177679
0.7350893
-1.933214
0.0076786
1
0.151887
0.247932
0.331655
0.789782
0
1.0380357
0.333044
1.251875
0.401419
N
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
112
Fore Limb
M R209H Mean
0.09525
38.39479167
0.07973958
31.809375
0.074625
29.796875
0.15432292
61.60520833
0.24967708
51.459375
48.540625
1.628125
6.1375
4.18645833
-1.11875
3.77708333
6.57083333
3.11458333
53.5625
1.13145833
12.44791667
56.60416667
24.5625
19.09291667
33.63541667
42.73958333
26.689375
SD
0.011893
3.422753
0.021724
6.68853
0.018382
5.594329
0.026341
3.422753
0.034296
9.584303
9.584303
0.247813
0.788636
0.590917
4.682854
2.962662
1.67997
2.298145
58.20288
0.449982
12.89053
70.76982
5.537076
9.18012
42.3313
59.83877
12.71546
0.2796875
0.036171
0.02072917
0.013396
1
1
1
9.67708333
0.9975
15.17510417
-3.645625
9.675248
0.044745
2.457478
1.177005
0
0
0
0.4609375
0.805625
-1.9984375
-0.02947917
1
0.193277
0.413488
0.388778
0.781835
0
177!
Table S3.6 Gait analysis parameters of female Gnao1 R209H mutant mice
Fore Limb
Hind Limb
Brake
Stride
Swing
Propel
Stance
X.BrakeStride
X.SwingStride
X.PropelStride
X.StanceStride
Stride.Frequency
Measured Parameters
X.BrakeStance
X.PropelStance
Stance.Swing
StrideLength
P value
0.000862
0.553568
0.257048
0.239615
0.062971
0.457782
0.052934
0.553568
0.009436
0.314746
0.314746
0.554245
0.920542
0.014077
0.094114
0.008759
0.273075
0.48535
0.829913
0.178551
0.620186
0.52429
0.134323
0.237484
0.865848
0.162584
0.294853
Paw.Area.at.Peak.Stance.in.sq..cm 0.00641
Paw.Area.Variability.at.Peak.Stan 0.166619
Hind.Limb.Shared.Stance.Time
Stride.Length.CV
Stance.Width.CV
Step.Angle.CV
Absolute.PawAngle
Paw.Angle.Variability
StanceWidth
StepAngle
Swing.Duration.CV
StepAngleVar
SLVar
SWVar
PawAngle
X.Steps
FDR
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
X..Shared.Stance
StanceFactor
Gait.Symmetry
MAX.dA.dT
MIN.dA.dT
Tau..Propulsion
Overlap.Distance
Ataxia.Coefficient
Midline.Distance
Axis.Distance
Paw.Drag
PawPlacementPositioning.PPP.
0.369433
0.140168
0.013856
0.020047
0.000061
0.110841
0.291223
0.495964
0.755914
No
No
No
No
Yes
No
No
No
No
P value
0.001686
0.784138
0.198976
0.003087
0.000237
0.000572
0.019444
0.784138
0.003393
0.001422
0.001422
0.625985
0.777101
0.002251
0.886736
0.141453
0.075906
0.502257
0.607524
0.006484
0.655617
0.895657
0.121155
0.006673
0.556665
0.685476
0.002003
0.66547
0.005094
0.537429
0.630463
0.629805
0.140168
0.798156
0.170644
0.758007
0.000061
0.110841
0.016224
0.002418
0.937074
0.000004
F WT Mean
0.088759
38.269277
0.0732651
31.40241
0.0713374
30.322289
0.1446145
61.730723
0.2334036
50.877108
49.122892
1.6361446
5.836747
4.5030121
-1.226506
3.673494
6.5048193
3.5240964
62.024096
1.0621687
14.644578
61.03012
27.427711
18.675843
50.23494
51.463855
25.371325
0.2454217
0.0223494
1
1
1
1
9.3192771
1.0078313
12.997831
-3.557771
0.9884337
0.4084337
0.7913855
-1.795783
0.003253
SD
0.011161
3.355496
0.016007
4.642243
0.017694
4.635664
0.026021
3.355496
0.033972
6.954237
6.954237
0.235657
0.776261
0.657122
4.09998
2.178745
1.980413
2.863991
67.35433
0.280301
15.3228
78.19598
6.803579
6.063927
55.10375
72.44272
7.757021
0.046964
0.0077
0
0
0
0
9.282136
0.026056
2.686344
0.714287
0.392521
0.147262
0.282622
0.305625
0.750176
N
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
Fore Limb
F R209H Mean
0.09418182
38.53295455
0.07582955
30.67613636
0.07560227
30.7875
0.15142045
61.46704545
0.24563636
49.93863636
50.06136364
1.61818182
5.82613636
4.28636364
-0.36022727
2.94204545
6.20113636
3.79545455
60.11363636
1.11465909
15.68181818
54.72727273
26.14772727
19.66806818
51.48863636
65.53409091
24.28715909
0.2625
0.02386364
1
1
1
1
8.26136364
1.01545455
13.86136364
-3.82125
1.23068182
0.44352273
0.83193182
-1.82375
-0.02784091
SD
0.013945
3.399754
0.019054
4.729754
0.016582
4.943423
0.027503
3.399754
0.038132
7.271317
7.271317
0.218955
0.859229
0.678264
3.519944
1.940655
2.301149
3.093037
67.41746
0.321265
16.8117
68.41115
5.759629
6.872459
58.28448
82.82733
7.973726
0.047398
0.009276
0
0
0
0
8.197973
0.055974
2.557019
1.069478
0.543744
0.197449
0.305505
0.321102
0.771926
N
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
SD
F WT Mean
0.0102
0.0841024
4.11783
36.083735
0.013145
0.0411506
4.881231
17.425904
0.021791
0.1098855
3.90007
46.489157
0.028399
0.1510843
4.11783
63.916265
0.03372
0.2352108
6.667062
27.063855
6.667062
72.936145
0.324599
1.8054217
0.758562
5.8759036
0.641689
4.4710843
15.44145
0.7493976
4.431629
14.766265
1.885737
4.7891566
6.89088
7.560241
45.03798
34.722892
0.275812
0.8433133
6.824598
6.560241
67.93058
52.662651
6.745719
27.274096
4.889888
14.471386
94.41713
72.174699
81.02243
67.572289
5.795205
20.003675
0.079416
0.5005422
0.016391
0.0453615
21.25847
17.277108
53.24314
97.849398
9.739514
10.120482
0.026056
1.0078313
34.648434
6.534348
-7.6283133 1.321913
61.46583
0.392521
0.147262
0.254344
0.233483
1.19335
69.54238
0.9884337
0.4084337
0.6390964
1.5896988
-0.0086145
142.39157
112
N
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
166
Hind Limb
F R209H Mean
0.08846591
35.93068182
0.03895455
15.58409091
0.12122727
48.48409091
0.16014773
64.06931818
0.24863636
24.28409091
75.71590909
1.82727273
5.90568182
4.21590909
0.45227273
15.65681818
5.24431818
8.19318182
37.97727273
0.97397727
6.97727273
53.84090909
25.94886364
16.61125
79.59090909
72
22.93125
0.49590909
0.05272727
19.07954545
101.2954546
10.76136364
1.01545455
34.42875
-7.84977273
109.4545455
1.23068182
0.44352273
0.72954545
1.69352273
-0.02136364
99.01136364
SD
0.010834
4.442386
0.012517
4.255784
0.025295
5.061345
0.030727
4.442386
0.035716
6.276995
6.276995
0.36632
0.864811
0.598059
16.46886
4.845256
2.029884
7.600451
53.14521
0.48285
7.549039
68.31551
5.887372
7.523195
97.67394
86.10726
9.096783
0.084386
0.024947
23.70665
56.13001
10.67169
0.055974
6.45687
1.00655
64.65877
0.543744
0.197449
0.331666
0.296365
1.27859
70.34978
N
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
88
FDR
Yes
No
No
No
Yes
Yes
No
No
No
Yes
Yes
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
No
Yes
No
No
No
No
No
No
No
No
No
Yes
No
No
Yes
No
Yes
178!
Table S3.7 Gait analysis parameters of male Gnao1 KO mutant mice
Fore Limb
Hind Limb
FDR
Yes
No
No
Yes
No
No
No
No
No
No
No
No
Yes
No
No
No
No
No
No
Yes
No
No
No
No
No
No
No
P value
<0.000001
0.000566
0.72381
0.098909
0.549673
0.030644
0.662124
0.000566
0.036704
0.430085
0.430085
0.000456
0.000016
0.029354
0.929857
0.000132
0.975957
0.968237
0.887753
0.001399
0.86119
0.083955
0.970756
0.1875
0.611842
0.668913
0.001224
FDR
Yes
Yes
No
No
No
No
No
Yes
No
No
No
Yes
Yes
No
No
Yes
No
No
No
Yes
No
No
No
No
No
No
Yes
0.055625
0.032442
0.974929
0.558834
0.277738
0.220542
0.282875
0.049741
0.236915
0.001913
0.840325
No
No
No
No
No
No
No
No
No
No
No
0.067872
0.000027
0.526683
0.010604
0.843972
0.558834
0.036413
0.000003
0.111301
0.282875
0.049741
0.394142
<0.000001
0.817383
0.000026
No
Yes
No
No
No
No
No
Yes
No
No
No
No
Yes
No
Yes
P value
0.000054
0.024865
0.165587
0.000746
0.011992
0.075845
0.224409
0.024765
0.017583
0.003525
0.003525
0.007551
0.000016
0.010131
0.4019
0.117131
0.033279
0.098559
0.582856
0.000188
0.954784
0.90312
0.908455
0.229963
0.57553
0.969952
0.041877
Measured Parameters
X.SwingStride
X.BrakeStride
X.PropelStride
X.StanceStride
Swing
Brake
Propel
Stance
Stride
X.BrakeStance
X.PropelStance
Stance.Swing
StrideLength
Stride.Frequency
PawAngle
StanceWidth
StepAngle
SLVar
SWVar
StepAngleVar
X.Steps
Stride.Length.CV
Stance.Width.CV
Step.Angle.CV
Absolute.PawAngle
Paw.Angle.Variability
Swing.Duration.CV
Paw.Area.at.Peak.Stanc
e.in.sq..cm
Paw.Area.Variability.at.P
eak.Stan
Hind.Limb.Shared.Stan
ce.Time
X..Shared.Stance
StanceFactor
Gait.Symmetry
MAX.dA.dT
MIN.dA.dT
Tau..Propulsion
Overlap.Distance
PawPlacementPositioni
ng.PPP.
Ataxia.Coefficient
Midline.Distance
Axis.Distance
Paw.Drag
M WT Mean
0.0966143
38.779286
0.0560429
22.397857
0.0979429
38.821429
0.1540143
61.220714
0.2506429
36.537143
63.462857
1.5964286
6.29
4.185
-0.705714
4.9457143
7.0635714
3.45
61.464286
1.4037143
13.928571
58.392857
21.817857
22.860071
46.407143
54.4
28.250429
SD
0.013541
3.410721
0.014514
4.576582
0.022044
4.457018
0.028645
3.410721
0.03881
6.893364
6.893364
0.227723
0.818869
0.630966
5.816768
3.114537
2.029963
2.661543
66.30954
0.373751
14.98519
74.79071
4.385313
7.555922
53.94267
73.56002
9.041509
0.2716429
0.051797
0.021
0.008591
0
0
0
1
1
1
8.7571429
1.0077143
16.484857
-4.639
9.097293
0.051682
2.680563
0.926215
1.3222857
0.336865
0.4799286
0.9550714
-2.694786
0.0115
1
0.173721
0.340194
0.350606
0.801008
0
SD
0.013372
4.330104
0.015854
5.745158
0.021646
5.447477
0.027824
4.331596
0.035769
8.483339
8.483339
0.304477
0.777601
0.644087
5.154065
3.095935
2.431709
2.092217
61.51358
0.305636
15.00567
73.8727
4.550977
6.790976
56.61555
72.00602
7.979169
0.055659
0.006557
0
0
0
8.903614
0.025839
3.247951
1.002793
0.340982
0.215823
0.333297
0.389434
0.768448
0
N
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
M WT Mean
0.09272143
37.1207143
0.03049286
12.1007143
0.12967857
50.775
0.16015
62.8792857
0.25293571
19.2214286
80.7785714
1.735
6.33285714
4.16285714
0.34071429
18.8221429
4.76857143
7.68571429
30.8785714
1.13464286
8.29285714
59.25
21.6285714
18.1392143
72.3
59.2642857
24.3625714
SD
0.010933
4.462917
0.00852
2.923768
0.029871
4.326131
0.034105
4.462917
0.040235
4.247196
4.247196
0.333269
0.759443
0.663579
19.33209
4.126107
1.893194
6.825772
42.43293
0.419421
10.34269
74.44153
4.351206
7.026218
91.3001
72.92655
8.268823
0.59078571
0.059483
0.05207143
0.027261
17.5357143
87.6285714
10.3857143
1.00771429
43.0139286
-8.8659286
110
1.32228571
0.47992857
0.77992857
1.45178571
-0.0047143
103.528571
21.94513
49.65526
10.55481
0.051682
4.791189
1.463116
64.09716
0.336865
0.173721
0.325529
0.368579
1.32741
66.43366
Hind Limb
M KO Mean
0.08395
35.062
0.03093
12.858
0.12735
52.082
0.15824
64.938
0.24218
19.738
80.262
1.899
5.899
4.352
0.127
16.617
4.761
7.65
31.66
0.9808
8.54
43.77
21.65
17.0179
66.41
63.41
21.0523
SD
0.010752
4.550764
0.010592
4.159477
0.029423
4.937619
0.032238
4.550764
0.037453
5.879957
5.879957
0.377524
0.740665
0.652653
17.32487
4.610157
1.949302
6.867248
41.96396
0.265626
11.37285
58.11359
4.60758
5.621839
84.49746
75.36405
6.891723
0.607
0.077401
0.0391
0.015641
19.41
104.62
10.12
1.011
44.5698
-7.9607
123.91
1.37
0.5296
0.7451
1.0712
0.035
139.88
23.43919
51.38344
9.93136
0.025839
6.666559
1.404932
69.66487
0.340982
0.215823
0.290925
0.43125
1.290374
62.16779
N
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
N
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
Fore Limb
M KO Mean
0.08936
37.65
0.05879
24.677
0.09069
37.679
0.1495
62.351
0.23888
39.468
60.532
1.689
5.828
4.401
-1.316
4.306
7.682
2.92
56.83
1.2313
14.04
59.58
21.885
21.718
50.45
54.04
25.9427
0.2851
0.0188
1
1
8.72
1.011
16.9022
-4.4848
1
1.37
0.5296
0.9027
-2.8457
0.0323
1
N
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
140
179!
Table S3.8 Gait analysis parameters of female Gnao1 KO mutant mice
N
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
M WT Mean
0.08811111
36.402381
0.03019841
12.4555556
0.12659524
51.1444444
0.15688095
63.5976191
0.24495238
19.5436508
80.4571429
1.78571429
6.0047619
4.31825397
-0.3563492
16.1230159
4.68571429
7.17460317
31.4126984
1.10555556
8.01587302
47.5238095
23.5277778
18.5309524
52.7698413
47.9444444
24.2536508
SD
0.011389
4.265311
0.009046
3.618786
0.030455
4.588133
0.033709
4.265311
0.040905
5.338532
5.338351
0.327344
0.663669
0.718265
16.56968
3.556811
1.67345
6.33603
41.97052
0.359134
9.890589
61.83885
4.458836
5.962569
74.45967
60.44056
6.894116
0.58753968
0.118644
0.05039683
0.021887
18.1746032
82.8174603
9.76984127
1.01571429
42.4457143
-8.7710318
93.0396825
1.31436508
0.39960317
0.82809524
1.37214286
-0.026746
96.7857143
21.7576
44.47514
9.806253
0.04399
8.392421
2.194612
54.28361
0.442389
0.21485
0.318601
0.462222
1.208638
59.58625
N
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
Hind Limb
M KO Mean
0.08773684
36.98947368
0.03043421
12.71184211
0.12134211
50.30657895
0.15181579
63.01052632
0.23951316
20.11447368
79.88552632
1.73552632
5.77763158
4.41315789
1.41184211
15.79342105
6.30394737
6.96052632
33.48684211
1.13855263
11.75
46.46052632
24.89473684
19.94105263
52.28947368
52.36842105
25.08039474
SD
0.011287
3.859001
0.0087
3.000665
0.026067
3.779959
0.030911
3.859001
0.038918
4.377875
4.377875
0.279859
0.581171
0.723573
16.49608
4.62412
4.855964
6.163204
44.48086
0.330473
15.73203
59.4231
5.08548
6.327376
70.78961
63.93452
7.679944
0.57828947
0.068729
0.05644737
0.031271
16.5
73.63157895
9.60526316
1.00421053
42.05407895
-8.63855263
100.7105263
1.19828947
0.41618421
0.90736842
1.26828947
-0.00013158
110.8289474
19.89874
38.92106
9.929188
0.050153
4.853255
1.626896
53.62383
0.412137
0.290181
0.298098
0.288543
1.174256
54.61755
N
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
76
1.3143651
0.442389
1.19828947
0.412137
Fore Limb
Hind Limb
P value
0.80571
0.727928
0.959871
0.766918
0.433018
0.611446
0.595756
0.727928
0.63591
0.68239
0.68239
0.692429
0.085256
0.630716
0.013715
0.044961
0.007312
0.297271
0.489835
0.565803
0.444068
0.705629
0.139258
0.955387
0.330411
0.612745
0.338005
FDR
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
P value
0.820639
0.327439
0.855721
0.604358
0.212013
0.18154
0.287305
0.327439
0.352323
0.432768
0.432118
0.26692
0.014479
0.365384
0.462617
0.570224
0.00075
0.814438
0.739738
0.515402
0.039537
0.904505
0.046748
0.113165
0.963959
0.622492
0.430043
FDR
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
0.040811
0.055277
0.175091
0.089351
0.088329
0.25101
0.06535
0.642852
0.406479
0.306676
0.630299
No
No
No
No
No
No
No
No
No
No
No
0.536284
0.108075
0.58501
0.138078
0.908552
0.089351
0.711101
0.648945
0.329558
0.06535
0.642852
0.080856
0.079655
0.878369
0.095768
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Measured Parameters
X.SwingStride
X.BrakeStride
X.PropelStride
X.StanceStride
Swing
Brake
Propel
Stance
Stride
X.BrakeStance
X.PropelStance
Stance.Swing
StrideLength
Stride.Frequency
PawAngle
StanceWidth
StepAngle
SLVar
SWVar
StepAngleVar
X.Steps
Stride.Length.CV
Stance.Width.CV
Step.Angle.CV
Absolute.PawAngle
Paw.Angle.Variability
Swing.Duration.CV
Paw.Area.at.Peak.Stanc
e.in.sq..cm
Paw.Area.Variability.at.P
eak.Stan
Hind.Limb.Shared.Stan
ce.Time
X..Shared.Stance
StanceFactor
Gait.Symmetry
MAX.dA.dT
MIN.dA.dT
Tau..Propulsion
Overlap.Distance
PawPlacementPositioni
ng.PPP.
Ataxia.Coefficient
Midline.Distance
Axis.Distance
Paw.Drag
M WT Mean
0.090881
37.835714
0.0605079
25.046825
0.0901111
37.116667
0.1505952
62.164286
0.2415397
40.286508
59.713492
1.6603175
5.9293651
4.3777778
1.4746032
4.2285714
6.8833333
3.7857143
54.34127
1.3600794
14.293651
49.730159
23.960317
23.392857
46.468254
47.706349
28.772381
SD
0.015084
3.303319
0.016707
4.950098
0.022099
5.123838
0.02981
3.303319
0.041883
7.425463
7.425463
0.239359
0.731253
0.727174
5.189311
3.331207
1.681987
2.986924
59.0929
0.343405
15.47285
62.78731
4.61762
6.995052
51.84063
61.74529
7.820674
0.2765079
0.054792
0.0228571
0.010648
1
1
1
10.134921
1.0157143
16.179365
-4.832778
10.84019
0.04399
3.068414
1.217865
0
0
0
0.3996032
1.0359524
-2.553175
0.013254
1
0.21485
0.359989
0.490671
0.758111
0
N
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
126
Fore Limb
M KO Mean
0.09034211
37.99342105
0.06063158
25.26184211
0.08768421
36.73684211
0.14834211
62.00657895
0.23867105
40.73947368
59.26052632
1.64736842
5.75657895
4.42894737
-0.54342105
5.20657895
7.89210526
3.35526316
48.53947368
1.3325
16.11842105
53.19736842
24.99342105
23.44684211
54.17105263
52.38157895
27.70763158
SD
0.015029
2.778601
0.017212
5.049963
0.01981
5.16622
0.02814
2.778601
0.041275
7.909034
7.909034
0.19898
0.608678
0.739336
6.194951
3.347649
3.577812
2.564912
55.40865
0.306725
17.7981
63.64171
5.068526
5.989497
58.31349
66.31239
7.309925
0.26118421
0.04472
0.02763158
0.024214
1
1
1
8.15789474
1.00421053
15.49092105
-4.62289474
8.423859
0.050153
2.176046
1.315192
0
0
0
0.41618421
1.07894737
-2.48618421
0.06552632
1
0.290181
0.348879
0.372673
0.727057
0
180!
Feng%at%al%Table&S5& &Benchling)off,target)list)for)Gnao1)G203)gRNA))
&
Table S3.9 Benchling off-target list for Gnao1 G203R gRNA
PAM& Score&
Chromosome& Mismatches&
0%
4)
4)
2)
4)
4)
4)
4)
4)
3)
4)
4)
4)
4)
4)
4)
4)
3)
4)
4)
4)
4)
4)
4)
4)
4)
4)
4)
3)
4)
GGG%
Sequence&
TGCAGGCTGTTTGACGTCGG%
GGCAAGCTGATTGACGTCTG) TAG)
TGGATGGTGTTGGACGTCGG) AAG)
TGCAGGCTGTTTGAAGTCTG) CAG)
GGTGGGCTGTTTGACGTGGG) AGG)
TTCAGGCTGAGTGACGTCAG) TGG)
AGCAGGCACTTTGAAGTCGG) AAG)
TTCAGTCTGTTAGACGTCTG) TAG)
TGCATGGGGTTTGACTTCGG) AGG)
TGCTGGCTGTTTGAGGTGGG) AAG)
TCCAGGCTGGTGGACGTGGG) CAG)
TGATGGCTGTTCGACTTCGG) GAG)
TACAGAATGTTTGACGTGGG) AGG)
TTCAGTCTGTTTGAGGTCGT) TGG)
AGCAGGCTGCTTGACATCGA) GAG)
TGCAAGCTGGTTGAGGTCAG) GGG)
TCCAGGATGTTTGATGCCGG) AAG)
TGCAGGCTGTCTGAAGTCTG) GGG)
GGCTGGCTGTTTGACCTCAG) AGG)
AGCAGCCTGTTTGAAGTCTG) TGG)
GGCAGGCTGTATGAAGGCGG) AGG)
TGGAGGCTGTTACACGTCAG) CAG)
TGCTGGCTATTTGAAGTCTG) AGG)
TGCTGGTTATTTGTCGTCGG) GAG)
TCCAGGCTGTCTGATGTCAG) GAG)
TTCAGGATGTTTGACGTATG) CAG)
TGCACGCTGTGAGACGTGGG) CGG)
TGCATGCTGTCTGAAGTCAG) AAG)
TGCAGGCTGTATGACCTCTG) GGG)
TGCAGTCTCTTTGACGACAG) TGG)
Gene&
chr8%
100% ENSMUSG00000031748%
) chr18)
0.6189)
0.5200) ENSMUSG00000041390) chr6)
) chr3)
0.5076)
0.3804)
) chr1)
0.3169) ENSMUSG00000032497) chr9)
) chr3)
0.2931)
0.1953)
) chr1)
0.1929)
) chr13)
) chr1)
0.1923)
0.1710)
) chr1)
0.1556) ENSMUSG00000086805) chr8)
0.1543) ENSMUSG00000057614) chr5)
0.1515)
) chrX)
) chr4)
0.1480)
0.1450)
) chr17)
) chr18)
0.1403)
0.1343) ENSMUSG00000026413) chr1)
0.1262)
) chrX)
) chr11)
0.1144)
0.1127)
) chr5)
) chr1)
0.1127)
0.1004)
) chr10)
0.1002)
) chr11)
) chrX)
0.0954)
0.0933)
) chr3)
0.0930) ENSMUSG00000020015) chr10)
0.0865)
) chrX)
0.0862)
) chr2)
) chr11)
0.0836)
181!
Table S3.9 (cont’d)
TGCATGCTGTAGGACCTCGG) AGG)
CGGAGGCTGTTTGACTTGGG) AGG)
TCCAGGCTGTTTCAGGACGG) AAG)
GGCAGCCTGTTTGACATCAG) GAG)
TGCAAGATGTTTGACCTCAG) AAG)
TGGAGGTTGTTTGAGGTAGG) AGG)
TGTGGGCTGTTTGACCTGGG) AGG)
GGCAGGCTGTTTGAAGCCAG) GGG)
TCCAGGCTGTTTGAGGGCTG) CAG)
TGCAGGCTGGCTGACGATGG) TGG)
TGCAGGATGCTTGACCTCTG) TAG)
TGCACTCTGTTTGAGGTTGG) AGG)
TCCAGGCTGTGTGAGGTGGG) AGG)
GGCAGGCTGTTGGAAGTAGG) GAG)
TGAAGGCTGTTCGAAGTGGG) GAG)
TGCAGGCTGATTGATGGCTG) GAG)
TACAGACTGTTTGACTTGGG) CAG)
TTCAGGCTGTTTTACTTCTG) AGG)
AGCAGGATGTTTGTCGTGGG) GAG)
AGCAGGCTGTGTGACCTGGG) AGG)
) chr4)
0.0771)
) chr5)
0.0754)
) chr8)
0.0745)
) chr17)
0.0744)
) chr19)
0.0720)
) chr2)
0.0704)
) chr19)
0.0693)
) chr9)
0.0669)
0.0647) ENSMUSG00000097637) chr8)
) chr8)
0.0611)
0.0604)
) chr2)
0.0599)
) chr10)
) chr9)
0.0574)
0.0520)
) chr2)
) chrX)
0.0480)
0.0470)
) chr7)
0.0430)
) chr3)
) chr15)
0.0422)
0.0420)
) chr1)
) chr6)
0.0385)
)))))))))))))))))))))Row)1)includes)the)on,target)gRNA)for)the)Gnao1)G203)site.)Off,target)hits)are)scored)and)ranked)by)an)
4)
4)
4)
4)
4)
4)
4)
4)
4)
4)
4)
4)
4)
4)
4)
4)
4)
4)
4)
4)
inverse)likelihood)of)off,target)binding.)If)an)off,target)is)predicted)to)occur)within)a)coding)region)of)a)
gene,)the)Ensembl)number)of)the)affected)locus)is)listed)in)the)Gene)column.)Analysis)was)performed)on)
the)Benchling)platform)using)reference)genome)GRCM38)(MM10,)Mus)Musculus),)guide)length)of)20bp,)
and)an)NGG)PAM.)
Row 1 includes the on-target gRNA for the Gnao1 G203 site. Off-target hits are scored
and ranked by an inverse likelihood of off-target binding. If an off-target is predicted to
occur within a coding region of a gene, the Ensembl number of the affected locus is
listed in the Gene column. Analysis was performed on the Benchling platform using
reference genome GRCM38 (MM10, Mus Musculus), guide length of 20bp, and an NGG
PAM.
182!
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CHAPTER 4: MICE WITH GNAO1-ASSOCIATED MOVEMENT DISORDER EXHIBIT
REDUCED INHIBITORY SYNAPTIC INPUT TO CEREBELLAR PURKINJE CELLS
Yukun, Y. did the recording for Figure S4.4 A-H.
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4.1 Abstract
GNAO1 encodes a heterotrimeric G protein subunit, Gαo, which belongs to the Gi/o
family. Mutations
in GNAO1 are associated with both early
infantile epileptic
encephalopathy 17 (EIEE17) and neurodevelopmental disorder with involuntary
movement (NEDIM). Our previous finding showed that gain-of-function (GOF) or
normal-function (NF) GNAO1 mutations, characterized by their inhibition of cAMP
production, are associated with movement disorder patients (Chapter 2) (Feng et al.,
2017). The majority of these patients present early onset dystonia or chorea/athetosis,
hypotonia, and developmental delay. Although NEDIM patients have been treated with
numerous available drugs, few proved to be effective. The pathological mechanisms of
this disorder also remain unclear. In this chapter, I provide the first data elucidating
neural mechanisms of a human GNAO1 mutant by investigating electrophysiological
effects in Gnao1+/G203R mice, a Gnao1-associated movement disorder mouse model.
These mice carry one of the most prevalent GNAO1 GOF mutations, G203R.
Patch clamp studies of cerebellar Purkinje cells showed significantly lower
frequencies of both action potential (AP) related (sIPSCs) and non AP-related (mIPSCs)
GABAergic responses in G203R mice. Amplitudes were not affected. Gαo inhibitors
reversed this reduction in inhibition significantly, and eliminated the difference between
WT and G203R mice. Furthermore, Gαo-coupled α2A adrenergic receptors played a
critical role in reducing the sIPSCs while mIPSCs events were regulated by GABAB
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receptors. The results prove G203R to be a bona fide GOF mutation in an in vivo context,
supporting
our
proposed mechanistic
genotype-phenotype
correlation
of
GNAO1-associated neurological disorders. Also, the identification of receptors that
regulate both mIPSCs and sIPSCs should facilitate the discovery of new drugs or drug
repurposing for GNAO1-associaed disorders.
4.2 Introduction
GNAO1 encodes the α subunit of a heterotrimeric G protein, Go, which is the most
abundant membrane protein in mammalian central nervous system. It participates in
multiple neural signaling pathways (Jiang & Bajpayee, 2009). Mutations in GNAO1 were
first found in early onset epileptic encephalopathy (EIEE17). However, since 2016, there
have been a growing number of reports on GNAO1 mutation-associated movement
disorders with/without seizures. This disorder was officially categorized by OMIM (Online
Mendelian Inheritance in Man) in 2017 as neurodevelopmental disorder with involuntary
movements (NEDIM). GNAO1-associated NEDIM is a rare neurogenetic disorder,
characterized by early onset of hypotonia, movement disorder and developmental delay.
Although numerous available drug treatments were tested on NEDIM patients, few
proved to be effective (Feng, Khalil, Neubig, & Sidiropoulos, 2018). The pathological
mechanisms of this disorder also remained unclear.
The Gαo protein functions as a messenger for a broad range of signaling pathways.
They include inhibition of cAMP (Levitt, Purington, & Traynor, 2011; Sunahara & Taussig,
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2002), inhibition of high-voltage-gated calcium channels (N- and P/Q-type calcium
channels) (Ikeda, 1996), and activation of G-protein regulated inward rectifying
potassium (GIRK) channels (Zhang, Dickson, & Doupnik, 2004). Our lab has previously
established a genotype-phenotype correlation of GNAO1-associated neurological
disorders based on Gαo’s canonical pathway of inhibiting cAMP production (Feng et al.,
2017). We proposed that loss-of-function (LOF) and partial-loss-of-function (PLOF)
GNAO1 mutations are found in epilepsy patients, while the gain-of-function (GOF) and
normal-function (NF) mutations are generally found in movement disorder patients (Feng
et al., 2017). We have also generated a novel animal model with a knock-in GOF
mutation G203R (Feng et al., 2019). Similar to human patients with GNAO1 G203R
mutation, this animal model exhibited movement abnormalities in a battery of behavioral
assessment including RotaRod, grip strength, and DigiGait (Chapter 3) (Feng et al.,
2019). Unlike most GOF GNAO1 mutations, patients with the G203R mutant allele also
exhibit early-onset epilepsy (Arya, Spaeth, Gilbert, Leach, & Holland, 2017; Feng et al.,
2019; Kelly et al., 2019; Nakamura et al., 2013; Saitsu et al., 2016; Schorling et al., 2017;
Xiong et al., 2018), validating the relevance of this animal model, which also showed an
increased sensitivity to pentylenetetrazole (PTZ) kindling.
Although the G203R animal model mimics the symptoms of human G203R patients,
the mechanisms by which these mice develop their movement disorder are still unclear.
Also, the GOF nature of the G203R mutation in a physiological environment has only
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been demonstrated for cAMP regulation in HEK293T cells. It is critical to know if Gαo
G203R also has GOF behavior in neurons. To answer these questions, I performed
patch clamp studies of Purkinje cells in cerebellar slices of WT and Gnao1+/G203R mutant
mice. The cerebellum has long been known for its critical regulation of motor
coordination. The evidence for a role in dystonia has begun to emerge. Deficiency in
cerebellar motor control may manifest as inaccuracies of visual guided movement,
speeded complex movement, loss of muscle tone, abnormal timing, and loss of
prediction and coordination (Gowen & Miall, 2007). Clinically, these features are
characterized as dysmetria (inaccurate movement), dysdiadochokinesis (inability to
execute rapidly alternating movements), hypotonia (reduced muscle tone), and
dyscoordination or ataxia (inability to perform smoothly coordinated voluntary movement)
(Gowen & Miall, 2007). Structural and/or functional abnormalities of the cerebellum are
also associated with dystonia (Bologna & Berardelli, 2018) and chorea (Walker, 2016).
Several reports elucidated the role of cerebellum in DYT1 hereditary dystonia (Fremont,
Tewari, Angueyra, & Khodakhah, 2017; Song, Bernhard, Hess, & Jinnah, 2014; Vanni et
al., 2015). Interestingly, dystonia and chorea/athetosis are the most commonly seen
involuntary movements in patients with the G203R mutation (Arya et al., 2017; Kelly et
al., 2019; Nakamura et al., 2013; Saitsu et al., 2016; Schorling et al., 2017). Structurally,
a core cerebellar circuit mediates all of its function (Eccles, 1967; Reeber, Otis, & Sillitoe,
2013). This circuit centers on Purkinje cells, which are the sole output of the cerebellar
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cortex (Brown et al., 2019). Purkinje cells receive input from several classes of
interneurons. Granule cells project parallel fibers that send excitatory signals to Purkinje
cells (Barbour, 1993; Eccles, Llinas, & Sasaki, 1966a, 1966b; Konnerth, Llano, &
Armstrong, 1990), while basket cells and stellate cells send inhibitory input to Purkinje
cell (Cesana et al., 2013; Hull & Regehr, 2012).
In this chapter, I report data on patch clamp recordings of the Purkinje cells in
cerebellar slices. I found a decreased frequency in both sIPSC and mIPSC events in
G203R mutant mice. A likely mechanism underlying this reduction in frequency is
enhanced signaling by the mutant Gαo to mediate presynaptic inhibition of GABA release.
Different receptors serve as the driving force for this inhibition. GABAB receptor-activated
Gαo mediates non-AP related mIPSCs while α2A adrenergic receptors stimulate AP
mediated sIPSCs. Although Gαo-mediated inhibition of high-voltage activated (N,
P/Q-type) calcium channels
is well-studied (Ikeda, 1996), only α2A adrenergic
receptor-mediated sIPSCs function through the activation of membrane-located,
voltage-gated calcium channels. GABAB receptor mediated inhibition of mIPSCs is likely
to involve Gβγ-mediated direct inhibition of synaptic vesicle release (Feng et al., 2018;
Zurawski et al., 2017; Zurawski, Rodriguez, Hyde, Alford, & Hamm, 2016).
4.3 Materials and Methods
4.3.1 Tissue preparation and solutions
All animal procedures complied with the National Institutes of Health of the USA
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guidelines on animal care and were approved by Michigan State University Institutional
Animal Use and Care Committee. Animal used for this chapter were between 5 to 10
weeks old. Only male animals were used due to the sex difference we have observed in
our previous study (Chapter 3) (Feng et al., 2019). Mice were sacrificed by direct cervical
dislocation and cerebellums were dissected and quickly mounted on a VibrotomeTM 1000
machine (Leica, Wetzlar, Germany). Sagittal cerebellar slices (250 µm) were prepared
according to methods previously described by Yuan Y et al (Yuan & Atchison, 1999,
2003, 2007, 2016). Briefly, the cerebellums were transferred into a chilled oxygenated
sucrose-based slicing solution and parasagittal cerebellar slices (250 µm thick) were cut
using the VibrotomeTM 1000 machine (Leica, Wetzlar, Germany). The slicing solution
contains (in mM): 125, NaCl; 2.5, KCl; 4, MgCl2; 1.25, KH2PO4; 26, NaHCO3; 0.5, CaCl2
and 25, D-glucose (pH 7.35–7.4 when saturated with 95% O2 /5% CO2 at room
temperature of 22–25°C). Slices were incubated in the pre-chilled and oxygenated
slicing solution for 15 min, and then transferred into standard artificial cerebrospinal fluid
(ACSF) solution at room temperature for 30 min. The standard ACSF contains: 125,
NaCl; 2.5, KCl; 1, MgCl2; 1.25, KH2PO4; 26, NaHCO3; 2, CaCl2 and 20, D-glucose (pH
7.35–7.4 saturated with 95% O2/5% CO2 at room temperature).
4.3.2 Electrophysiological recording
Whole-cell patch clamp recording methods were detailed in previous publications
(Yuan & Atchison, 1999, 2003, 2007, 2016). Slices were placed in a recording chamber
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and perfused with standard ACSF bubbled with 95% O2/5% CO2. Individual neurons
were visualized with a Nomarski 40X water immersion lens with infrared differential
interference contrast optics using a Nikon E600FN upright microscope. Recording
electrodes were fire polished and had a resistance of 3–7(MΩ when filled with pipette
solution. For recording sIPSCs and mIPSCs, the pipette solution consisted of (in mM)
140, CsCl; 0.4, GTP; 2, Mg-ATP; 0.5, CaCl2; 5, Phosphocreatine Na2; 5, EGTA-CsOH;
10, HEPES (pH 7.3 adjusted with CsOH). For recording sEPSCs and mEPSCs, the
pipette solution consisted of (in mM) 140, K-Gluconate; 0.4, GTP; 2, Mg-ATP; 0.5, CaCl2;
5, Phosphocreatine Na2; 5, EGTA-CsOH; 10, HEPES (pH 7.3 adjusted with KOH). The
holding potential was −70(mV for recording of both IPSCs and EPSCs. For recording
inhibitory currents, 6-Cyano-7-nitroquinoxaline-2,3-dione
(CNQX, 10(µM) and
amino-5-phosphonopentanoic acid (APV, 100(µM) were added to the external solution to
block glutamate receptor-mediated sEPSCs. For recordings of miniature IPSCs
(mIPSCs), 0.5(µM tetrodotoxin (TTX) was added to the external solution in addition to
CNQX and APV. For recording sEPSCs, bicuculline (10 µM) was added to the external
solution to block GABAergic receptor-mediated sIPSCs. For recording of miniature
EPSCs (mEPSCs), the external solution was supplemented with 0.5(µM TTX in addition
to bicuculline. Whole cell currents were filtered at 2–5(kHz with an 8-pole low-pass
Bessel filter and digitized at 10–20(kHz for later off-line analysis using the pClamp 9.0
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program (Molecular Devices, Inc., Sunnyvale, CA). All experiments were carried out at
room temperature of 22–25°C.
4.3.3 Pharmacology
The following agents were used: CNQX disodium salts (Sigma-Aldrich, St. Louis,
MO), DL-2-Amino-5-phos-phon-o-pent-anoic (APV) acid solid (Sigma-Aldrich, St. Louis,
MO), tetrodotoxin (TTX) (Tocris, Bristol, UK), pertussis toxin (PTX) (List Biological
Laboratories, Campbell, CA),
baclofen
(Sigma-Aldrich, St.
Louis, MO),
N-ethylmalaeimide (NEM) (Sigma-Aldrich, St. Louis, MO), UK14,304 (Sigma-Aldrich, St.
Louis, MO), CGP36216 (hydrochloride) (Cayman Chemical, Ann Arbor, MI), BRL44408
(Sigma-Aldrich, St. Louis, MO), cadmium chloride (Sigma-Aldrich, St. Louis, MO). All
drugs were made up as 1000 x concentrated stock solutions in distilled water, aliquoted
and stored at ~20oC. Aliquots were thawed and dissolved in oxygenated ACSF
immediately prior to use.
4.3.4 SDS Page and Western Blots
Male mice (6-8 weeks old) were sacrificed and their brains were dissected into
different regions and flash-frozen in liquid nitrogen. For Western Blot analysis, tissues
were thawed on ice and homogenized for 5 min with 0.5 mm zirconium beads in a Bullet
Blender (Next Advance; Troy, NY) in RIPA buffer (20mM Tris-HCl, pH7.4, 150mM NaCl,
1mM EDTA, 1mM β-glycerophospate, 1% Triton X-100 and 0.1% SDS) with a protease
inhibitor cocktail (Roche/1 tablet in 10 mL RIPA). Homogenates were centrifuged for 5
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min at 4°C at(13,000 G. Supernatants were collected and protein concentrations
determined using the bicinchoninic acid method (BCA method; Pierce; Rockford, IL).
Protein concentration was normalized for all tissues with RIPA buffer and 2x SDS
sample buffer containing β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO) was added.
Thirty µg of protein was loaded onto a 12% Bis-Tris gel (homemade), and samples were
separated for 1.5 hrs at 160V. Proteins were then transferred to an Immobilon-FL PVDF
membrane (Millipore, Billerica, MA) on ice either for 2 h at 100 V, 400 mA or overnight at
30 V, 50 mA. Immediately after transfer, PDVF membranes were washed and blocked in
Odyssey PBS blocking buffer (Li-Cor, Lincoln, NE) for 40 min at RT. The membranes
were then incubated with anti-Gαo (rabbit; 1:1,000; sc-387; Santa Cruz biotechnologies,
Santa Cruz, CA) or anti-Gβ (recommended for detection of Gβ1, Gβ2, Gβ3 and Gβ4;
mouse; 1:1000; sc-378; Santa Cruz biotechnologies, Santa Cruz, CA) and anti-actin
(goat; 1:1,000; sc-1615; Santa Cruz) antibodies diluted in Odyssey blocking buffer with
0.1% Tween-20 overnight at 4oC. Following four 5-min washes in phosphate-buffered
saline with 0.1 % Tween-20 (PBS-T), the membrane was incubated for 1 hr at room
temperature with secondary antibodies (1:10,000; IRDye® 800CW Donkey anti-rabbit;
IRDye® 800CW Donkey anti-mouse; IRDye® 680RD Donkey anti-goat; LI-COR
Biosciences) diluted in Odyssey blocking buffer with 0.1 % Tween-20. The membrane
was subjected to four 5-min washes in PBS-T and a final rinse in PBS for 5 minutes. The
membrane was kept in the dark and the infrared signals at 680 and 800nm were
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detected with an Odyssey Fc image system (LI-COR Biosciences). The Gαo polyclonal
antibody recognizes an epitope located between positions 90-140 of the Gαo protein
(Santa Cruz, personal communication).
4.3.5 Statistical Analysis
Electrophysiological data analysis was performed as described previously (Yuan &
Atchison, 1999, 2003, 2007, 2016). The individual performing the analysis was blinded to
the genotype of the sample until all results were recorded. In brief, spontaneous synaptic
currents were first screened automatically using MiniAnalysis software (Synaptosoft Inc.,
Decatur, GA) with a set of pre-specified parameters. They were accepted or rejected
manually with an event detection amplitude threshold at 5(pA for sIPSCs/mIPSCs and
3pA for sEPSCs/mEPSCs as well as the kinetic properties (fast rising phase and slow
decay phase) of the spontaneous events. Unless otherwise specified, synaptic events
per cell collected over a 2-min period were averaged to calculate the frequency and
amplitude of spontaneous synaptic currents. Amplitudes of currents were measured after
subtraction of the baseline noise. MiniAnalysis-derived results were plotted in GraphPad
Prism (GraphPad; LaJolla, CA). Results from more than one neuron from a single animal
were averaged prior to statistical analysis. Some graphs, when indicated, do show points
for each individual neuron while bar graphs and error bars are calculated from the per
animal data. Data are presented as mean value ± SEM, where n=number of animals.
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Statstical significance was determined using unpaired Student’s t-test unless stated
otherwise. A p value < 0.05 was deemed as significant.
Quantification of infrared (IR) Western blot signals was performed using Image
Studio Lite
(LI-COR Biosciences).
Individual bands were normalized
to
the
corresponding actin signals, and WT Gαo was set as control for each blot. All data were
analyzed using GraphPad Prism 7.0 (GraphPad; LaJolla, CA).
4.4 Results
Purkinje cells mediate the entire output of the cerebellar cortex; therefore any
mechanisms able to modulate the firing pattern of Purkinje cells will influence cerebellar
function. Purkinje cells fire spontaneously, even in the absence of glutamate input, and
the pattern of firing is strongly influenced by GABAergic input. At least under experiment
conditions, two types of inhibitory interneurons, the basket cells and the stellate cells
largely convey this inhibitory input onto the Purkinje cells (Donato et al., 2008).
Presynaptic neurotransmitter release is strongly regulated by G-protein-coupled
receptors (GPCRs). Many GPCRs in the central nervous system are coupled to the Gαo
protein, which belongs to the Gαi/o family. The activation of Gαo protein by GPCRs leads
to the inhibition of voltage-gated calcium channels, inhibition of cAMP production,
activation of G-protein coupled inward rectifying potassium channels (GIRKs) and also
inhibition of synaptic vesicle release, all of which can be possible mechanisms of
GPCRs-mediated inhibition of GABA release.
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4.4.1 Presynaptic GABA release is suppressed in the cerebellar Purkinje cells of
Gnao1+/G203R mice
Baseline recording of sIPSCs, which are due to AP-dependent GABA release, was
isolated by adding 10 µM CNQX and 100 µM AP-V in standard ACSF. Interestingly,
CNQX and AP-V significantly increased IPSC events in Purkinje cells (data not shown),
which is consistent with previous observations (Brickley, Farrant, Swanson, &
Cull-Candy, 2001). There is a significant decrease in sIPSC frequency in G203R mice
comparing to that of their WT sibling (WT: 21.0 ± 1.7 Hz; G203R: 12.7 ± 2.5 Hz; Figure
4.1C & 4.1D), but no difference is detected in sIPSC amplitude (WT: 41.8 ± 8.5 pA;
G203R: 36.7 ± 7.4 pA; Figure 4.1E & 4.1F). Data were recorded from 25 cells of 13 mice
for WT and 21 cells from 9 mice for G203R.
mIPSCs, which are due to AP-independent GABA release in the Purkinje cells were
investigated by the additional application of 0.5 µM TTX. As reported previously in
cerebellar Purkinje cells, TTX reduced mean IPSC frequency and amplitude to isolate
mIPSCs (Bardo, Robertson, & Stephens, 2002; Boxall, 2000; Harvey & Stephens, 2004;
Yuan & Atchison, 2003). Slices from G203R mice exhibited a marked reduction in
mIPSC frequency compared to that of WT mice (Figure 4.1G - 4.1L). The effect on
mIPSC frequency was greater than that on sIPSCs (75% vs 40% decrease). Data were
recorded from 25 cells of 13 mice for WT and 21 cells from 9 mice for G203R.
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(A, B) Representative
Figure 4.1 Cerebellar Purkinje cells in brain slices from 4-6 week-old G203R mice
display reduced GABAergic spontaneous synaptic currents (sIPSCs) and reduced
miniature synaptic currents
recording of
spontaneous inhibitory postsynaptic currents in a cerebellar Purkinje cell from a 4
week-old mouse in the presence of 10 µM of CNQX and 100 µM of AP-V at a holding
potential of -70 mV. (C, D) G203R mice showed a decrease in the frequency of sIPSCs.
(E, F) No significant difference is observed in the amplitude of sIPSCs between WT and
G203R mice. Unpaired Student’s t-test; **p=0.0086 WT (n=13 mice), G203R (n=9 mice).
(G, H) Representative recording of spontaneous miniature inhibitory postsynaptic
currents in a cerebellar Purkinje cell from a 4 week-old mouse in the presence of 10 µM
(mIPSCs).
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Figure 4.1 (cont’d) of CNQX, 100 µM of AP-V and 0.5 µM TTX at a holding potential of
-70mV. (I, J) G203R mice showed a decrease in the frequency of mIPSCs. (K, L) No
significant difference is observed in the amplitude of mIPSCs between WT and G203R
mice. Unpaired Student’s t-test; **p=0.0011; WT (n=13 mice), G203R (n=9 mice).
Recordings from each cell are shown as a data point but the bar graph, error bars, and
statistical analysis was averaged data per animal.
4.4.2 Gαo blockers can reverse the enhanced inhibition of mIPSC frequency in
G203R mice
To investigate whether the reduced frequency of sIPSCs and mIPSCs is due to an
enhanced signaling by the G203R mutant Gαo, we examined the effect of sulphydryl
alkylating agent NEM, which uncouples pertussis toxin-sensitive Gαi/o subunits from
receptors by modifying cysteine residues (Aktories, Schultz, & Jakobs, 1982). Similar to
the effects of NEM on GABAergic mIPSCs in other brain slice preparations, 50 µM NEM
significantly increased the frequency of mIPSCs in both WT and G203R cerebellar slices
(WT: from 4.19 ± 0.57 Hz to 22.3 ± 0.4 Hz; G203R: from 1.52 ± 0.35 Hz to 20.8 ±1.4 Hz;
Figure 4.2D & 4.2G) but did not affect the amplitude (WT: 11.1 ± 1.7 pA to 19.1 ± 3.1 pA;
G203R: 16.0 ± 1.9 pA to 30.8 ± 5.5 pA; Figure 4.2F & 4.2H). Also, NEM eliminated the
difference in the mIPSC frequency between WT and G203R mice (WT: 22.3 ± 0.4 Hz;
G203R: 20.8 ±1.4 Hz; Figure 4.2D & 4.2F).
Considering that NEM is not a selective Gαo protein blocker, we also examined the
effects of PTX incubation on the AP-dependent and AP-independent IPSCs in cerebellar
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Purkinje cells. PTX catalyzes the ADP-ribosylation of the α subunit of the heterotrimeric
Gi/o family, thereby preventing the G proteins from interacting with GPCRs (Mangmool &
Kurose, 2011). All slices in this study were subject to incubation in 1 µg/mL PTX for more
than 6 hours before recording. To maintain comparable conditions to compare data with
and without PTX, separate slices were used as control so that both groups underwent
the 6-hour incubation. PTX incubation significantly increased the mIPSC frequency in
slices from G203R mice but had no effects on WT mice (WT: 4.10 ± 0.70 Hz to 3.40 ±
0.68 Hz; G203R: 1.31 ± 0.16 Hz to 2.29 ± 0.30 Hz; Figure 4.3C & 4.3G). The amplitude
of mIPSCs was not changed in either WT or G203R mice after PTX incubation (WT: 12.1
± 1.6 pA to 9.7 ± 1.4 pA, G203R: 19.6 ± 1.6 pA to 15.8 ± 2.9 pA; Figure 4.3D & 4.3H). In
contrast to mIPSCs, sIPSC frequency was not significantly affected by PTX in either WT
or G203R mice (Frequency: WT: 13.8 ± 1.7 Hz to 10.9 ± 3.6 Hz; G203R: 9.1 ± 1.4 Hz to
12.5 ± 2.3 Hz; Amplitude: WT: 18.9 ± 3.4 pA to 19.2 ± 8.9 pA; G203R: 29.2 ± 4.1 pA to
24.4 ± 4.5 pA; Figure 4.3E-4.3F, 4.3I-4.3J).
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Figure 4.2 The frequencies of mIPSCs were sensitive to NEM, an inhibitor of Gαi/o
proteins. (A, B) Representative recordings showing mIPSCs traces in (A) WT and (B)
G203R mice with the presence of 50 µM NEM. (C, D) NEM eliminated the difference in
mIPSC frequency between WT and G203R. (E, F) No significant difference was
observed in amplitude of mIPSCs after adding NEM. (G) NEM significantly increased the
frequency of mIPSCs, (H) but with minor influence in the amplitude of mIPSCs. Unpaired
Student’s t-test; WT (n=8 mice), G203R (n=7 mice).
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Figure 4.3 A selective inhibitor of Gi/o, pertussis toxin (PTX), increased the
frequency of mIPSCs in G203R but not WT mice. Slices were incubated in 1 µM/ml of
PTX for >6 hrs pre-recording. Representative traces showed the example recordings of
(A) mIPSCs and (B) sIPSCs in WT and G203R mice before and after PTX incubation. (C,
G) PTX incubation significantly relived the Go mediated inhibition of mIPSC frequency in
G203R mice, but not WT mice. Unpaired Student’s t-test; WT (n=5 mice), G203R (N=6
mice); **p=0.006. (D, H) PTX did not change the mIPSC amplitude of either G203R or
WT mice. Neither frequency (E, I) nor amplitude (F, J) of sIPSCs was affected by PTX
incubation. Unpaired Student’s t-test; WT (n=5 mice), G203R (n=6 mice); *p=0.03
between WT vs. G203R mice without PTX incubation. Results between WT and G203R
were not significant.
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4.4.3 Presynaptic glutamate release is not affected by the G203R mutation in Gαo
protein
Purkinje cells receive glutamatergic inputs at their extremely elaborated dendrites
from parallel fibers at the molecular layer of the cerebellar cortex and send GABA
outputs to the deep nuclei (Tian & Zhu, 2018). To investigate whether the GOF mutation
G203R also affects excitatory inputs on Purkinje cells, we recorded sEPSCs and
mEPSCs from Purkinje cells. The AP-dependent excitatory postsynaptic currents
(sEPSCs) were isolated with 10 µM bicuculline and AP-independent mEPSCs were
recorded with the addition of 0.5 µM TTX. Although the GOF Gαo protein significantly
decreased sIPSCs and mIPSCs frequency, EPSCs are not seemingly affected by the
G203R mutation in Gαo. Neither frequency nor amplitude of sEPSCs and mEPSCs
showed significant differences between WT and G203R slices (Figure 4.4; sEPSC
frequency: WT: 1.50 ± 0.20 Hz vs. G203R: 1.36 ± 0.43 Hz; sEPSC amplitude: WT: 7.52 ±
0.92 pA vs. G203R: 6.42 ± 0.50 pA; mEPSC frequency: WT: 1.05 ± 0.16 Hz vs. G203R:
0.99 ± 0.46 Hz; mEPSC amplitude: WT: 6.39 ± 0.91 pA; G203R: 6.24 ± 0.60 pA).
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Figure 4.4 G203R mutant slices show no difference in either spontaneous
excitatory postsynaptic currents (sEPSCs) or miniature excitatory postsynaptic
currents (mIPSCs). (A) sEPCSs were recorded from Purkinje cells at a holding potential
of -70mV in the presence of 10 µM bicuculline. (B) 0.5 µM TTX was then added to the
bath in order to record mEPSCs. (C, D & G, H) No significant difference in either
frequency or amplitude was observed between WT and G203R. (E, F & I, J) No
significant difference between WT and G203R was observed in mEPSCs either.
Unpaired Student’s t-test; WT (n=5), G203R (n=5).
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4.4.4 Effects of G-protein coupled GABAB receptors on AP-independent GABA
release onto Purkinje cells
The effects of baclofen on AP-independent mIPSCs, isolated by the application of
0.5 µM TTX, were investigated. After recording baseline mIPSCs, baclofen (10 µM) was
applied to the bath. Baclofen caused a clear reduction in mean mIPSC frequency in
slices from both WT (from 5.47 ± 0.80 Hz to 1.19 ± 0.25 Hz, 78% inhibition) and G203R
mice (from 1.24 ± 0.20 Hz to 0.65 ± 0.11 Hz, 48% inhibition). Baclofen was typically
applied for 4 to 8 min in this and subsequent experiments. After baclofen application, the
difference in mIPSC frequency still remains between WT (1.19 ± 0.25 Hz) and G203R
(0.65 ± 0.11 Hz) mice (Figure 4.5C & 4.5E). This suggests a role of GABAB receptors in
regulating AP-independent GABA release. Mean mIPSC amplitude was unchanged by
baclofen (Figure 4.5F; WT: 12.8 ± 2.3 pA to 13.6 ± 4.3 pA; G203R: 17.8 ± 1.9 pA to 17.3
± 1.6 pA) (Figure 4.5D). Interestingly, the application baclofen did not affect either
frequency or amplitude of sIPSCs (Figure S4.2), suggesting that GABAB receptors do
not regulate the AP-dependent inhibitory neurotransmitter release.
To confirm that Gαo causes the baclofen-induced inhibition of mIPSC frequency as
previously reported (Harvey & Stephens, 2004), PTX was used to block Gαo protein. The
Gαo antagonist, 1 µg/mL PTX eliminated baclofen-induced inhibition of mIPSC frequency
(Figure 4.5I & 4.5K & 4.5M), while exhibiting no effects on mIPSC amplitude (Figure 4.5J,
4.5L, 4.5N). PTX increased mean mIPSC frequency from 1.31 ± 0.16 Hz to 2.29 ± 0.30
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Hz in G203R mice but did not change that of the WT mice (non-PTX: 4.10 ± 0.70 Hz;
PTX: 3.40 ± 0.68 Hz). These data are consistent with a presynaptic role of Gi/o subunit in
baclofen-induced inhibition of AP-independent GABA release onto Purkinje cells.
Figure 4.5 Activating GABAB receptor with baclofen reduces mIPSC frequency but
not amplitude. PTX incubation eliminates baclofen-induced inhibition of mIPSC
frequency in WT and G203R mice. Representative traces showing the reduced mIPSC
responses before and after adding baclofen (10 µM) in WT (A) and G203R (B) mice w/o
PTX or baclofen. (C, E, G) Baclofen significantly decreased the frequency of mIPSC and
the difference between WT and G203R remains though baclofen was present. Unpaired
Student’s t-test; WT (n=6), G203R (n=6); ****p<0.001, ***p=0.003, *p=0.029 (WT vs.
G203R), *p=0.014 (G203R w/o baclofen). (D, F, H) Amplitude remained unchanged
regardless of the existence of baclofen. No significant change was observed in the (I, K,
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Figure 4.5 (cont’d) M) frequency or the (J, L, N) amplitude of mIPSCs after adding
baclofen in both WT and G203R mice. Unpaired Student’s t-test; WT (n=5), G203R
(n=6).
4.4.5 Effects of G-protein coupled α2A adrenergic receptors on AP-dependent
GABA release onto Purkinje cells
Adrenoceptors are divided into three subtypes, α1, α2, and β receptors, which are
coupled to Gq/11-, Gi/o- and Gs-proteins respectively (Kobilka et al., 1987; O'Rourke,
Iversen, Lomasney, & Bylund, 1994). Previous publications reported the dual regulation
of AP-dependent GABA release modulated by both Gi/o-coupled α2 receptors and
Gq-coupled α1 receptors (Hirono & Obata, 2006). Here, we investigated whether
Gi/o-coupled α2 receptors plays a role in regulating AP-dependent GABA release. We
used a selective α2 receptor agonist UK14,304 to confirm whether α2 receptors drives the
decrease in sIPSC frequency and whether G203R mutant enhanced this reduction in
sIPSC frequency. We applied 10 µM UK14,304 in bath perfusion. UK14,304 greatly
inhibited AP-dependent GABAergic IPSC (sIPSC) frequencies of both WT (from 10.7 ±
2.6 Hz to 4.48 ± 1.65 Hz) and G203R (from 3.38 ± 0.99 Hz to 1.03 ± 0.14 Hz) mice
(Figure 4.6C & 4.6E & 4.6G). Interestingly, a significant difference remained between
WT and G203R mice in mIPSC frequency after the application of UK14,304 (Figure 4.6E;
WT: 4.48 ± 1.65 Hz vs. G203R: 1.03 ± 0.14 Hz). Like baclofen, the amplitude of mIPSC
was not affected by the application of UK14, 304 (Figure 4.6D & 4.6F &4.6H).
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Figure 4.6 The frequency of sIPSCs
is modulated by α2AR receptors.
Representative recordings showing the sIPSCs of (A) WT and (B) G203R mice before
and after adding the selective α2AR receptor agonist UK14,304 (10 µM). (C, E, G) UK14,
304 significantly reduced the frequency of sIPSCs in both WT and G203R mice, and the
frequency of sIPSCs remained lower in G203R mice after UK14,304 treatment
comparing to WT. (D, F, H) The amplitude of sIPSCs was unaffected with UK14, 304
treatment. Unpaired Student’s t test; WT (n=8 mice), G203R (n=9 mice); *p<0.05
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4.4.6 α2A adrenergic receptor-induced inhibition of sIPSC frequency depends on
activation of voltage-gated calcium channels
It has been established that Gβγ subunits can act on multiple types of voltage-gated
calcium channels to inhibit calcium influx from the extracellular space and decrease
neurotransmitter release (Currie, 2010; Zamponi & Currie, 2013). For AP-dependent
GABA release, high voltage activated calcium channels (N-type and P/Q-type calcium
channels) are first activated by depolarization from AP-stimulated influx of sodium ions.
Following the activation of calcium channels, GPCRs stimulate Go, which releases the
Gβγ subunit. Since the neurotransmitter release is directly proportional to the extent of
calcium influx from the extracellular space and the resultant changes in the intra-terminal
calcium concentration
(Wu & Saggau, 1997), we examined whether
the
UK14,304-induced inhibition of GABAergic IPSCs is dependent on the extracellular
calcium concentration. To verify effects of
low extracellular calcium on
the
UK14,304-induced inhibition of GABAergic IPSCs, we recorded baseline sIPSCs and
UK14,304-induced inhibition of sIPSCs in the presence of 100 µM cadmium chloride
(CdCl2). Cd2+ greatly decreased sIPSC frequency in both WT (from 17.3 ± 1.6 Hz to 1.01
± 0.22 Hz) and G203R (from 10.9 ± 2.3 Hz to 1.46 ± 0.27 Hz) mice (Figure 4.7Aa-b,
4.7Ba-b & 4.7C & 4.7E). It also eliminated the inhibition of sIPSC frequency induced by
application of 10 µM UK14,304 (Figure 4.7Ab-c, 4.7Bb-c & 4.7C & 4.7E). The amplitude
of sIPSCs showed a trend toward a decrease with application of Cd2+ but the change
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was not significant (Figure 4.7D & 4.7F). Interestingly, UK14,304 did not affect the
frequency of mIPSCs in either WT or G203R mice (Figure S4.2).
However, since mIPSCs do not depend on the activation of extracellular calcium
channels, Cd2+ does not affect the frequency and amplitudes of mIPSCs in either WT or
G203R mice (Figure 4.8). Moreover, baclofen induced decrease of mIPSC frequency
was not affected by the inhibition of membrane calcium channels either (Figure 4.8).
Although the difference between mIPSC frequency of WT and G203R is not significant in
this figure, it is understandable since there is fewer n numbers for this experiment shown
in Figure 4.8 comparing to experiment in Figure 4.5.
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Figure 4.7 Cadmium-block of extracellular calcium
influx suppresses the
frequency of sIPSCs in both WT and G203R mice. Representative recordings
showing the sIPSCs of (A) WT and (B) G203R in (a) ASCF with 100 µM AP-V and 10 µM
CNQX, (b) Cd2+(100 µM)-ASCF with AP-V and CNQX, (c) Cd2+ (100 µM)-ASCF with
AP-V, CNQX and 10 µM UK14,304. (C, E) 100 µM Cd2+ significantly reduced sIPSC
frequency in both WT and G203R mice and blocks inhibition of sIPSC frequency induced
by UK14, 304. (D, F) 100 µM Cd2+ did not affect amplitudes of sIPSCs. Unpaired
Student’s t-test; WT (n=5 mice), G203R (n=5 mice); ****p<0.0001, ***p<0.001.
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Figure 4.8 Cadmium-block of extracellular calcium influx does not affect the
frequency and the amplitude of mIPSCs
in both WT and G203R mice.
Representative recordings showing the sIPSCs of (A) WT and (B) G203R in (a) ASCF
with 100 µM AP-V, 10 µM CNQX and 0.5 µM TTX; (b) Cd2+(100 µM)-ASCF with AP-V,
CNQX and TTX; (c) Cd2+ (100 µM)-ASCF with AP-V, CNQX, TTX and 10 µM baclofen.
(C, E) 100 µM Cd2+ did not reduce the frequency of mIPSCs in either WT or G203R mice.
Baclofen (10 µM) reduced the frequency of mIPSC with the presence of 100 µM Cd2+. (D,
F) Similarly, 100 µM Cd2+ does not affect amplitudes of sIPSCs. Unpaired Student’s
t-test; WT (n=5 mice), G203R (n=5 mice); *p<0.05.
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4.4.7 G203R mice exhibit decreased Gαo protein expression but no change in Gβ
levels
Gαo G203R mutation led to a reduction in Gαo protein expression in transiently
transfected HEK293T cells (Feng et al., 2017). To see if this still stands in vivo, we tested
Gαo protein expression in whole brain and also selected brain regions WT and G203R
mice. Results showed a significant reduction in Go protein expression (50% of WT) in the
whole brain (Figure 4.9A, 4.9C). The decrease in Go protein expression was significant in
cerebellum, cortex, hippocampus and striatum of the G203R mice (Figure 4.9B, 4.9D).
No significant change was observed in the brain stem and the olfactory bulb between the
WT and G203R mice.
The Gβγ subunits not only support the role of GPCR-Gα interaction, but also act
directly and independently to regulate downstream signaling. The number of identified
effectors of Gβγ has grown in recently years. They include some important targets like
the GIRK channel, P/Q and N-type calcium channels, and the SNARE protein complex
(Blackmer et al., 2005; Herlitze et al., 1996; Qin, Platano, Olcese, Stefani, & Birnbaumer,
1997; Wells et al., 2012; Zhang et al., 2004; Zurawski et al., 2017). The current model of
heterotrimeric G protein function hypothesizes that the conformational changes in Gα
lead to its dissociation from Gβγ to expose effector interaction surfaces on Gβγ. In fact,
many of the GPCR-dependent physiological process inhibited by PTX (Go/i family) are
mediated by the Gβγ subunits rather than the Gα subunits (Ikeda, 1996; Logothetis,
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Kurachi, Galper, Neer, & Clapham, 1987; Stephens et al., 1994; Welch et al., 2002).
Thus, the gain-of-function mutation G203R in Gαo may function though Gβγ subunits to
regulate the neurotransmitter release. The expression level of Gβ did not change in
G203R mutant mice (Figure 4.10). However, G203R is a GOF mutation with a decreased
Gαo protein expression level and a normal Gβ protein level. This could lead to a
constitutive increase in Gβγ protein function in G203R mice.
Figure 4.9 G203R mice showed a significant decrease in Gαo protein expression.
(A, C) Whole brain Gαo expression level decreased to about 50% in G203R mice’s brain
lysates. (B, D) This reduction in expression was most significant in specific regions like
cerebellum (CERE), cortex (CTX), hippocampus (HIP) and striatum (STR), while
remained unaffected in brain stem (BS) and olfactory bulb (OB). All expression levels
were normalized to that of WT accordingly. Unpaired Student’s t-test; WT (n=8), G203R
(n=8); ****p<0.0001, *p<0.05.
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Figure 4.10 G203R mice did not show any significant changes in Gβ expression in
the brain. (A) A representative gel shows the Gβ protein expression patterns in each
individual brain region, including olfactory bulb (OB), brain stem (BS), striatum (STR),
hippocampus (HIP), cerebellum (CERE) and cortex (CTX). (B) Quantification of the
protein expression levels is unchanged in G203R mice brain lysates comparing to those
of their WT siblings. Unpaired Student’s t-test; WT (n=4), G203R (n=4).
4.5 Discussion
G203R was deemed a gain-of-function mutation in our previous report, where it
showed an enhanced ability to support α2A adrenergic receptor mediated inhibition of
cAMP production (Feng et al., 2017). Patients with G203R all develop severe movement
disorders and seizures at an early age (Arya et al., 2017; Kelly et al., 2019; Nakamura et
al., 2013; Saitsu et al., 2016; Schirinzi et al., 2019; Schorling et al., 2017; Xiong et al.,
2018). Previously, we reported that G203R mice, especially male mice, exhibit
abnormalities in a battery of motor and behavioral tests (Feng et al., 2019). In this
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chapter, we explore possible physiological mechanisms of GNAO1-related movement
disorders.
Purkinje cells function as the sole output of cerebellar neural signaling transduction.
They synapse onto the deep nuclei of the cerebellum and release inhibitory
neurotransmitter to control their interaction with the thalamus. Therefore, the altered
excitatory/inhibitory
regulation
received by Purkinje cells can
reflect possible
abnormalities in the cerebellum. The cerebellum is well-known for playing a role in motor
coordination and control of movement. Recent research has also linked disturbed
cerebellar function to ataxia and dystonia (Bologna & Berardelli, 2018; Garcia et al.,
2017; Marsden, 2018). Consequently, I started my investigation by examining neural
control of cerebellar Purkinje cells.
I discovered that the Gnao1 G203R mutation decreased both AP-dependent sIPSC
frequency and AP-independent mIPSC
frequency. Amplitudes were unaffected.
Inhibition of Go signaling with NEM or PTX increased the frequency of mIPSCs. This
finding suggests that the G203R mutation enhanced inhibition of GABA release through
a pre-synaptic mechanism (Figure 4.11). Since PTX only increased the IPSC frequency
in G203R but not WT slices, this suggests that G203R is a bona fide gain-of-function
mutation. As such, the mutant Gαo protein has enhanced inhibition compared to a
normal-functioning WT protein. Moreover, we have also confirmed that mIPSCs and
sIPSCs are likely mediated by different receptors through different mechanisms.
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AP-independent mIPSCs are mainly mediated by GABAB receptors (Figure 4.11C &
4.11D). However, GABAB receptors likely modulate spontaneous GABA release by
inhibition of synaptic vesicle fusion through actions of Gβγ rather than inhibition of
membrane calcium channels (Figure 4.11C & 4.11D). In contrast, AP-dependent sIPSCs
are regulated by α2A adrenergic receptors through inhibition of voltage-gated calcium
channels (Figure 4.11A & 4.11B). The identification of the relevant GPCRs provides a
possible direction for new drug discovery and drug repurposing. Antagonists with
combined effects on GABAB receptors and α2A adrenergic receptors may be an effective
strategy for suppressing GNAO1-associated movement disorders.
Figure 4.11 Models of GABABR and α2AR mediated inhibition of GABA release. (A)
α2AR agonist activates α2AR, which results in the separation of Gαo and Gβγ. Gβγ inhibits
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Figure 4.11 (cont’d) calcium influx from membrane calcium channels activated by AP.
(B) G203R mutant Gαo protein enhances the suppression of calcium influx, which lead to
a reduction in GABA release. (C) Spontaneous GABA release without AP stimulation is
regulated by the activation of GABABR, which inhibits synaptic vesicle fusion. (D) G203R
mutant Gαo further inhibits the synaptic vesicle release.
However, it is not clear whether the G203R mutant Go heterotrimer inhibits the
neurotransmitter release via the mutated Gαo subunit or by the released free Gβγ protein.
Activated Gαo protein can expose the surface on Gβγ to form a core site for effector
binding and effector activation (Smrcka, 2008). The G203R mutant mice exhibited a
reduced mIPSC frequency in cerebellar Purkinje cells in the absence of any external
GPCR agonists. This suggests three possibilities. First, endogenous agonists in the
cerebellar slices may activate Go-coupled GPCRs. Second, sufficient free Gβγ subunits
may exist, due to the reduced amount of mutant Gαo, and may cause pre-synaptic
inhibition of GABA release. This is plausible considering that G203R mutant mice did not
show any reduction in Gβ protein (Figure 4.10) but had a significant decrease in Gαo
protein expression (Figure 4.9). Third, the G203R mutation may consecutively activate
Gαo signaling pathways. This could activate both Gαo and also release free Gβγ subunits
to mediate Gβγ signaling. There has been reported GNAO1 mutation that is
consecutively active: Q205L (Ram, Horvath, & Iyengar, 2000). Structurally, G203 and
Q205 are located close together; therefore it is reasonable to suspect that G203R may
be a constitutively active GNAO1 mutation that does not require the activation by a
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GPCR. Moreover, the Gαo G203R mutant showed a more rapid GDP release than the
WT - as measured by the binding of a fluorescent GTP analog (personal communication).
That suggests that the GOF effect of G203R may provide free Gβγ subunits more quickly
than the WT Gα.
Figure 4.12 Gβγ may play a major role in the regulation of IPSCs. (A) Activation of
GPCRs leads to the separation of Gα-GTP and Gβγ. They may carry on content
dependent activation or inhibition of the downstream signaling targets. (B) G203R
mutant Gαo protein may contribute to an enhanced function of Gβγ, which leads to an
increased inhibition of Gβγ-mediated inhibition of N- and P/Q-type calcium channels, AC,
and synaptic vesicle fusion. But G203R may tamper the signaling pathway mediated by
Gαo-GTP, like Gαo-activated neurite outgrowth.
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A confusing aspect of the GOF GNAO1 mutations in children and in our mouse
models has been that these mutants causing the dual phenotypes of MD and epilepsy.
One potential explanation for this could be that GNAO1 G203R mutant mice may have
context-dependent GOF and LOF in for different signal outputs (Figure 4.12) or in
different brain regions (Figure 4.13). Despite the preliminary biochemical data showing
G203R mutant Gαo protein’s rapid GDP release, we also found that protein expression of
the G203R mutant is significantly lower that normal in cerebellum, striatum, cortex, and
hippocampus (Figure 4.9). If one signal (e.g. neural migration) was mediated by Gα and
the other (e.g. VGCC inhibitor) was mediated by Gβγ, the signal mediated by Gαo could
be reduced (i.e. Gαo mediated activation of neurite outgrowth), while there is more free
Gβγ causing GOF for the inhibition of AC, VGCC or vesicle release (Figure 4.12). There
is one precedent for this with a human Gαs mutant that causes increased signaling in the
testis but reduced signaling in the pituitary (Turan & Bastepe, 2015). In a similar aspect,
the reason why G203R and R209H mutant mice showed different behavioral results can
be attributed to that the different Gnao1 mutations tilt the balance between excitatory and
inhibitory effects in different brain regions (Figure 4.13). If the G203R GOF mutant has a
stronger influence on neurotransmitter release in brain regions that are closely related to
epileptogenesis (i.e. cortex and hippocampus), while the R209H mutant with NF
behavior does not, then it is more likely for G203R mutant mice to develop a higher
susceptibility to seizures.
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More research needs to be done to assess whether the G203R mutation could lead
to different signaling outcomes in different brain regions. Future directions should focus
on identifying the link between the cell-types and signal outputs that can test this model
we proposed.
Figure 4.13 GNAO1 mutations may have region specific effects, which cause an
imbalance between excitatory and inhibitory neurotransmitters. Under normal
condition, Go mainly inhibits the inhibitory neurotransmitter release to keep a fine-tuned
balance between inhibitory and excitatory effects. However, G203R mutant and R209H
mutant may reduce the inhibitory effects, hence overexcite the brain. Considering the
difference in the presence of human symptoms, it is likely that G203R and R209H
mutants affects brain regions that control movements like cerebellum or basal ganglia,
but G203R mutant can further affect hippocampus and cortex therefore lead to the onset
of epilepsy in both animals and humans.
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APPENDIX
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APPENDIX
SUPPLEMENTAL DATA
Figure S4.1 Despite the hypothesis that α2A receptor antagonist yohimbine (10µM)
could reverse the inhibition of sIPSC frequency induced by UK14, 304, the
application of yohimbine further reduced the sIPSC frequency with the application
of UK14, 304. Representative traces are shown here with WT (A) and G203R (B) mice
in the recoding of baseline level sIPSCs (a), sIPSCs with the application of UK14, 304 (b),
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Figure S4.1 (cont’d) and sIPSCs with the application of both UK14, 304 and yohimbine
(c). (C, E) Although not significant, yohimbine seems further reduced the sIPSC
frequency that has already been decreased by the application of UK14, 304. It is highly
possible since yohimbine is not a highly selective α2A receptor antagonist. It also
antagonizes multiple serotonin receptors (Papeschi, Sourkes, & Youdim, 1971; Winter &
Rabin, 1992), which also plays a role in regulating cerebellar GABA release and
development (Nichols, 2011; Oostland & van Hooft, 2013). (D, F) Neither UK14, 304 nor
yohimbine has any effects on the amplitudes of sIPSCs in WT and G203R mice.
Unpaired Student’s t-test; WT (n=5), G203R (n=5).
Figure S4.2 Baclofen does not affect either frequency or amplitude of sIPSCs, and
UK14,304 does not affect mIPSC frequency or amplitude. Recording of sIPSCs are
not affected by baclofen (10 µM) in either frequency (A) or amplitude (B). (WT: n=2 mice;
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Figure S4.2 (cont’d) G203R: n=2 mice). Amplitudes (C) or frequency (D) of mIPSCs are
not decreased by UK14, 304 (10 µM) either. (WT: n=2 mice; G203R: n=2 mice).
Figure S4.3 Heterozygous G184S (GOF) mice also showed a low Gαo protein
expression level. In whole brain lysates, (A) representative gel and (B) quantification of
the relative protein level both showed that G184S mice had a reduced Gαo protein
expression level (80% expression) comparing to WT (100%) and heterozygous KO mice
(50%). Similar to the protein expression pattern seen in G203R mice, the Gαo protein
levels were different in different brain regions. Onw-way ANOVA; +/+ (n=9), +/- (n=9),
+/G184S (n=9); ****p<0.001, **p<0.01. (C, D) Heterozygous KO mice exhibited a
reduced Gαo protein level in all brain regions tested: hippocampus (HIP), cortex (CTX),
striatum (STR) and cerebellum (CERE). However, heterozygous G184S mice showed
selective Gαo protein reduction in hippocampus and cerebellum but not in cortex or
striatum. Two-way ANOVA; +/+ (n=6), +/- (n=6), +/G184S (n=6); ***p<0.001, **p<0.01.
233!
Figure S4.4 Female Gnao1+/G184S mice showed reduced sIPSC frequency in
hippocampal pyramidal cells, cortical layer II/IV pyramidal cells but not in
cerebellar Purkinje cells. (A-D) Frequency of sIPSCs was significantly different in
Gnao1+/G184S mice in the hippocampal CA1 pyramidal neurons (B, D), but amplitude
showed no difference between WT and G184S mice (A, C). Unpaired Student’s t-test;
WT (n=6), G184S (n=9); **p<0.01. (E-H) The same trend of a reduced sIPSC frequency
was also seen in the cortical layer II/IV pyramidal neurons in the G184S mice (E, G), and
the amplitudes of those two groups were not significantly different, although a trend of
decreased amplitude can be seen in the G184S mice (F, H). Unpaired t-test; WT (n=8),
234!
Figure S4.4 (cont’d) G184S (n=13); **p<0.01. (I, J) There is no significant difference in
either frequency or amplitude in sIPSCs between WT and G184S mice’s cerebellar
Purkinje cell. Unpaired Student’s t-test; WT (n=9), G184S (n=9).
Figure S4.5 Brain Gαo expression does not change in R209H mice’s brain lysates.
(A) Representative gel showing the Gαo protein expression in different brain regions of
both WT (+/+) and R209H (+/R209H) mice. (B) Quantification of Gαo protein expression
shows that there is no significant difference in all 6 brain regions tested between WT and
R209H mice. CERE: cerebellum; OB: olfactory bulb; STR: striatum; HIP: hippocampus;
BS: brain stem; CTX: cortex; Unpaired Student’s t-test; WT (n=7), R209H (n=7).
235!
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CHAPTER 5: CONCLUSION AND FUTURE DIRECTIONS
244!
5.1 General conclusion
It has been six years since
the
first
reported cases of
the GNAO1
mutation-associated neurological disorders in 2013 (Nakamura et al., 2013). There is a
rapid increase in the number of GNAO1 mutations reported. Advances in neurological
genetics and the growing interest in genetic counseling have pushed the increased
interest in GNAO1 mutation-related movement disorders and/or epilepsy. Recently, The
Bow Foundation (www.gnao1.org) has been founded in support of research for
understanding GNAO1-associated neurological disorders.
We are one of the first labs that took an interest in GNAO1-related neurological
disorders. Through
five years of research on my dissertation,
I
identified a
genotype-phenotype correlation between the in vitro function of GNAO1 mutations and
the nature of patients’ neurological symptoms (Chapter 2). Our lab has also established
three animal models that phenocopy human GNAO1 patients and I have, in collaboration
with Cassie Larrivee and Jeffrey Leipprandt, characterized their movement abnormalities
and seizure propensity (Chapter 3). Furthermore, I have obtained electrophysiological
data establishing altered cerebellar signaling in mice with the Gnao1 G203R mutation
which causes a movement disorder and verified that G203R is a bona fide GOF mutation
in the neural context (Chapter 4).
Although I established that all functioning GNAO1 mutations (GOF and NF mutations)
are associated with movement disorder patients, and all non-functioning mutations (LOF
245!
and PLOF mutations) are related to epilepsy patients (Feng et al., 2017), this
genotype-phenotype correlation was created based on a human engineered system with
transiently transfected Gao mutants and the α2AR in HEK293T cells. To test this
correlation in a physiological background, we have selected mutations that are either the
most prevalent (GOF: G203R and NF: R209H) or related to the most severe epilepsy
(LOF; ΔT191F197) to verify our genotype-phenotype correlation model. Interestingly,
mice with the LOF mutation ΔT191F197 were abnormally small and developed severe
behavioral seizure at around day 7 of life (P7). All mice with the ΔT191F197 mutation
died before P16 and the strain was lost. Like human patients with G203R mutations,
mice heterozygous for the G203R mutations (Gnao1+/G203R or G203R mice) behaved
abnormally in our behavioral tests of movement and also showed heightened sensitivity
to seizures, which was assessed by a PTZ kindling study (Feng et al., 2019).
Comparably, the NF mutation R209H is only associated with movement disorders in both
humans and mice (Larrivee et al., 2019); they do not show an epilepsy pattern. These
behavioral tests established that our genotype-phenotype correlation stands in a
physiological context across both mice and humans. However, there is an obvious sex
difference in our animal models that has not been consistently observed in patients,
perhaps due to the relatively small size of human GNAO1 patient population. Also in
humans, the GNAO1 mutations could cause prenatal death of male embryos. G203R
mice showed a male-dominant movement abnormality, while R209H mice have
246!
symptoms that are equally severe in both male and female mice. Since our animal
models exhibit symptoms similar to human GNAO1 patients, they make it possible to use
those animal models to study the mechanisms of how GNAO1 mutations could lead to
the onset of neurological disorders. Specifically, I used the patch clamp technique to
measure both the excitatory and inhibitory neurotransmitter release in cerebellar slices of
G203R mice and discovered that G203R mice exhibited decreased GABA release while
glutamate release was unaffected. Also, it is possible to use the animal models to test
new compounds or to repurpose drugs that are specifically effective for GNAO1
mutation-related disorders.
While my work has covered preliminary aspects of GNAO1 mutation-associated
neurological disorders, more research needs to be done to address unanswered
questions that are beyond the scope of this dissertation. First, we have not tested the
functions of mutant Gαo in any neuronal cell line or used different canonical pathways
(such as Ca++ or K+ channel regulation) for characterization. Second, we have not
explained why NF mutations also cause neurological disorders. It is possible that NF
GNAO1 mutations could lead to other disturbed downstream signaling pathways but do
not affect inhibition of cAMP. Third, since the majority of patients with GNAO1 mutations
present with developmental delay and hypotonia at birth, it is yet to be established that
GNAO1 mouse models (G203R and R209H) also present developmental issues. One
other interesting question on development is how this disorder will progress when our
247!
mutant mice become older. There haven’t been any reported human GNAO1 patients
over 45 years old; therefore understanding the progression of this disorder could
potentially prepare patients for any future complications. Last but not least, the sex
difference in expression of neurological disorders, as mentioned before, needs to be
verified as larger patient populations are reported. In our animal model, there is an
obvious sex difference in the movement disorders or epilepsy but it differs among
genotypes.
In this chapter, I will discuss some major directions this project could take and
provide some analysis for the development of each direction. Many of these ideas are
based on preliminary data that I collected but did not have time to develop into a
complete story. Those results are presented as an Appendix to this chapter
5.2 Testing the functional changes of a growing variety of GNAO1 mutations
Our previous model of genotype-phenotype correlation was established on
Gαo-mediated inhibition of cAMP production in HEK293 cells. However, the Gαo protein,
either functioning by itself or through interaction with the Gβγ protein, regulates multiple
essential intracellular effectors in its functional signaling pathways. Therefore, cAMP
cannot be the sole evaluation upon which the GNAO1 mutations are examined.
5.2.1 Do GNAO1 mutations affect Go’s inhibition of high-voltage gated calcium
channels (N- type & P/Q- type calcium channels)?
Go’s inhibition of Ca2+ channels has received extensive scrutiny. Intracellular calcium
248!
levels are important for neuronal signal transduction and neuronal development.
Numerous hormones or neurotransmitters suppress Ca2+ channel currents (Dunlap &
Fischbach, 1978). Later, Dunlap and colleagues showed that treating dorsal root
ganglion with PTX blocks noradrenaline and GABA-mediated inhibition of Ca2+ channels
(Holz, Rane, & Dunlap, 1986). Moreover, GTPγS, a non-hydrolysable GTP analog that
binds to and activates G proteins, irreversibly potentiates agonist-mediated inhibition of
Ca2+ channels (Holz et al., 1986; Scott & Dolphin, 1986) while GDPβS, a stable form of
GDP, blocks it. Specifically, opioids activate their receptors in dorsal root ganglion
neurons to suppress N-type Ca2+ channels (Jiang et al., 1998). Neurons lacking Go
protein lose the opioid inhibitory effect (Jiang et al., 1998). As mentioned in Chapter 1,
LOF mutations in CACNA1A (which encodes the P/Q type Ca2+ channel subunit) and
CACNA1B (which encodes the N-type Ca2+ subunit) and GNAO1 GOF mutations lead to
similar neurological symptoms.
There are several ways to test how mutations in GNAO1 affect Go’s inhibition of
calcium channels. The most traditional way is to measure the Ca2+ currents with the
patch clamp technique. This was also used in the first published GNAO1 mutation case
report in 2013 (Nakamura et al., 2013). In preliminary data, I transfected a previously
established HEK293 cell line stably expressing the three subunits of N-type Ca2+
channels (G1A1 cell line with α1B-1, α2Bδ, β1B subunits) with plasmids for the α2AR and
WT Gαo. In this system, I found that norepinephrine could inhibit calcium currents (Figure
249!
S5.1) (Bleakman et al., 1995; McCool, Pin, Brust, Harpold, & Lovinger, 1996). Using this
approach, it should be possible to transiently transfect these cells with all of the GNAO1
mutants and test their ability to inhibit the calcium currents with the patch clamp
technique.
However, with the ever-growing number of the GNAO1 mutations, patch clamp
methods may be a time-consuming procedure for this aim. An alternative to patch clamp
studies is to use the high-throughput calcium mobilization assay. There have been
multiple reports describing high-throughput assays with multiple available calcium dyes
to screen for N-type calcium channel blockers (Lubin et al., 2006; Zamponi, Striessnig,
Koschak, & Dolphin, 2015; Zhang, Kauffman, Yagel, & Codd, 2006). Using this strategy,
we could use either the N-type channel expressing G1A1 cell line or a neuronal cell line
to screen the GNAO1 mutants’ effects on calcium currents in a relatively short time. A
preliminary study with the Fluo-4 NW dye and the Hamamatsu’s FDSS µCell imaging
system in the MSU Assay Core confirmed that G1A1 cell line does express N-type
calcium channels (Figure S5.2A). Also the SH-SY5Y human neurobastoma cell line is a
good candidate for this screening (Figure S5.3B). Evaluation of whether GNAO1
mutations alter membrane calcium channel function is extremely important. The findings
would not only broaden our understanding of the genotype-phenotype correlation of the
GNAO1 mutation-related neurological disorders, but also could determine the most
predictive functional assay for drug repurposing or development.
250!
5.2.2 Do GNAO1 mutations affect Go’s activation of G protein-regulated inward
rectifying potassium (GIRK) channels?
Potassium channels on the plasma membrane are another intracellular effector of
Go-mediated signaling. Not only have multiple studies documented the importance of this
pathway, mutations in potassium channels were also reported to cause movement
disorders as discussed in Chapter 1 (Luscher & Slesinger, 2010). In hippocampal
pyramidal cells, serotonin and the selective GABABR agoinst baclofen hyperpolarize
cells by increasing K+ channel conductance. The serotonin and baclofen responses are
also ablated in PTX-treated cells (Andrade, Malenka, & Nicoll, 1986). Addition of GDPβS
reduces the cell’s response to serotonin and baclofen, while GTPγS mimics the action of
serotonin and baclofen (Andrade et al., 1986). Moreover, both purified bovine brain Go
proteins as well as a recombinant form of Gαo proteins activate K+ channels in
membranes from hippocampal pyramidal cells (Jiang & Bajpayee, 2009; Peleg, Varon,
Ivanina, Dessauer, & Dascal, 2002; VanDongen et al., 1988). However, studies later
focused on the role of Gβγ complex’s ability to activate the K+ channels while the Gα
subunit mostly modulates the channel kinetics (Corey & Clapham, 2001; Huang, Jan, &
Jan, 1997; Lei et al., 2000; Logothetis, Kurachi, Galper, Neer, & Clapham, 1987;
Reuveny et al., 1994). Mutations in the Gαo protein may affect its role as a chaperone to
release the Gβγ complex.
251!
Apart from traditionally used patch clamp techniques, using high-throughput
screening with thallium ions as a surrogate for potassium ions was also developed for
screening potassium ion channel blockers (Beacham, Blackmer, M, & Hanson, 2010).
Cell lines stably expressing GIRK channels (Lei et al., 2000) or primary hippocampal
neurons are both available and good candidates for screening the effects of GNAO1
mutation on Go-mediated activation of GIRK channels (for both Gαo and Gβγ
mechanisms).
5.2.3 How do GNAO1 mutations affect G protein-regulated neurite outgrowth?
The initial formation of neurites during neuronal differentiation is commonly referred
to as “neurite outgrowth”. This is the beginning point for neurogenesis, which is a crucial
but long and winding journey in the development process. The Gαo protein is not only the
most abundant membrane protein in the mammalian central nervous system, but is also
highly enriched in neuronal growth cones. Elucidation of the effects of GNAO1 mutations’
on regulation of neurite outgrowth can be an essential step towards understanding
GNAO1 mutation-associated developmental delay. Recent studies showed that the Gαo
protein might directly stimulate neurite outgrowth. First, both Gαo and one of its
interactors GRIN (G protein-regulated inducer of neurite outgrowth) are largely enriched
in the growth cones, and activation of both can induce neurite outgrowth (Chen, Gilman,
& Kozasa, 1999; Hwangpo et al., 2012; Strittmatter, Fishman, & Zhu, 1994; Strittmatter,
Valenzuela, Kennedy, Neer, & Fishman, 1990). Second, dopamine-activated D2
252!
receptors, which couple to the Gαo protein, induce neurite outgrowth in cortical neurons
(Reinoso, Undie, & Levitt, 1996). Also, activation of CB1 receptors leads to the activation
of downstream signaling converging on STAT3, which induces neurite outgrowth in
Neuro2A cells (He et al., 2005; Jordan et al., 2005). Additionally, collapse of growth
cones, induced by contact between neurites and a variety of molecules, can be inhibited
by pertussis toxin (Igarashi, Strittmatter, Vartanian, & Fishman, 1993). Moreover, Gβγ is
also involved in regulating neurite outgrowth. Research showed that Nerve Growth
Factor (NGF) promoted Gβγ’s interaction with microtubules and stimulated microtubule
assembly (Sierra-Fonseca et al., 2014). Also, GRK2i, which sequesters Gβγ, inhibited
neurite formation, disrupted microtubules and led to neurite damage, while the Gβγ
activator mSIRK stimulated neurite outgrowth (Sierra-Fonseca et al., 2014).
A neurite outgrowth assay has been performed with PC12 cells (Figure S5.3)
(Strittmatter, Fishman, et al., 1994; Traina, Petrucci, Gargini, & Bagnoli, 1998), Neuro2A
cells (Georganta, Tsoutsi, Gaitanou, & Georgoussi, 2013; He et al., 2005), SH-SY5Y
cells (Figure S5.4) (Paik, Somvanshi, & Kumar, 2019) and with primary isolated neurons
(Lotto, Upton, Price, & Gaspar, 1999; Reinoso et al., 1996). A high-throughput neurite
outgrowth assay kit has also been developed to reduce the labor of neurite staining and
counting (Yeyeodu, Witherspoon, Gilyazova, & Ibeanu, 2010). Whether mutations in
GNAO1 would affect its role in neurite outgrowth remains unanswered.
253!
Additionally, for neurite outgrowth, one inevitable question is how activation of the
Gαo protein (Strittmatter, Fishman, et al., 1994) and increases in cAMP levels (Aglah,
Gordon, & Posse de Chaves, 2008) both lead to neurite outgrowth. These two
competitive pathways may take control during different stages during neurite extension.
It would be interesting to see how GOF GNAO1 mutations, which lead to enhanced
suppression of cAMP production, regulate neurite outgrowth in vitro.
Table 5.1 Comparison of clinical patterns of G203R and R209H patients
Case No.
Sex
Age of onset
Seizures
Hypotonia
Developmental
Delay
Chorea/athetosis
1
F
7 mo
+
+
+
2
F
7 d
+
+
+
Dystonia
Severe EEG
++
++
diffuse
irregular
spike-and-
slow-wave
complex at
5 yr
++
EEG
Severe MRI
slow-wave
bursts,
migrating
focal
epileptiform
discharges
++
delayed
myelination
at 1 yr, 3
mo;
reduced
cerebral
white
matter, thin
corpus
callosum at
4 yr, 8 mo
Nakamura
et al 2013
progressive
cerebral
atrophy
with
delayed
myelination
at 14 mo
Saitsu et al
2015
MRI
Reference
3
F
9 d
+
+
not
+
impressive
movement
disorder but
characterized
delta and
theta activity
and rare multi-
regional, bi-
hemispheric
epileptic
activity
+
mild atrophy
Dietel et al
2016
4
M
1 mo
+
+
+
++
G203R
5
F
3 mo
+
+
+
+
6
F
birth
+
+
+
+
++
multifocal
and diffuse
discharges,
along with
generalized
-onset
seizures
++
multifocal
sharp
waves, left
temporal
seizure
pattern
+
background
slowing
++
7
F
+
birth (deceased
at 12 mo)
+
++
hypsarrhythmia
2
M
18 mo
+
+
+
3
M
2 y
+
+
9
M
12 d
+
+
+
+
1
M
1 y
+
+
+
5
M
3 y
+
+
+
R209H
4
M
10 mo
+
+
+
+
6
F
7
M
8
F
6 mo
6 mo
6 mo
+
+
+
+
+
+
+
+
+
+
8
F
birth
+
+
+
+
+
++
multifocal
paroxysmal
activities in
both
temporal
hemispheres
+
NA
+
normal
no
no
irregularities
other than
irregularities
other than
diffuse
slowing
diffuse
slowing
NA
NA
++
normal
++
NA
normal
progressive
diffuse
cerebral
atrophy and
volume loss
atrophy, thin
corpus
in
callosum (2
atrophy (10
mild
mo)
cerebellum
Arya et al
2017
y)
Schorling et
al 2017
Schorling
et al 2017
normal
Xiong at al.
2018
thin corpus
callosum
Schirinzi et
al 2018
hypomyelination
and atrophy
Schirinzi et al
2018
normal
Menke et
al 2016
normal
Kulkarni et
al 2015
normal
Kulkarni et
al 2015
normal
Dhamija et
al 2016
global
atrophy at
15 yr
Ananth et
al 2016
13 mo:
frontal
lobe
volume
loss
normal
Kelly et al
Blumkin et
2019
al 2018
frontal
lobe
volume
loss (13
mo)
Kelly et
al. 2018
5.3 Comparison between R209H and G203R mouse models
We have studied behavioral abnormalities using our mouse models with the R209H
and G203R Gnao1 mutations (Chapter 3) and also explored electrophysiological
characteristics of cerebellar Purkinje cells in the G203R mouse model (Chapter 4).
Needless to say, the two models exhibit some differences and similarities that may help
254!
us understand the differences between patients with NF GNAO1 mutations (i.e. R209)
and patients with GOF mutations (i.e. G203R). A comparison between patients with
R209H and G203R mutations is shown in Table 5.1. All patients present with
developmental delay from an early age. However, the G203R patients all exhibited
seizure episodes while R209H patients very seldom developed seizure events. In
addition, G203R patients are more likely to develop severe brain malformations, which
can be seen from their MRI results. It is hard to say, however, if those malformations
were caused by or contributed to epileptogenesis in those patients. In this section, I will
discuss some ideas for future directions generated from the similarities and differences
between R209H and G203R human patients and animal models.
5.3.1 Do G203R and R209H mouse models exhibit delayed development?
Among reported cases with GNAO1-associated neurological disorders, all G203R
patients (Arya, Spaeth, Gilbert, Leach, & Holland, 2017; Dietel, 2016; Nakamura et al.,
2013; Saitsu et al., 2016; Schorling et al., 2017; Xiong et al., 2018) and R209H patients
(Ananth et al., 2016; Dhamija, 2016; Kulkarni, Tang, Bhardwaj, Bernes, & Grebe, 2016;
Menke et al., 2016) exhibit developmental delay. Although we have assessed movement
abnormalities of adult mice, we are still unclear about whether our mouse models
replicate this seemingly universal symptom for human G203R and R209H patients.
Heyser (Heyser, 2004) published a very detailed milestone assessment for rodents that
can be adopted by this project. Understanding the role of Gαo role in neural development
255!
is crucial for understanding GNAO1-associated disorders, since developmental delay
seems to be unrelated to the genotype-phenotype correlation that we established
between epilepsy and movement disorders. Patients with both GOF/NF and LOF
GNAO1 mutations exhibit developmental delay. So far, apart from Gαo’s regulation of
neurite outgrowth (Strittmatter, Fishman, et al., 1994) and growth cone collapse (Igarashi
et al., 1993) in vitro, there has been little research done on the role of Gαo in mammalian
neuronal development (Tanaka, Treloar, Kalb, Greer, & Strittmatter, 1999).
In addition to confirming whether G203R and R209H mice have developmental delay,
how Gαo might play a role in regulating neuronal development should also be addressed.
At the in vitro level, primary neurons from brains of G203R and R209H mutant mice can
be isolated for assessing neurite outgrowth, axonal elongation, and growth cone
development. Mechanistically, one interesting question is how G203R and R209H
mutations in Gαo affect its interaction with GAP-43. GAP-43 (also called neuromodulin or
B57) is a “growth” or “plasticity” related presynaptic protein that plays a key role in
modulating growth cone signal transduction (Strittmatter, Valenzuela, Vartanian, et al.,
1991), axonal growth and guidance (Goslin, Schreyer, Skene, & Banker, 1988), and
synapse formation (Holahan, 2017). Homozygous mice lacking GAP-43 die in the early
postnatal period (Strittmatter, Fankhauser, Huang, Mashimo, & Fishman, 1995) and
heterozygous GAP-43 deficient mice survived but suffered from neuronal developmental
defects that last through adulthood (Latchney et al., 2014). The amino-terminal domain
256!
of GAP-43 promotes release of GDP from and binding of GTP to Gαo (Strittmatter,
Igarashi, & Fishman, 1994; Strittmatter et al., 1990; Strittmatter, Valenzuela, Sudo,
Linder, & Fishman, 1991). Co-expression of Gαo and GAP-43 can also be seen
throughout mouse embryo development stages (Schmidt, Zubiaur, Valenzuela, Neer, &
Drager, 1994). GAP-43 increases the GTPγS binding activity of Gαo (Jiang & Bajpayee,
2009; Yang, Wan, Song, Wang, & Huang, 2009). It is possible that GNAO1 mutants
affect the Gαo and GAP-43 interaction by changing the guanine nucleotide exchange
rate.
Another interactor of Gαo protein that may be of interest here is GRIN1 (G
protein-regulated inducer of neurite outgrowth 1) encoded by the GPRIN1 gene. GRIN1
binds to both Gαi and Gαo protein through the GRIN1 carboxyl-terminal region (Chen et
al., 1999). Co-expression of GRIN1 and constitutively active Gαo protein (Q205L)
induces neurite extensions in Neuro2A cells through the activation of Cdc42 (Nakata &
Kozasa, 2005). Like GAP-43, GRIN1 also co-localizes with Gαo protein expression at
neuronal dendrites and axons in different regions of adult mouse brains (Masuho et al.,
2008).
There are other possibilities for how Gαo protein may play a role in mouse
development. Understanding the mechanisms of GNAO1-related neurodevelopmental
disorders might provide unique insights into mechanisms of GNAO1-related movement
disorder and epilepsy after birth.
257!
5.3.2 How does the G203R mutation in Gao lead to epileptogenesis?
One puzzle between the patients with R209H and G203R mutations is why R209H
patients very seldom exhibit epilepsy, while all of the G203R patients present with both
epilepsy and movement disorders (Table 5.1) (Feng, Khalil, Neubig, & Sidiropoulos,
2018). This difference was also confirmed in our animal models with the PTZ kindling
study (Chapter 3) where male G203R mutants have enhanced kindling responses to
PTZ. We did not observe spontaneous seizures by G203R mice but we have not done
EEG recordings so spontaneous seizures are not entirely ruled out. Three of the
Gnao1+/G203R mutant mice did die in adulthood (Figure 3.1C) - similar to the G184S
mutant mice that do have rare spontaneous seizures (Kehrl et al., 2014).
The mechanisms of epileptogenesis in the G203R mutant are unclear. Since Gαo
and Gβγ are involved in multiple aspects of neurobiology, possible mechanisms of
G203R mutation-induced epileptogenesis includes: 1) altered neurotransmitter release;
2) a loss of subset of neurons; 3) altered neurite density and/or synaptogenesis; 4)
changed membrane properties (lower
threshold
for activation); 5) altered cell
morphology; and/or 6) malformation of cortical/hippocampal development.
Activation of Gαo and Gβγ is well-studied for regulation of neurotransmitter release
pre-synaptically (Stephens, 2009) and membrane potential post-synaptically (Beckstead
& Williams, 2007; Newberry & Nicoll, 1985). It would not be surprising if the G203R GOF
mutant has a stronger influence on neurotransmitter release in brain regions that are
258!
closely related to epileptogenesis, while the R209H mutant with NF behavior does not. In
addition, due to the role of Gαo and Gβγ in regulating neuronal development, it is
possible that the Gao protein with the G203R mutation leads to malformation during one
or more neural developmental stages. This hypothesis can be tested by carefully
monitoring the developmental states of G203R mice at the behavioral, morphological
and cellular levels. Malformations of cerebral cortical development are common causes
of neurodevelopmental delay and epilepsy (Barkovich, Guerrini, Kuzniecky, Jackson, &
Dobyns, 2012). The alteration of one or several developmental steps, including
proliferation of neural progenitors, migration of neuroblasts, layer organization, or
neuronal maturation may all lead to cortical malformation (Pang, Atefy, & Sheen, 2008).
Previously, I have stained and observed the cerebellum region of adult Gnao1+/G184S
mice, which exhibited similar behavioral abnormalities with Gnao1+/G203R mice (Chapter
3). There were no major abnormalities in the morphology of the cerebellum in the G184S
mice expect for a slight decrease in lobule number (Figure S5.7). No staining for the
G203R mutant mouse brain was done for the purpose of observing the gross
morphology.
Activated Gαo and Gβγ both play a role in regulating these developmental steps. The
literature shows that stimulation of Gαo inhibited neuronal migration of the EP cells,
which are a set of ~300 gut neurons begin to express Gαo at the time coincident with
their migration along the stereotyped pathways (Copenhaver & Taghert, 1989; Horgan &
259!
Copenhaver, 1998; Horgan, Lagrange, & Copenhaver, 1994). Also, Gβ1 knockout
mouse embryos developed neural tube defects, abnormal actin organization, and
microcephaly and then died at P2 (Okae & Iwakura, 2010). In the G203R mice or human
patients, the G203R mutant may trigger an abnormal formation at one or multiple
embryonic stages in cortex or hippocampus, resulting in susceptibility to epilepsy. For
example, mutant Gαo may cause the failure of GABAergic neurons to migrate toward the
cortex thus altering the excitatory/inhibitory balance. This could result in network
hyperactivity (Wonders & Anderson, 2006). Or mutant Gαo may cause abnormal
SNAP-25 function (Zurawski, Rodriguez, Hyde, Alford, & Hamm, 2016), which could lead
to derangements of synaptic transmission in the hippocampus.
5.3.3 How do G203R and R209H mice differ in movement disorder phenotypes?
Although dystonia and chorea/athetosis are both seen in G203R and R209H patients,
G203R mice and R209H mice exhibit a striking difference in their movement disorder
phenotypes. G203R mice were less able to remain on the RotaRod, had decreased
capability of lifting up heavy weights, and had more abnormal gait characteristics like
human patients with G203R mutation. They did not exhibit abnormalities in the open field
test. In contrast, R209H mice mainly exhibited increased locomotor activity in the open
field arena, which has not been seen in any previous Gnao1 mouse model. While it is
understandable that mouse models will not precisely reflect human symptoms, it is still
an interesting question on how mutations in the same gene cause different movement
260!
phenotypes in rodents. One hypothesis is that R209H and G203R mutations affect
different brain regions.
Gαo expression seems to be ubiquitous in mammalian brains, however, due to the
complex and diverse neural networking system in different regions, it is possible that
G203R and R209H mutations have effects distinct from each other. Gαo staining is
enriched in the cerebral cortex (particularly the molecular layer), the neuropil of the
hippocampal formation, and in the striatum, substantia nigra pars reticulata, and in the
molecular layer of the cerebellum (Worley, Baraban, Van Dop, Neer, & Snyder, 1986).
Coincidently, dystonia is commonly linked with injury to the basal ganglia, thalamus,
brainstem, and the cerebellum. Chorea is associated with disorders of the cerebral
cortex, basal ganglia, cerebellum and thalamus (Sanger et al., 2010). Both disorders are
seemingly associated with brain areas with significant Gαo protein expression levels.
In chapter 4, I showed that the G203R mutation causes a reduced frequency of
GABA release in cerebellar slices from G203R mice. However, we have not yet tested
changes due to the G203R mutation in other brain regions, so we are not sure whether
signaling in the striatum is also affected. The brain regions affected by the R209H
mutation are not yet known. To quickly locate the brain regions related to the movement
phenotype, we can do regional injection of oxotremorine (Pelosi, Menardy, Popa, Girault,
& Herve, 2017) or pertussis toxin (PTX) to help exclude the irrelevant brain regions. This
will be a crucial finding to help guide follow-up research on more detailed mechanisms of
261!
the movement disorder and
to
find more
targeted
therapeutic methods
for
GNAO1-associated movement disorders.
5.3.4 How is sex involved in abnormalities of the Gnao1 mutants?
GNAO1-associated neurological disorders are more prevalent in females than males
overall according to currently published reports (Chapter 1), but it may also only be due
to relatively small patient numbers. Alternatively, a more severe male phenotype would
cause a premature death of the fetus. However, in animal models we have observed
striking sex-related phenotypes. In G184S GOF mouse models, female mice are more
prone to both abnormal movements and seizures; while in G203R mouse models, male
mice developed more a significant phenotype compared to female for both movement
disorders and epilepsy. Interestingly, R209H mice do not seem to differ much in
sex-related phenotypes. Although sex differences in human GNAO1 patients remains
unclear, it is still interesting to consider the sex differences of the Gnao1 mutant animals
in terms of their behavioral abnormalities.
Biological differences between the male and female sexes contribute to many
sex-specific illness and disorders. These differences are not only due to gonadal
hormone secretion-related events, such as differences in neuroanatamy, synaptic
patterns, and neuronal density, but also due to non-hormonal related aspects,
particularly direct gene products mediated by genes located on the X- and Y-
chromosomes (Ngun, Ghahramani, Sanchez, Bocklandt, & Vilain, 2011).
262!
Most researchers attribute the sex differences in neurological disorders to the
actions of estrogens, progestins, and androgens. After all, those hormones regulate
early neurodevelopment to program the brain to be sexually bimorphic, and later activate
circuitries that trigger adult behaviors after puberty (Kight & McCarthy, 2014). The
actions of gonadal hormones lead to differences in brain structure (Farrell, Gruene, &
Shansky, 2015; Phan et al., 2012), connectivity (Ingalhalikar et al., 2014), signaling
(Harte-Hargrove, Varga-Wesson, Duffy, Milner, & Scharfman, 2015; Skucas et al., 2013),
responsivity (Garrett & Wellman, 2009), plasticity (Gould, Woolley, Frankfurt, & McEwen,
1990; Greenough, Carter, Steerman, & DeVoogd, 1977; Parducz et al., 2006), and even
adult neurogenesis (Galea, Spritzer, Barker, & Pawluski, 2006; Livneh & Mizrahi, 2011;
Vivar, Peterson, & van Praag, 2016). This makes hormone levels and functions an
important aspect to consider for the mechanisms of sex differences in our Gnao1 mutant
mouse models. Gαo may directly play a role in this hormonal regulation. Estrogen
attenuates the reuptake of both endogenous and exogenous dopamine in the striatum
and nucleus accumbens by altering the D2 receptor responsiveness (Thompson &
Certain, 2005). Estrogen also destabilizes GABAB, 5-HT1a, 5-HT1b and CB1 receptors
after a short exposure (Mize & Alper, 2000). Progesterone activates progestin
membrane receptors to down-regulate adenylyl cyclase activities, which can be blocked
by PTX (Thomas et al., 2007). Nuclear localization of androgen receptors is also
controlled by Gi-specific RGS proteins (Rimler, Jockers, Lupowitz, & Zisapel, 2007).
263!
Emerging research has also shown that sex differences are mediated by
mechanisms other than action of the hormone secretions. If hormones do not explain the
sex differences, then we should consider the different effects of XX versus XY sex
chromosome complement. After all, every neuron, glia, or other cell type carries either
the male chromosomes (XY) or female chromosomes (XX), but not both (Arnold &
Burgoyne, 2004). Although the traditional view attributes sex differences in neuronal
development to different hormonal exposure, recent research shows that some
non-gonadal tissues, including the brain, are sexually dimorphic even when they develop
in a similar endocrine environment (Arnold & Burgoyne, 2004). For example, primary cell
cultures harvested from the XX and XY mesencephalon and diencephalon before the
differentiating actions of gonadal hormones are present can develop into different
numbers of dopamine neurons or prolactin neurons, respectively (Beyer, Kolbinger,
Froehlich, Pilgrim, & Reisert, 1992; Beyer, Pilgrim, & Reisert, 1991). Another example is
the fact that male and female mammalian embryos develop at different rates at ages
before the onset of gonadal differentiation. There is evidence supporting that genes on
the Y chromosome enhance the rate of embryonic development (Burgoyne, 1993;
Burgoyne et al., 1995); while X chromosome genes slow down development (Thornhill &
Burgoyne, 1993). Another aspect is the presence of the SRY gene on the
Y-chromosome. Research shows that animals with Sry gene are associated with a
higher number of tyrosine hydroxylase (TH+) cells compared to those without Sry (Ngun
264!
et al., 2011). In humans, SRY expression is seen in both adult and fetal brains (Clepet et
al., 1993; Mayer, Lahr, Swaab, Pilgrim, & Reisert, 1998). Also Sry has a direct effect on
the expression of TH in the substantia nigra in the rat (Dewing et al., 2006). Effects of the
SRY gene are suspected to contribute to the susceptibility of men to Parkinson’s disease
(PD).
Previous research has not yet established the interconnection between functions of
Gαo and other embryonic developmental events in mammals; therefore it is hard to
determine whether Gαo plays a role in the embryonic development before or after
gonadal hormones are present, or both. Besides, two different GOF mutations, G184S
and G203R, seem to affect a different sex in terms of the severity of the motor behavior
and seizures in mice (Chapter 3). This observation provides additional evidence of a
pathophysiological mechanism more complex than just Gαo-regulated inhibition of cAMP.
The sex differences in the G184S and G203R mice should each be established as a
separate project with a deeper understanding of the differences between the G184S and
G203R mutant mice. Here, I will mainly focus on the discussion of the male-dominant
phenotype in the G203R mutant mice.
From a hormonal secretion aspect,
it
is possible
that estrogen exerts a
neuroprotective effect in the female mice (Brann, Dhandapani, Wakade, Mahesh, &
Khan, 2007; Green & Simpkins, 2000); therefore the male G203R mice have a more
severe genotype. Research showed that estrogen reduced dopamine D2 receptors by 20%
265!
to 25% (Chavez et al., 2010), which could counterbalance the GOF mechanisms
induced by the G203R mutant in female mice, and leave the male mice affected. Another
possibility would be that Gαo with the GOF G203R mutation affects the nuclear
localization of androgen receptors in the hippocampus (Rimler et al., 2007), which
reduces the serum androgen level, and consequently lead to reduction of the GABAergic
inhibition (Frye, 2006; Reddy & Jian, 2010). This would expose the male G203R mice to
the risk of disease such as temporal lobe epilepsy (Harden & MacLusky, 2004, 2005;
Herzog, 1991).
From a non-hormonal perspective, we should look into the embryonic developmental
stages of the G203R mutant mice to see if it correlated with the Y-chromosome
regulated increase in rate of development. Otherwise, we should also consider the SRY
gene’s modulation of dopamine biosynthesis and motor function. SRY, as a transcription
factor, can directly activate the TH promoter, which enhances expression of tyrosine
hydroxylase, the rate-limiting enzyme of dopamine synthesis (Czech et al., 2012).
Although it is yet to be established whether the G203R mutation could affect dopamine
transmission or the survival of dopamine neurons in the brain, the presence of the SRY
gene on the Y-chromosome in the male G203R mutant mice could potentially
exacerbate the effect of increased dopamine in causing abnormal movements (Cepeda,
Murphy, Parent, & Levine, 2014).
266!
While we are not yet sure of the effect of the R209H mutation on Gαo’s functional
pathways, R209H mutants may not exhibit as extensive an effect as G203R mutants do.
This may be why we do not see a significant sex difference in the R209H mutant mice.
5.4 Development of a high-throughput assay for drug repurposing or drug
development
Calcium ions participate in a variety of physiological and pathological mechanisms.
In presynaptic neurons, calcium levels regulate neurotransmitter release. Detectable
calcium signaling results from a complicated interaction between activation and
inactivation of both intracellular and extracellular calcium channels. This interaction can
be observed with calcium-sensing probes such as Fura-2 or Fluo-4. Neural calcium
signaling often results in sequential regenerative discharges of stored calcium, a process
referred to as calcium oscillation (Dupont, Combettes, Bird, & Putney, 2011). Calcium
oscillations can be categorized into two classes based on the involvement of intracellular
calcium storage. The intracellular release of calcium most commonly derives from the
endoplasmic reticulum (ER) driven by inositol 1,4,5-trisphosphate (InP3) (Streb, Irvine,
Berridge, & Schulz, 1983). However, in excitable cells like neurons, the increase of
intracellular calcium levels can also be initiated by activation of membrane channels that
lead to the influx of calcium ions from the extracellular space (Tsien et al., 1986). This
may further facilitate intracellular calcium release from the endoplasmic reticulum (ER)
into the cytoplasm (Fabiato, 1983).
267!
Dissociated mouse cortical neurons can re-associate in vitro to form connected
synaptic networks. This method provides an assay to reduce the expense and labor of
testing drugs in vivo and provides a potential approach for high-throughout screening.
Previous research has shown that neural calcium oscillations involve the activation of
NMDA, AMPA/kainate receptors, and mGluR (Dravid & Murray, 2004; Nash et al., 2002;
Robinson, Kawahara, et al., 1993). Also, the rising phase of each calcium spike is
usually coincident with a brief burst of action potentials (Murphy, Blatter, Wier, &
Baraban, 1992). Additionally, calcium oscillations measured from dissociated rat cortical
neurons are dependent on the influx of extracellular calcium rather than mobilization
from intracellular stores (Wang & Gruenstein, 1997). Oscillating calcium activity in
dissociated neurons is also temporally correlated with the maturity of neurons and the
time in culture to allow connections between neurons. Generally, synchronized calcium
oscillations occur in around DIV7-DIV10 after plating and they plateau at DIV13 (Pacico
& Mingorance-Le Meur, 2014). This is coincident with a burst of synapse formation that
happens around DIV14 (Ichikawa, Muramoto, Kobayashi, Kawahara, & Kuroda, 1993).
Thus, the synchronized calcium-spiking events seem to be an emergent property due to
the formation of a large number of glutamatergic synapses. In agreement with earlier
studies (Robinson, Kawahara, et al., 1993; Robinson, Torimitsu, Jimbo, Kuroda, &
Kawana, 1993; Wang & Gruenstein, 1997), my preliminary data showed that removal of
extracellular Mg2+ induces cultured mouse neurons to undergo synchronized calcium
268!
oscillations (Figure S5.5). Calcium oscillation activities are also dependent on
extracellular calcium concentrations (Figure S5.5). Although previous studies showed
that inhibition of voltage-gated K+ channels induces calcium oscillation (Wang &
Gruenstein, 1997), we found that high levels of extracellular K+ also suppress calcium
oscillation activity (Figure S5.5). Since the calcium oscillations shown here are uniform
and stationary, the analysis can be accomplished by Fast Fourier Transform (FFT) that
will identify both the frequency and the amplitude of these oscillations (Barhoumi, Qian,
Burghardt, & Tiffany-Castiglioni, 2010; Vajda, Donnan, Phillips, & Bladin, 1981).
Here, we intend to use this technique to explore whether there is any difference
between WT and G203R mice in the calcium oscillatory activity. As discussed previously,
the Gαo protein mainly functions to inhibit synaptic neurotransmitter release. However,
since Gαo is present at both excitatory and inhibitory synapses, it is hard to predict the
end result of abnormalities in Gαo regulation. Based on what we observed in Chapter 4,
the G203R GOF Gαo protein mainly enhanced presynaptic inhibition of GABA release
but has less to no inhibitory effect on glutamate release. Therefore, we hypothesize that
the GOF G203R mutant enhances suppression of GABA release thereby increasing
excitability of the neural network, which leads to increased calcium spiking events. Our
preliminary study confirmed our hypothesis that G203R mutant animal cortical cell
cultures (both Gnao1+/G203R and Gnao1G203R/G203R) have more calcium spikes (Figure
S5.6). However, it has been challenging to optimize this technique for high-throughput
269!
screening. The data generated between experiments are highly variable; therefore
comparison between plates in multiple experiments done at different times requires a
standard control for each plate. Another issue is the variability between animals, which is
hard to avoid but can be minimized by increasing the n number for experiments. With
further optimization, calcium oscillation can utilize primary neurons from different brain
regions to address the different oscillatory activities between WT and mutant mice.
Ultimately, this may be used in high throughput methods to identify candidate drugs or
receptor targets for drug repurposing or new drug development.
5.5 Impact of work in this thesis on the field
This thesis is the summary of the first project to explore the GNAO1-related rare but
serious neurologic disorders in children. I defined the biochemical correlation between
the functional changes of the human GNAO1 mutants and the patients’ neurological
symptoms. In addition, our work also provided the first human avatar Gnao1 mutant mice
and verified their value in studying the neurophysiological mechanisms of GNAO1
mutations. Subsequent mechanistic and intervention studies should greatly enhance the
development of potential therapeutic strategies for these devastating childhood
neurologic disorders. In a broader aspect, due to the ubiquitous expression of Gαo
(encoded by GNAO1) and its multiple effectors, our study may enhance understanding
of neurological disorders that involve the same pathways shared by the Gαo protein.
Similar pharmacological interventions may also be valuable for genetic conditions
270!
involving GNB1, ADCY5, PDE10A and GNAL. Furthermore, our study may serve as a
prototype for other correlations between reported monogenic mutations and human
neurological disorders.
271!
APPENDIX
272!
APPENDIX
SUPPLEMENTAL DATA
Figure S5.1 α2AR activates Gαo, which inhibits N-type calcium channels in G1A1
cells. GOF mutation G184S enhance the inhibition of calcium currents. (A) G1A1
cells stably express N-type calcium channels, which can be blocked by MVIIa but not
entirely. Previous report showed G1A1 cells have endogenous L-type calcium channels.
All membrane calcium channels can be blocked by Cd2+ ions and enhanced by Ba2+ ions.
(B) G1A1 cells have negligible amount of Gαi/o proteins, therefore activating α2AR alone
cannot inhibit the calcium currents. (C) G1A1 cells with transiently transfected Gαo
273!
Figure S5.1 (cont’d) protein and α2AR can lead to the inhibition of calcium current when
α2AR is activated by norepinephrine (NE). (D) GOF GNAO1 mutation G184S causes a
slight enhanced inhibition of calcium current immediately after adding selective α2AR
agonist UK14, 304, however, the maximum inhibition between WT and G184S is not
significant. WT (n=6), G184S (n=7), pcDNA (n=3). (E) G184S (n=4) significantly
enhanced inhibition of N-type calcium channels comparing to WT (n=4). Unpaired
Student’s t-test; p=0.0431.
Figure S5.2 Both G1A1 and SH-SY5Y cells are good candidates to study mutant
Gαo’s effects on N-type calcium channels with Fluo 4-NW dye in a Hamamatsu
µCELL plate reader. (A) N-type calcium channels in G1A1 cells are activated by 90 mM
274!
Figure S5.2 (cont’d) KCl with the presence of 5 mM CaCl2. (B) SH-SY5Y cells have
more types of endogenous calcium channels. All calcium currents can be induced by 90
mM KCl. N-type calcium channels can be blocked by 250 nM MVIIa, and L-type calcium
channels can be inhibited by 10 µM Nifedipine.
Figure S5.3 Neurite outgrowth in rat pheochromocytoma cells (PC12) can be
induced by 50 ng/mL Nerve Growth Factor (NGF) in normal growth medium with
reduced serum. (A) Representative pictures showing PC12 cells growth after 48 hrs in
(a) normal growth medium, (b) normal growth medium with reduced FBS (3% FBS), and
(c) 50 ng/mL NGF in normal growth medium with 3% FBS. (B) NGF significantly
promotes the percent of cells with visible neurites (n=3; Unpaired Student’s t-test;
p<0.001). Spontaneous neurite outgrowth does occur without any NGF. (C) NGF also
significantly increases the neurite length from PC12 cells (n=3; Unpaired Student t-test;
p<0.0001).
275!
Figure S5.4 Neurite outgrowth can be induced by 10 µM retinoid acid (RA) in
normal growth medium with reduced serum (30% FBS) in human neuroblastoma
cells (SH-SY5Y). (A) SH-SY5Y cells are largely non-differentiated in normal growth
medium with 10% FBS (a); reduced serum induces morphological changes and neurite
outgrowth in SH-SY5Y cells (b); 10 µM RA in 3% FBS medium induces the majority of
cells to differentiated (c). (B) Preliminary studies show that RA increases the percent of
cells with neurites, (C) but does not seemingly affect the average neurite length. Since
the n number is small, there is no significance in comparison but the trend of change is
obvious.
276!
Figure S5.5 Representative traces of modulations in calcium oscillations with
different ion concentration in mixed cortical cultures from a WT mouse. (A)
Extracellular calcium concentration is crucial for calcium spiking events. The frequency
of calcium oscillation increases with lower calcium concentrations but the amplitudes of
calcium spikes peaks with a calcium concentration between 6.3 mM and 3.1 mM. (B)
Higher concentrations of magnesium inhibit calcium oscillatory activities and lower
magnesium concentrations induce them. All magnesium concentration responses were
done with 5 mM calcium present. (C) When potassium concentration is above 15 mM,
calcium oscillatory activities are completely quiescent. Lower concentrations of
potassium facilitate calcium oscillation. All potassium concentration responses were
done with 5 mM calcium present.
277!
Figure S5.6 High-throughput assessment of neural excitability in cortical cultures.
Cortical cultures (neurons and glia) from P0 Gnao1+/G203R mutant mice and WT
littermates (triplicate wells shown) were prepared in 96‐well plates, allowed to form
278!
Figure S5.6 (cont’d) connections (14 DIV), labeled with Fluo-4-NW calcium indicator,
then read in a Hamamatsu Cell reader for 10 minutes. Power spectra ‐ (A) (all) & (B)
(mean ± SD) of 10 WT (96 wells), 10 heterozygous (Het 66 wells), and 2 homozygous
(Homo 24 wells) pups under conditions showing differences in epilepsy studies (CaCl2 5
mM and KCl 7.5 mM), (C) AUC of Ca2+ power spectra for all wells. Due to the non‐
normal distribution, statistical analysis used the non‐parametric Kolgov‐Smirnov test of
cumulative distributions (GraphPad Prism 8.1). (D) Heat map of Ca2+ levels ‐ 3 wells per
mouse (4 WT & 2 Het) at CaCl2 5 mM and MgCl2 7.5 mM.
Figure S5.7 Nissl staining compares gross morphological changes in cerebellum
from Gnao1+/G184S and Gnao1+/+ mice at age 8 weeks old. (A) No obvious gross
difference in cerebellum region of Gnao1+/G184S and Gnao1+/+ mice. (B) Lobule number is
slightly lower in Gnao1+/G184S mice (4 slices were measured from each mice; Gnao1+/+:
n=8, Gnao1+/G184S: n=7). (C) Molecular layer thickness of IV, V, VIa and VIb in the
cerebellar region of Gnao1+/G184S and Gnao1+/+ mice does not show significant difference
279!
Figure S5.7 (cont’d) (12 repeated measurements were obtained from 4 slices for each
region per mouse; Gnao1+/+: n=8, Gnao1+/G184S: n=7).
280!
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