ANALYSIS OF MICE CARRYING HUMAN GNAO1 MUTATIONS AS A MODEL TO STUDY ASSOCIATED MOVEMENT DISORDERS By Casandra Lynn Larrivee A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Comparative Medicine and Integrative Biology—Master of Science 2019 ABSTRACT ANALYSIS OF MICE CARRYING HUMAN GNAO1 MUTATIONS AS A MODEL TO STUDY ASSOCIATED MOVEMENT DISORDERS By Casandra Lynn Larrivee Due to the increased availability of genetic screening, patients with idiopathic epilepsy and movement disorders are being identified with mutations in the GNAO1 gene. The GNAO1 gene encodes a heterotrimeric G protein subunit, Gαo, abundantly found within the brain. Patients with de novo mutations in GNAO1 specifically may have early onset seizure disorders and/or involuntary movements. These two phenotypes were later classified as early infantile epileptic encephalopathy (EIEE17) and neurodevelopmental delay with involuntary movements (NEDIM) respectively. Previous work in our lab uncovered a pattern between the in vitro function of mutations and the type of disorder observed in patients. Loss-of-function mutations associated with EIEE17 while gain-of-function mutations or proteins with essentially normal function were seen in NEDIM. To determine whether this pattern could be replicated in vivo, heterozygous mutant mice were created using CRISPR/Cas9. Here we report the first mouse models of GNAO1 disorders, Gnao1+/G203R and Gnao1 +/R209H. Using a variety of behavioral battery tests including open field, rotarod and digigait, we were able to show distinct behavioral patterns between the mutant mice. Using these models we began to explore preclinical drug repurposing and neural mechanisms. ACKNOWLEDGEMENTS I had a wonderful mentor in Dr. Richard Neubig, for whom I owe my deepest appreciation. His guidance was supportive, inspiring and ever-present. He always encouraged and challenged me to think past my limits, making me a stronger scientist. This project was made possible in large part to Huijie Feng and Jefferey Leipprandt; However, all the members of Neubig lab at one point or another helped me on my way. To all of you – thank you. Thanks to Dr. Nara Parameswaran, Dr. Brian Schutte, and Dr. Florian Kagerer each of whom served as my professors and as my committee members. Their expertise challenged me and extended my own knowledge base. Their input was invaluable and essential to this project and my own academics. Within my department of Comparative Medicine and Integrative Biology, Dr. Vilma Yuzbasiyan-Gurkan and Dimity Palazzola supported and aided me every step of the way. They were an anchor during my time in graduate school, I always knew I had a team of amazing women on my side. iii TABLE OF CONTENTS LIST OF TABLES ................................................................................................................................ v LIST OF FIGURES ............................................................................................................................. vi KEY TO ABBREVIATIONS .............................................................................................................. viii CHAPTER 1: INTRODUCTION .......................................................................................................... 1 Background of GNAO1 research ....................................................................................... 2 EIEE17 ................................................................................................................................ 6 NEDIM ................................................................................................................................ 7 Genetics of GNAO1 ............................................................................................................ 8 Statement of Purpose ........................................................................................................ 9 CHAPTER 2: MOUSE MODELS OF GNAO1-ASSOCIATED MOVEMENT DISORDER: ALLELE- AND SEX-SPECIFIC DIFFERENCES IN PHENOTYPES .............................................................................. 11 Statement of Contribution ............................................................................................... 12 Abstract ............................................................................................................................ 13 Introduction ..................................................................................................................... 15 Materials and Methods .................................................................................................... 18 Results .............................................................................................................................. 29 Discussion ........................................................................................................................ 43 CHAPTER 3: BEHAVIORAL ASSESSMENT OF MICE WITH A COMMON GNAO1 MOVEMENT DISORDER VARIANT R209H ......................................................................................................... 48 Statement of Contribution ............................................................................................... 49 Abstract ............................................................................................................................ 50 Introduction ..................................................................................................................... 51 Materials and Methods .................................................................................................... 54 Results .............................................................................................................................. 63 Discussion ........................................................................................................................ 74 CHAPTER 4: CONCLUSIONS AND FUTURE DIRECTIONS ............................................................... 77 APPENDICES ................................................................................................................................. 81 APPENDIX A: Chapter 2 .................................................................................................... 82 APPENDIX B: Chapter 3 .................................................................................................... 94 REFERENCES ................................................................................................................................ 96 iv LIST OF TABLES Table 2-1. Location, sequence and genotyping of gRNA targets in Gnao1 locus ......................... 20 Table 2-2. Scoring scale for postural and motor abnormalities .................................................. 25 Table 2-3. GNAO1 G203R patient classification .......................................................................... 40 Table 3-1. Location and sequence of gRNA and ssODN template for CRISPR-Cas targeting Gnao1 locus; primers and genotyping method for Gnao1+/R209H mice .................................................... 55 Table 3-2. GNAO1 R209H patient classification .......................................................................... 71 Supplemental Table 2-1. Gait analysis parameters Male Gnao1 G203R mutants ....................... 88 Supplemental Table 2-2. Gait analysis parameters Female Gnao1 G203R mutants ................... 89 Supplemental Table 2-3. Gait analysis parameters Male Gnao1 G184S mutants ....................... 90 Supplemental Table 2-4. Gait analysis parameters Female Gnao1 G184S mutants .................... 91 Supplemental Table 2-5. Benchling off-target list for Gnao1 G203 gRNA ................................... 92 v LIST OF FIGURES Figure 1-1. Functions of Gαo protein .............................................................................................. 3 Figure 1-2. Conformational changes of GPCR ................................................................................ 4 Figure 1-3. GNAO1 allelic variants ................................................................................................. 5 Figure 2-1. Development of Gnao1+/G203R mouse model ............................................................. 19 Figure 2-2. The timeline for utilizing animals in this study .......................................................... 22 Figure 2-3. Female Gnao1+/G184S mice and male Gnao1+/G203R mice show reduced time on RotaRod and reduced grip strength ............................................................................................. 31 Figure 2-4. G184S mutant mice showed reduced activities in Open Field Test but G203R mutants do not ............................................................................................................................ 33 Figure 2-5. DigiGait Imaging System reveals sex-specific gait abnormalities in Gnao1+/G184S mice and Gnao1+/G203R mice .................................................................................................................. 36 Figure 2-6. Gnao1+/G203R male mice have an enhanced Pentylenetetrazol (PTZ) Kindling response ..................................................................................................................................................... 38 Figure 2-7. Induction of abnormal movements and postures by oxotremorine ......................... 39 Figure 2-8. Treatment with trihexyphenidyl on Gnao1+/G203R mice ............................................ 41 Figure 2-9. Brain neurotransmitter analysis of Gnao1+/G203R mice .............................................. 42 Figure 3-1. Targeting of the mouse Gnao1 locus ......................................................................... 55 Figure 3-2. Gnao1+/R209H exhibited a developmental delay during the negative geotaxis assessment .................................................................................................................................. 64 Figure 3-3. Gnao1 +/R209H shows significant hyperactivity and reduced time in center in the open field arena ................................................................................................................................... 66 Figure 3-4. Male and Female Gnao1+/R209H mice shows gait abnormalities in different tests on the DigiGait imaging system ........................................................................................................ 68 vi Figure 3-5. Gnao1 +/R209H mice do not have an enhanced Pentylenetetrazol (PTZ) kindling response ...................................................................................................................................... 69 Figure 3-6. Risperidone treatments decreases hyperkinetic movements in Gnao1+/R209H ......... 72 Figure 3-7. Western blot shows no statistical difference in Gαo protein between Gnao1+/R209H and WT mice ............................................................................................................................... 73 Supplemental Figure 2-1. RotaRod test was conducted with 5 training sessions and 1 test session over two consecutive days .............................................................................................. 83 Supplemental Figure 2-2. Time spent at the center in the Open Field Tests .............................. 84 Supplemental Figure 2-3. RotaRod learning curve was collected in 10 consecutive tests with a 5- min break between each test ...................................................................................................... 85 Supplemental Figure 2-4. False discovery rate (FDR) calculation probed through all the parameters given by DigiGait in Gnao1+G184S mice ...................................................................... 86 Supplemental Figure 2-5. False discovery rate (FDR) calculation probed through all the parameters given by DigiGait in Gnao1+/G203R mice ..................................................................... 87 Supplemental Figure 2-6. GABA and glutamate show no significant differences between Gnao1+/R209H and wildtype littermates ......................................................................................... 93 Supplemental Figure 3-1. WT and Gnao1+/R209H mice show no difference in percent suppression of locomotion after oxotremorine treatment ............................................................................. 95 vii KEY TO ABBREVIATIONS 5-HT 5-Hydroxytryptamine AC Adenylyl Cyclase cAMP Cyclic adenosine monophosphate DA Dopamine DSB Double strand DNA break EEG Electroencephalogram EIEE17 Early infantile epileptic encephalopathy GABA Gamma-Aminobutyric acid GDP Guanine diphosphate GNAO1 Guanine nucleotide-binding protein, alpha-activating activity polypeptide O GOF Gain-of-function GPCR G-protein coupled receptor GTP guanine triphosphate GWAS Genome wide association studies HET Heterozygous HPLC High performance liquid chromatography LOF Loss-of-function NE Norepinephrine NEDIM Neurodevelopmental disorder with involuntary movements OXO Oxotremorine viii PAM Protospacer adjacent motif PTX pertussis toxin PTZ Pentylenetetrazol RGS regulators of g-proteins ssODN Single stranded oligodeoxynucleotide THP Trihexyphenidyl WT Wildtype ix CHAPTER 1: INTRODUCTION 1 Background of GNAO1 research Two neurological conditions, epilepsy and movement disorders, have both been linked to mutations within the brain abundant protein Gαo [1, 2]. The Gαo protein belongs to the Gi/Go subfamily of Gα proteins. Within the α family there are roughly 21 subunits, and can be grouped by the Gαi/o family as well as Gαs, Gα12/13 and Gαq. Encoded by the GNAO1 gene, the function of Gαo is widely characterized by its inhibition of adenylyl cyclase preventing production cyclic adenosine monophosphate (cAMP) and its sensitivity to pertussis toxin (PTX). Gαo was first identified in 1984 when researchers [3, 4] were looking to isolate Gαi from the brain instead finding another inhibitory Gα protein, naming Gαo for ”other”. Gα proteins are well known for their coupling to G-protein coupled receptors (GPCR) to aid in eliciting intercellular effects. Gαo couples to a wide range of GPCRs including, GABAB receptors, α2-adrenergic receptors, and D2 dopamine receptors. Activation of these receptors leads to a decrease in cAMP as mentioned above, through Gαo. However, Gαo signal transduction can also function to inhibit sodium and calcium ion channels, as well as activating potassium channels (Figure 1-1) [5, 6]. These functions lead to a decrease in neuronal excitability and allow receptors coupled to Gαo to regulate release of neurotransmitters, as well as other functions. 2 cyclase in producing cAMP. The Gαo and βγ dimer the associated by dimer complex also can Figure 1-1. Functions of Gαo protein Gαo proteins canonically function to inhibit adenylyl also activate potassium channels and inhibit activation of calcium channels. [7] (reproduced with permission under the Creative Commons Attribution License). Gαo can only cause its effects in its active state when it is bound to guanine triphosphate (GTP). During basal conditions the Gα subunit is bound to guanine diphosphate (GDP), and to a βγ dimer, composing the heterotrimeric G-protein. After receptor activation when Gα proteins are bound to GTP, it continues signal transduction, but once GTP is hydrolyzed back to GDP, the receptor is inactivated. This can happen spontaneously through intrinsic activity of the Gα protein or by specialized GTP accelerating proteins (GAPs) known as regulators of G-proteins (RGS). RGS proteins can bind to Gα proteins, stimulate GTP hydrolysis to GDP which will cause inactivation and the re-association of the Gα to the βγ dimer (Figure 1-2). 3 of the heterotrimeric G-protein. The Gα protein dissociates from the βγ dimer, both subunits can Figure 1-2. Conformational changes of GPCR Activation of GPCR cause conformational change then bring about effector functions until GTP is hydrolyzed to GDP by either intrinsic GTPase activity or by RGS proteins. [7] (reproduced with permission under the Creative Commons Attribution License). To assess the role of Gαo, , early studies mutated Gα proteins with a G184S point mutation which rendered Gαo insensitive to RGS proteins causing prolonged activation [8, 9]. After 2013, Nakamura et. al established a link between GNAO1 and cases of early onset epilepsy[10] researchers realized their G184S GOF mouse model could be used to study GNAO1 associated disorders including early infantile epileptic encephalopathy (EIEE17). Consistent with patient symptomology they were able to show heterozygous G184S mice were more sensitize to pentylenetetrazol (PTZ) kindling studies [11]. PTZ is a GABAA agonist, and in high doses it can cause a convulsion but in lower frequent doses it is causes an electrophysiological change in the brain decreasing the threshold of excitability, making it a model for epileptogenisis [12]. While the Gnao1+/G184S mouse model showed kindling sensitivity correlative with the epilepsy observed in patients, this specific GNAO1 G184S variant has not been seen any patients, which is why recent research has focused specifically on patient variant models. 4 In the past five years the number of pathological variants within GNAO1 have accumulated, to date there are 78 published cases[13-16] and 34 different variants (Figure 1-1.) While the variants were initially identified in children with early onset epilepsy[10] further reports discussed presence of developmental delay and movement disorders[17, 18] expanding the phenotype of GNAO1 mutations. It was later uncovered that certain variants more commonly associated to epilepsy while others linked to movement disorders[19]. This was largely dependent on functionality of the GNAO1 protein Gαo. Thrr191_phe197 del, 1 Y291N, 1 723 +1G->T, 1 E246K, 9 G203R, 8 R209C, 7 R209H , 7 Q233P, 1 R209G, 1 S207Y, 1 S47G, 1 Q52P, 1 N270H, 1 L199P, 1 R349_G352, 1 L284S, 1 I56T, 1 I344Del, 1 E246G, 1 D273V, 1 G45E, 1 G204R, 1 D270H, 1 A227V, 1 A221D, 1 Y231C, 2 R209L, 2 D174G, 1 723+1G - >A, 1 G40W, 2 G40E, 2 I279N, 3 F275S, 2 G40R, 3 E237K, 5 Figure 1-3. GNAO1 allelic variants To date there are 34 different causative variants that have been found within GNAO1. Many of the variants occur infrequently, presenting in only one or two patients, a few of the variants are found more frequently. Some of the mutation hotspots include; E246K, G203R, R209 (C,H) and E237K. 5 EIEE17 Epilepsy is the one of the common neurological conditions in the United States [20], characterized by multiple unpredictable seizures that may or may not be visible through electrical recording of the brain. Severity and types of seizures range between cases. Early infantile epileptic encephalopathy is a severe form of epilepsy categorized by tonic seizures that occur early in life, with the presence of observable EEG abnormalities. There is a wide range of genetic heterogeneity within EIEE, with over 50 different associated etiologies [21]. While many of the associated genes are only linked with the epilepsy syndrome, mutations in a few causative genes, including GNAO1, give rise to multiple syndromes. The CACNA1A gene, which encodes a subunit of a voltage gated P-type calcium channel, not only causes EIEE but is also linked to ataxia, a motor disorder characterized by spells of imbalance and a loss in coordination [22]. Possibly not surprising is that the regulation of P-type voltage gated calcium channels is also a role of Gαo proteins[6]. As calcium plays a vital role in the release of neurotransmitters and mutations in both genes cause epilepsy and a movement disorder it is likely that a causative mechanism behind one or both of the phenotypes is neurotransmitter release. Large scale sequencing efforts in patients with epilepsy have identified several other mutations within GNAO1. Cell based assays then classified the known mutations in terms of Gαo protein ability to inhibit cAMP through adenyl cyclase [19]. They found a correlation that those mutations with a decreased amount of cAMP inhibition, classified as a loss-of-function (LOF) mutations, were more commonly seen in patients with EIEE17. They also showed that mutations 6 with an increased amount of cAMP inhibition, gain-of-function (GOF) mutations, associated with the patients who had neurodevelopmental disorder with involuntary movements (NEDIM) [19]. NEDIM While some patients present with both epilepsy and movement disorders [17] those with mutations classified as GOF more commonly presented with neurodevelopmental disorder with involuntary movements (NEDIM). In addition to the presentation of involuntary movement disorders, patients with GNAO1 mutations also present with hypotonia – low muscle tone -- and developmental delay [23]. This consistent grouping of symptoms in GNAO1 patients is what lead to the classification of NEDIM. While NEDIM is a blanket name for presence the of movement disorders, there are more detailed clinical profiles of the movement abnormalities displayed in patients. Commonly seen disorders include chorea, athetosis, dystonia and dyskinesias [24]. Each of which explain a specific type of involuntary movement. From the Greek word for “dance” chorea is classified by brief and abrupt movements that seem to flow between body parts. Athetosis involves slow involuntary writhing movements. Dystonia involves sustained muscle contractions that can lead to repetitive or twisting movements. However, diagnosing movement disorders is quite complex as they often present together or with other comorbidities, such as epilepsy. Making genetic analysis an invaluable tool for determining etiologies. The GNAO1 GOF correlation with NEDIM is consistent with other rare monogenic disorders that disrupt cAMP. For example, a mutation in the gene that encodes an adenylyl cyclase, ADCY5, has been linked to dyskinesia. Mutations in ADCY5 have also been found to 7 increase cAMP [25], similar to GOF mutations in GNAO1. Mutations in GPR88, and GNAL are also genes involved in regulation of cAMP that cause movement disorders [26]. Movement is largely controlled by areas of the brain including the basal ganglia and the cerebellum, motor pathways largely involve the synaptic transition between these areas of the brain and the cortex. As Gαo is widely abundant in these regions [1] it is likely these regions may be mechanistically important within the pathophysiology of GNAO1 associated movement disorders. Genetics of GNAO1 There are now roughly 35 published variants of GNAO1, most of which are de novo missense mutations. While many of the mutations only have been found in one or two patients, there are a few mutational hotspots, these include G203R, R209C, and R209H (Figure 1-1). The high frequency of Arg209 mutations (R209C and R209H) and G203R are not surprising when we look at the specific base changes and the surrounding sequence. R209C and R209H mutations are due to 625 cytosine to thymine transitions (C >T) and 626 guanine to adenine (G > A) transitions respectively. The genetic sequence at these sites show a cytosine followed by a guanine (CpG dinucleotide). It is well known that sequences that mutate at a higher rate are CpG dinucleotides because cytosine is vulnerable to methylation and subsequent deamination resulting in a transitional mutation of C >T [27]. In the case of R209H mutation it is the complementary strand transition from C >T, which will result in a G -T mismatch at site 626 of GNAO1. DNA repair mechanisms will then change the guanine to an adenine resulting in the G>A 8 transition we observe in the R209H mutation. The G203R base change is a c. 607 G >A, at position at 606 is a cytosine, therefor we again observe another CpG dinucleotide. Moreover, DNA methylation has also been linked to other neurological conditions such as Huntington’s, and Rett syndrome [28] both of which have phenotypic similarities to GNAO1. All of the GNAO1 mutations are de novo, implying a germ cell (sperm or egg) mutation in one of the parents. Research has found a link between de novo mutations and age of fathers, possibility due to the fact that sperm cells generate high levels of mutation [29-31]. While such correlations may not be directly useful to treatments as CpG mutations may not be preventable, research is being done on possible epigenetic therapies such as preventing DNA demethylation [28, 32]. Further, this information might be useful for parents of patients with GNAO1 mutations considering having more children. De novo mutations in general are not preventable, however, it is possible to test for germ-cell mutations within sperm cells themselves. Therefore, fathers who have a child with a GNAO1 mutation might consider genetic testing to assess for possible germ-line mutations that could be passed onto to any future offspring. Statement of Purpose Clinically, scientists and physicians are able to correlate the presence of EIEE17 and NEDIM with mutations in the GNAO1 gene. While we are starting to understand the etiology, quality of life for these patients is still quite low. Both seizure disorders and involuntary movements are difficult to control and impair daily tasks. While there are a wide variety of therapeutic options, patients must try many different agents before they show some 9 improvement, many still do not have their symptoms completely under control [24, 33]. The work in this thesis looks primarily at the use of animal models as a means to understand the GNAO1 mutations. Using the phenotype-genotype correlation we begin to assess different variants in a CRISPR/Cas9 mouse model to assess their predictive nature. From there this work is able to study the models and assess outcome to pharmacological treatments in an allele-specific personalized medicine approach. 10 CHAPTER 2: MOUSE MODELS OF GNAO1-ASSOCIATED MOVEMENT DISORDER: ALLELE- AND SEX-SPECIFIC DIFFERENCES IN PHENOTYPES Reprinted and adapted with permission under the Creative Commons Attribution License 11 Statement of Contribution My role in the following chapter first published in PLoS ONE on 2019 January 25 was the following. I planned, performed and analyzed, open field, and rotarod studies for both of the Gnao1+/G184S and Gnao1 +/G203R animal models. I performed all initial data analysis for these experiments and wrote up my experimental protocols that would be used in writing the paper. I also performed some additional experiments that were not in the initial publication. These include; neurotransmitter analysis and testing the effects of oxotremorine and trihexyphenidyl on the Gnao1 +/G203R model. The didigait, grip strength and kindling studies were done by Huijie Feng. Genotyping and breeding of mice was done by Jefferey Leipprandt. Elena Demireva and Huirong Xie at the MSU transgenic core generated the mutant mouse models with CRISPR/cas9 technology. 12 Abstract Infants and children with dominant de novo mutations in GNAO1 exhibit movement disorders, epilepsy, or both. Children with loss-of-function (LOF) mutations exhibit Epileptiform Encephalopathy 17 (EIEE17). Gain-of-function (GOF) mutations or those with normal function are found in patients with Neurodevelopmental Disorder with Involuntary Movements (NEDIM). There is no animal model with a human mutant GNAO1 allele. Here we develop a mouse model carrying a human GNAO1 mutation (G203R) and determine whether the clinical features of patients with this GNAO1 mutation, which includes both epilepsy and movement disorder, would be evident in the mouse model. A mouse Gnao1 knock-in GOF mutation (G203R) was created by CRISPR/Cas9 methods. The resulting offspring and littermate controls were subjected to a battery of behavioral tests. A previously reported GOF mutant mouse knock-in (Gnao1+/G184S), which has not been found in patients, was also studied for comparison. We also tested the effects of multiple pharmacologic agents on the Gnao1 +/G203R mouse model. Gnao1+/G203R mutant mice are viable and gain weight comparably to controls. Homozygotes are 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. Movement phenotype in the Gnao1+/G203R model was exacerbated after administration of oxotremorine, a cholinergic agonist. However, treatment with a cholinergic antagonist did not alleviate motor impairment. Mice with a G184S GOF knock- 13 in also showed movement-related behavioral phenotypes but females were more strongly affected than males. Gnao1+/G203R mice phenocopy children with heterozygous GNAO1 G203R mutations, showing both movement disorder and a relatively mild epilepsy pattern. This mouse model should be useful in mechanistic and preclinical studies of GNAO1-related movement disorders. 14 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 in GNAO1 [19]. The GNAO1 gene has also been associated with early infantile epileptic encephalopathy 17 (EIEE17; OMIM 615473). However, 36% of patients showed both epilepsy and movement disorder phenotypes (G40R, G45R, S47G, I56T, T191_F197del, L199P, G203R, R209C, A227V, Y231C and E246G) [34]. GNAO1 encodes Gαo, the most abundant membrane protein in the mammalian central nervous system[35]. 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 receptor [35-38]. 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 cAMP and N-type calcium channels and activation of G-protein activated inward rectifying potassium channels (GIRK channels)[36]. Go is expressed mainly in the central nervous system and it regulates neurotransmitter release by modulating intracellular calcium concentrations in pre-synaptic cells [37]. It has also been suggested that Go plays a role in neurodevelopmental processes like neurite outgrowth and axon guidance [38, 39]. 15 Consequently, Go is an important modulator of neurological functions. In this report we began to assess differences in neurotransmitter levels within the brains of Gnao1 +/G203R mice. Previously, we defined a functional genotype-phenotype correlation for GNAO1 [19]. GOF mutations are found in patients with movement disorders, while loss-of-function (LOF) mutations are associated with epilepsy [19]. An updated mechanistic review of this genotype-phenotype correlation was recently published [34]. The experimental study of mutant alleles, however, was done with human GNAO1 mutations expressed in HET293T cells, which lack a complex physiological content. Therefore, it would be 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 preclinical testing model for possible new therapeutics. Previously, we studied heterozygous Gnao1+/G184S mice carrying a human-engineered GOF mutation (G184S). This mutation blocks the binding of the regulation of G protein signaling (RGS) proteins and results in GOF [8, 40]. Those mice showed heightened sensitization to pentylenetetrazol (PTZ) kindling and had an elevated frequency of interictal epileptiform discharges on EEG [41]. In this report, we tested whether the Gnao1+/G184S mice also exhibit movement disorders. The G184S is a GOF mutation but has not been found in human. G203R is a GOF mutation that is one of the more common GNAO1 mutations found clinically [10, 17, 33, 42, 43]. Most patients with this mutation exhibit both seizures and movement disorders [10, 17, 33, 42, 43]. We wanted to develop a mouse model with that mutation (Gnao1+/G203R) to see if it replicated the clinical phenotype of GNAO1 G203R-associated 16 neurological disorders. If so, it would be a valuable tool to understand neural mechanisms underlying the complex phenotypic spectrum of patients with GNAO1 mutations. In this report, we show that mice carrying two Gαo GOF mutations Gnao1+/G203R and Gnao1+/G184S have sex-specific motor impairment and seizures. We show that motor impairment can be extenuated with a nonselective cholinergic agonist. These two mouse models present the possibility of studying GNAO1-associated neurological defects in animal models. 17 Materials and Methods 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 and trained by the Michigan State University Institutional Animal Care and Use Committee. 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. Generation of Gnao1 mutant mice Gnao1+/G184S mutant mice were generated as previously described [8, 9, 11, 19] 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 (Fig 2-1A). Synthetic single-stranded DNA oligonucleotides (ssODN) were used as repair templates carrying the desired mutation and short homology arms (Table 2-1). 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 (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 [44], using a Genome Editor 18 electroporator (GEB15, BEX CO, LTD). C57BL/6NCrl embryos were implanted into pseudo- pregnant foster dams. Founders were genotyped by PCR (Table 2-1) followed by T7 endonuclease I assay (M0302, New England BioLabs) and validated by Sanger sequencing. Figure 2-1. 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 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. 19 Table 2-1. Location, sequence and genotyping of gRNA targets in Gnao1 locus DSB location gRNA target ssODN PCR primers Gnao1 G203R chr 8: 93,950,314 5’ TGCAGGCTGTTTGACGTCGG GGG 3’ (+) 5’ ATGGCCGTGACATCCTCAAAGCAGTGGATCCAC TTCTTGCGTTCAGATCGCTGGCCGCGGACGTCAAA CAGTTTGCAGGGAGTCAGGGAAAGCTGT 3’ Fwd: 5’ GACAGGTGTCACAGGGGATG 3’ Rev: 5’ TCCTAGCCAAGACCCCAACT 3’ PCR product = 462bp Genotyping SacII site created by G203R mutation 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 specificity scores from 0 to 100 (100=best) to rank gRNAs by specificity with respect to off-target modifications occurring [45-47]. The gRNA target used for this experiment has a specificity score of 94, which is the highest seen in over 40 similar targeting experiments done by the MSU Transgenic and Genomic Editing Facility. This greatly reduces the probability of off-target edits. After examining the off- target lists (S5 Table), 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 that we employed to deliver CRISPR reagents to mouse embryos further lowers the risk of off-target events [48]. Nevertheless, we directly validated several predicted off-target loci for the G203 gRNA target (TGCAGGCTGTTTGACGTCGG GGG) that occur within coding regions. One potential off-target site 20 with 4 mismatches and a score of 0.52 was validated for locus ENSMUSG00000041390. We also analyzed two other off-target candidates with 4 mismatches ENSMUSG00000086805 and ENSMUSG00000097637 and scores of 0.15 and 0.069 respectively. They were predicted to occur on the same chromosome (chr 8) as Gnao1. To test these 3 off-target candidates, DNA from WT and founder animals was 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). 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 stranded break. PAM – protospacer adjacent motif. Genotyping and Breeding 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 [49]. 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: 0.8µl template, 4µl 5x Promega PCR buffer, 0.4µl 10mM dNTPs, 1µl 10µM Forward Primer, 1µl 10µM Reverse Primer, 0.2µl Promega GoTaq and 12.6 µl DNase free water (Promega catalog # 21 M3005, Madison WI). Samples were denatured for 4 minutes at 95 oC then underwent 32 cycles of PCR (95 oC for 30 seconds, 60oC for 30 seconds, and 72 oC for 30 seconds) followed by a final extension (7 minutes at 72oC). After PCR, samples were incubated with Sac II restriction enzyme for 2 hrs. 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 2-2. Two female experimenters conducted all behavioral studies. Figure 2-2. 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. After completion of the motor behavior studies, animals were used for the PTZ kindling study. 22 Open Field The Open Field test was conducted in a 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), 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. 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 [50]. 23 Grip Strength Mouse grip strength data was collected following a protocol adapted from Deacon et al [51] 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. 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 10 g 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 [51]. The final score was normalized to the body weight of each mouse, which was measured before the trial. DigiGait Mouse gait data were collected using a DigiGait Imaging System (Mouse Specifics, Inc., Framingham, MA) [52]. The test is used for assessment of locomotion as well as the integrity of the cerebellum and muscle tone/equilibrium [53]. 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 [53]. For each speed, left & right paws were averaged for each animal while fore and hind paws were evaluated 24 separately. Stride length was normalized to animal body length. We eliminated data points at speed 36 cm/s since many mice cannot run at that speed, which increased the variability. Tests of Oxotremorine on Motor Behavior Male 8-12 week old mice Gnao1 +/G203R and Gnao1 +/+ littermates were allowed to habituate in the testing room for ten minutes. Mice were then treated with a single intraperitoneal (IP) dose of either 0.1mg/kg oxotremorine methiodide (Cayman Chemical) dissolved in DI water, or vehicle control. After twenty minutes they were evaluated for postural abnormalities every ten minutes using a scoring scale [54] (Table 2-2) Table 2-2. Scoring scale for postural and motor abnormalities Scoring scale 0. Normal Motor behavior 1. No gait changes, but slowed movement 2. Mild impairments: slow walk, occasional postural abnormalities 3. Moderate impairment: frequent abnormal postures, limited ambulation 4. Severe impairment: sustained abnormal postures with no ambulation or upright position 25 Tests of Trihexyphenidyl Treatment We tested the effects of trihexyphenidyl treatment on Gnao1+/G203R mice using our rotarod protocol. Male 8-12 week old mice Gnao1 +/G203R and Gnao1+/+ littermates were allowed to habituate in the testing room for ten minutes. On day one, mice were trained on the accelerating rotarod as normal. On day two mice, were then treated with a single intraperitoneal (IP) dose of either 5 mg/kg or 10 mg/kg trihexyphenidyl (Cayman Chemical) dissolved in DI water, or vehicle control. Ten minutes following injection mice were tested using the day 2 rotarod protocol. The third test on day two was analyzed. PTZ Kindling Susceptibility A PTZ kindling protocol was performed as described before [11] 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 signs of behavioral seizures as described [11, 55, 56]. 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 the kindled state 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. 26 Brain Neurotransmitter Analysis Gnao1 +/G203R Mice and their control littermates, ages 8-12 weeks old, were euthanized. Immediately following brain tissue was dissected and left hemisphere was collected, flash frozen until stored in -80 until samples were processed for HPLC. High-Performance Liquid Chromatography Measurements of monoamines was followed as described here [57]. Striatum and Cerebellum tissue was homogenized in 4 times volume of 0.1 m perchloric acid, centrifuged and then filtered through a 30 kDa tube. To analyze the filtrates, we used a HtuPLC system with a Coulochem III electrochemical detector set at -300 mV (Thermo Fisher Scientific; Waltham, MA, USA). Analytes were separated at 35˚C on a HR-80 reverse-phase column (Thermo Fisher Scientific) with Cat-A-Phase II (Thermo Fisher Scientific), a mobile phase with a flow rate of 1.1 mL min-1. Standards were run every 5th sample to confirm peak location on the chromatogram. The limit of detection for NE, DA, DOPA was 0.1 ng mL-1, for DA, and 0.5 ng mL-1 for 5-HT. HPLC data are reported as the mean ± SEM monoamine content in each tissue normalized to initial weight of sample. Amino Acid Neurotransmitters Amino acids were measured as there OPA/BME derivatives according to [58, 59]. OPA/BME stock solutions were prepared by dissolving 27mg OPA in 1 mL methanol, then 5ul of BME and 9 ml tetraborate buffer were then added. To make the working OPA/BME derivatizing reagent 2.5mL of the stock OPA/BME derivatizing reagent was mixed with 7.5ml Tetraborate 27 buffer. 50ul of the working reagent was combined with 20-25ul tissue extract which was deproteinized in 0.1M perchloric acid 1:10 (wet weight : acid). Stock solutions for amino acid standards were prepared in 50% for a level of 1mg/ml. Derivatization was performed by the 542 autosampler using the 4- line method. Line 1 contained 30ul from reagent, line 2 mix 4 cycles with 30ul line 3 wait 1 min line 4 End. We used a Waters Xterra MS Column. Mobile phase consisted of 100mM Disodium Hydrogen phosphate; 20% methanol; 3.5% acetonitrile and a mobile phase flow rate 0.6mL/min at 30˚C. Injection of 20ul in partial loop mode with 17ul flush volume. The detector used Model 5600A. HPLC data are reported as the mean ± SEM amino acid content in each tissue normalized to initial weight of sample. 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 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. 28 Results 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 2-1B & 2-1D) and they had relatively normal survival. There were two spontaneous deaths (~5-7 weeks) seen for Gnao1+/G203R mice out of 33 (Fig 2-1C). This is reminiscent of the spontaneous deaths seen previously with the Gnao1+/G184S GOF mutant mice [11]. Gnao1+/G203R mice did not exhibit any obvious spontaneous seizures or abnormal movements. Female Gnao1+/G184S and male Gnao1+/G203R mice show impaired motor coordination and reduced grip strength. Since GOF alleles of GNAO1 in children result primarily in movement disorder, we tested motor coordination in two mouse lines. One carried an engineered GOF mutant G184S, designed to block RGS protein binding [8, 40, 54]. The other is the G203R GOF mutant, which has been seen in at least 7 children (1, 2). First, we used a two-day training and testing procedure on the RotaRod (Figure 2-3A & B). 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 29 accelerating RotaRod (unpaired t-test, p<0.001, Figure 2-3A) 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 2-3B) while female Gnao1+/G203R mice did not show any abnormalities. Results from all the RotaRod training and testing sessions are shown in S1 Fig. Neither Gnao1+/G184S nor Gnao1+/G203R mice showed a significant difference in learning rate on RotaRod (S3 Fig), suggesting that the differences we observed in the RotaRod study were due to movement deficits rather than learning difficulties. Grip strength was assessed as described [51]. This test is widely done in combination with the RotaRod motor coordination test. This may be relevant to the hypotonia, seen in many GNAO1 patients [14-18, 23, 33, 60-67]. Similar to the RotaRod results, female Gnao1+/G184S mice also showed reduced forepaw grip strength compared to their littermate controls (unpaired Student’s t-test, p<0.05, Figure 2-3C) while males did not exhibit a significant difference (Figure 2-3C). In contrast, both male and female Gnao1+/G203R mice displayed reduced forepaw grip strength (unpaired t-test, p<0.05, Figure 2-3D). 30 Figure 2-3. 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 body weight. (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. 31 Gnao1+/G184S mice show reduced activity in the open field arena. 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 [68], however, environmental salience may reduce the impact of the motor impairment on behaviors [69]. Therefore, we divided the 30-min open field measurements into two periods, with the first 10 minutes assessing activity in a novel environment and the 10-30 minute period designated as sustained activity (Figure 2-4C & 2-4D). 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 2-4C). Female, but not male, Gnao1+/G184S mice showed reduced activity in the sustained phase of open field testing (Figure 2-4C, 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, Fig 4A & 4C). Neither male nor female Gnao1+/G203R mice performed differently in the open field arena compared to their littermate controls (Figure 2-4B & 2-4D). No significant difference was observed in the time mice spent in the center of the arena (S2 Fig). 32 Figure 2-4. 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 a Novelty (0-10 min) and a Sustained (10-30 min) period. (A) Representative heat map of overall activity comparing Gnao1+/+ and Gnao1+/G184S mice of 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 of 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. 33 Female Gnao1+/G184S mice and male Gnao1+/G203R mice exhibit markedly abnormal gaits. 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 [70, 71]. The multiple parameters assessed in DigiGait allow it to pick up subtle neuromotor defects and make 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 (S2-4 and S2-5 Fig, S2-1-S2-4 Tables). Gnao1+/G184S female mice showed 22 significant differences (Q<0.01) and males showed 8 (S2-4 Fig, S2-3 and S2-4 Table). 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 (S2-5 Fig, S2-1 and S2-2 Table). 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, Fig 2-5A) and increased paw angle variability (2-way ANOVA, p<0.0001, Fig 2-5E) 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 (Fig 2-5C & 2-5G). These results are consistent with the results of RotaRod and grip strength measurements in that female 34 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, Fig 2-5D) and increased paw angle variability (2-way ANOVA, p<0.05, Fig 2- 4H). In contrast, female Gnao1+/G203R mice did not show any significant differences in stride length or paw angle variability (Fig 2-5B & 2-5F). 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, Fig 2-5J). For reasons that are not clear such a difference was not seen for Gnao1+/G184S mice (Fig 2-5I). 35 Figure 2-5. 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 not different 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 36 showed significantly 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). Male Gnao1+/G203R mice are sensitized to PTZ kindling. Epilepsy has been observed in 100% of patients with GNAO1 G203R mutations [10, 17, 33, 34, 42, 43]. Also in the Gnao1+/G184S GOF mutant mice, we previously reported spontaneous lethality as well as increased susceptibility to kindling by the chemical anticonvulsant PTZ for both males and females [11]. Kindling is a phenomenon where a sub-convulsive stimulus, when applied repetitively and intermittently, leads to the generation of full-blown convulsions [72]. 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 of the mice kindled at 4 and 8-10 injections for females and males, respectively (Fig 2-6A & 2-6B). 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 (Fig 2-6A). On the contrary, male Gnao1+/G203R mice were more sensitive to PTZ kindling than controls (Fig 2-6B, 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 [11]. We cannot, however, attribute those deaths to seizures at this point. 37 . Figure 2-6. Gnao1+/G203R male mice have an enhanced Pentylenetetrazol (PTZ) Kindling response (A) Female Gnao1+/G203R mice did not show heightened sensitivity to PTZ injection. (B) Male Gnao1+/G203R mice developed seizures earlier than WT littermates after repeated PTZ injections (Mantel-Cox Test; p<0.05). Gnao1+/G203R mice show increased sensitivity to oxotremorine Gnao1+/G203R mice do not display any overt movement or postural abnormality at baseline. However, previous studies used oxotremorine, a muscarinic cholinergic agonist, to induce movement abnormalities in a LOF Gnal model [73]. The GNAL gene, which is linked to dystonia, encodes Gaolf an isoform of Gas which functions antagonistically of Gao to stimulate AC to 38 produce cAMP. GNAL LOF mutations decrease cAMP similar to GOF GNAO1 mutations. Therefore, we reasoned that Gnao1 +/G203R mice would display a similar phenotype. In response to 0.1 mg/kg of the cholinergic agonist oxotremorine, wildtype mice displayed some abnormal movements, characterized by slow movements and some abnormal postures. (Figure 2-7). Gnao1 +/G203R mice also displayed higher abnormal movement scores than vehicle control groups., however, at thirty minutes post injection Gnao1 +/G203R mice displayed a higher abnormal movement score compared to wildtype mice treated with oxotremorine. This phenotype was mainly characterized by the mouse standing vertically on hindlimbs for sustained periods of longer than >10s. Figure 2-7. Induction of abnormal movements and postures by oxotremorine (A) Starting at 20 minutes following injection of cholinergic agonist, oxotremorine, behavior was scored for abnormal movements and postural differences. Oxotremorine treated WT and Gnao1 +/G203R male mice show higher abnormal movement scores than vehicle control littermates. Gnao1+/G203R mice show higher sensitivity to oxotremorine at T=30 compared to WT mice. 2-Way ANOVA with Bonferonni correction: p<0.0001 **** , p<0.001 ***, p < 0.01**, p < 0.05 * 39 Trihexyphenidyl treatment on movement disorders Patients with GNAO1 G203R mutations have shown some benefits to oral therapeutics (Table 2-3) including trihexyphenidyl (THP), a cholinergic antagonist. As Gnao1 +/G203R mice displayed a greater sensitivity to oxotremorine, a cholinergic agonist, we had reason to believe the Gnao1+/G203R motor coordination impairments displayed on the rotarod would be alleviated following administration of trihexyphenidyl. In initial pilot studies of a single 5 mg/kg or 10 mg/kg doses of THP, both WT and Gnao1+/G203R mice showed small but non- significant reductions in rotarod times Gnao1 +/G203R mice (Figure 2-8.). Table 2-3. GNAO1 G203R patient classification 40 Figure 2-8. Treatment with trihexyphenidyl on Gnao1+/G203R mice Thirty minutes following injection of cholinergic antagonist, trihexyphenidyl, motor coordination was assessed on the accelerating rotarod. Trihexyphenidyl treatment showed no alleviation of motor abnormalities on the rotarod. 2-Way Anova: non-significant Neurotransmitter levels GNAO1 functions to regulate neurotransmitter release through multiple mechanisms[19]. Certain neurotransmitters, such as glutamate and γ-aminobutyric acid (GABA), and dopamine are shown to play a direct role in regulating movement control [74]. We reasoned that Gnao1+/R209H would show differences in neurotransmitter levels. To test this, we analyzed the left hemisphere of the brains of Gnao1 +/G203R mice for catecholamine and amino acid neurotransmitter levels via HPLC. The amino acid neurotransmitters GABA and glutamate showed no significant differences between mutant and wildtype mice in either group (Figure 2-6A and Supplemental Figure 2-6). Compared to wildtype mice we also saw no significant differences between L-3,4- 41 dihydroxyphenylalanine (DOPA), dopamine, and 5-HT. However, there was a significant increase in the amount of norepinephrine within the brains of Gnao1+/G203R mice (Figure 2-8). Figure 2-9. Brain neurotransmitter analysis of Gnao1+/G203R mice (A-C,E&F) HPLC analysis of brain DOPA, dopamine, 5-HT and amino acid neurotransmitters GABA and glutamate within the left hemisphere of Gnao1+/G203R showed no significant differences compared to WT littermates. Students unpaired t-test showed brain levels of norepinephrine had significantly higher levels within Gnao1+/G203R mice compared to WT littermates. 42 Discussion In this report, we describe the first mouse model carrying a human GNAO1 mutation associated with disease and we provide evidence to support the concept that GOF mutations are associated with movement disorder [19]. Heterozygous mice carrying the G203R mutation 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 choreoathetotic movements [10, 17, 33, 34, 42] . Also, we examined a possible movement phenotype in mice carrying the RGS-insensitive GOF mutant (Gnao1+/G184S) that we reported previously to have a mild seizure phenotype [11]. This mutation has not been reported in humans to our knowledge. As predicted from our mechanistic model [19, 34], the Gnao1 G184S mutant mice also show movement abnormalities. 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 [75, 76], however they are often informative about mechanism and therapeutics. For the patient-derived Gnao1+/G203R mutant mouse, neither the seizure propensity nor the movement abnormality was obvious without a stress being applied. Male Gnao1+/G203R mice showed decreased motor ability on RotaRod, decreased fore paw strength, and gait abnormalities at higher speeds of walking/running. No spontaneous seizures were observed but there was a substantial increase in sensitivity to PTZ- induced seizures in the kindling model in males. This very closely replicates the mild seizure phenotype of female Gnao1+/G184S mice [11]. We now show that the female Gnao1+/G184S mice also exhibit gait and motor abnormalities. 43 Both the GNAO1 G203R and the G184S mutations show a definite but modest GOF phenotype in biochemical measurements of cAMP regulation [19]. In each case, the maximum percent inhibition of cAMP is not greatly increased but the potency of the α2A adrenergic agonist, used in those studies to reduce cAMP levels, was increased about 2-fold. This effectively doubles signaling through these two mutant G proteins at low 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, of which the GNAO1 gene product, Gαo, is the defining subunit, can signal to many different effector mechanisms [34, 38, 77] We recently reviewed the mutations associated with genetic movement disorders and identified both cAMP regulation and control of neurotransmitter release as two GNAO1 mechanisms that seem highly likely to account for the pathophysiology of GNAO1 mutants [34]. Since many Go signaling effectors (including cAMP and neurotransmitter release) can be mediated by the Gβγ 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 Gαo may be involved [78]. 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. We observed a striking sex difference in the phenotypes of our two 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. Only G184S mutants showed significant changes in 44 open field tests while only the G203R mutants showed the striking reduction in ability to walk/run at higher treadmill speeds. For both mutant alleles, the seizure phenotype was also worse in the sex with the more prominent movement disorder. GNAO1 encephalopathy is slightly more prevalent (60:40) in female than male patients [34]. It is not uncommon to have sex differences in epilepsy or movement disease progression. One possible explanation is that estrogen prevents dopaminergic neuron depletion by decreasing the uptake of toxins into dopaminergic neurons in Parkinson’s disease (PD) animal model induced by neurotoxin [79]. The Gi/o coupled estrogen receptor, GPR30, also contributes to estrogen physiology and pathophysiology [80]. PD is more common in male than female human patients [81], therefore, the pro-dopaminergic properties of estrogen may exacerbate conditions mediated by hyper-dopaminergic symptoms like chorea in Hungtington’s disease [HD; 79]. Chorea/athetosis is the most prevalent movement pattern seen in GNAO1-associated movement disorders [34] 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 [82], but it is beyond the scope of this report to explain how the molecular differences contribute to the distinct behavioral patterns. Here we show that Gnao1 GOF mutation G203R impairs motor coordination however the model does not display any obvious movement abnormalities at baseline. We were able to show that Gnao1+/G203R mice displayed overt postural and motor abnormalities consistent with patient movement disorders after a dose of oxotremorine, a cholinergic agonist. We also showed the Gnao1+/G203R had an increase susceptibility to oxotremoine at 30 minutes, compared to WT mice. However, our model showed no reprieve or motor abnormalities on the rotarod following injection of OXO that has proved efficacious in one of the patients with G203R variant. A possible 45 explanation is the receptor selectivity between the drugs. Oxotremorine is a nonselective muscarinic receptor agonist, trihexyphenidyl however is selective to M1 receptors antagonism. Therefore, it is likely that M1 is not the receptor of interest in the GNAO1 mechanism. Gαo proteins have been known to couple to M2 receptors. It would be interesting to test an M2 selective cholinergic antagonist in our model. Since GNAO1 encephalopathy is often associated with developmental delay and cognitive impairment [34], it would be interesting to see whether the movement phenotype we have seen in female Gnao1+/G184S and male Gnao1+/G203R mice is due to a neurodevelopmental malfunction or to ongoing active signaling alterations. Go coupled GPCRs play an important role in hippocampal memory formation [83, 84]. 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 mutant mouse model 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. In this study we began initial tests looking at neurotransmitter levels with whole brain of Gnao1 +/G203R mice. We only showed significant differences in the amount of norepinephrine. Elevated norepinephrine has been heavily linked to being anticonvulsant, however there is also evidence for norepinephrine being proconvulsant as well [85]. To test if the high elevation of NE might be a mechanism behind the kindling phenotype observed in our model it would be interesting to test our PTZ model following administration of propranolol, an adrenoreceptor antagonist blocking the effects of 46 norepinephrine. While these results might explain the seizure phenotype they do not explain the movement disorder. In regards to this we hypothesized that neurotransmitters associated with movement control, such as dopamine or GABA and glutamate, would be decreased in the brain tissue of the Gnao1 +/G203R mice. While we did not see these in our analysis, we still can not rule this out. Gao is ubiquitously expressed within the brain but evidence shows it is very abundant within the striatum and cerebellum[1]. Both of these regions are widely known to be involved in motor control. It is possible in our analysis using the whole brain was too generalized. Further studies should be done to look at the more specific regions relevant to our model. Taken together this animal models can be used to further study mechanisms of GNAO1 associated epilepsy and movement disorders. While we did not show THP as an effective treatment this model can still be used for preclinical drug testing and may permit a true allele- specific personalized medicine approach in drug repurposing for the associated movement disorders. 47 CHAPTER 3: BEHAVIORAL ASSESSMENT OF MICE WITH A COMMON GNAO1 MOVEMENT DISORDER VARIANT R209H 48 Statement of Contribution In the following chapter my role was the following. I planned, performed and analyzed the open field, rotarod, grip strength and risperidone studies for the Gnao1+/R209H mouse model. The experiments on developmental milestones were performed by Alex Roy. For all of the studies mentioned above I performed the formal data analysis as well. The data curation and formal analysis for the digigait, kindling studies and western blot experiment was performed by Huijie Feng. Genotyping and breeding of mice was done by Jefferey Leipprandt. Elena Demireva and Huirong Xie at the MSU transgenic core generated the mutant mouse model with CRISPR/cas9 technology. The following chapter was put together and written by myself with edits from Elena Demireva, Huijie Feng and Richard Neubig. 49 Abstract Neurodevelopmental delay with involuntary movements (NEDIM) is characterized by a delay in psychomotor development, hypotonia and early onset of hyperkinetic involuntary movements. Heterozygous de novo mutations in the GNAO1 gene are the cause of NEDIM in patients. Gao, the gene product of GNAO1 is the alpha subunit of the heterotrimeric Gi/o family of G-proteins. It is abundantly found throughout the brain. However, the pathophysiological mechanisms linking Gao functions to GNAO1 clinical manifestations are still poorly understood. In order to begin to understand why mutations in GNAO1 cam cause NEDIM, models need to be validated as predictive. Heterozygous GNAO1 R209H mutant mice were created using CRISPR/Cas9 to assess whether the mice could replicate aspects of NEDIM clinical patterns. The R209H mutation altered development, increased locomotor activity, and displayed gait differences. This allowed us to explore possible treatments. One drug that has proven effective in a patient with the R209H mutation is risperidone, an atypical neuroleptic. Here we showed that administration of risperidone alleviated the hyperlocomotion observed in our animal model. The present results show that Gnao1+/R209H mice mirror some aspects of the patient phenotype but also mirror a response to a pharmacological agent. 50 Introduction Gao is the alpha subunit of the heterotrimeric G-protein and is the most abundant heterotrimeric G protein in brain, comprising 1% of the mammalian brain membrane protein. Therefore, it is no surprise that mutations in its gene, GNAO1, have been linked to neurological conditions. Since an initial report in 2013 [87] when four children with epileptic-encephalopathy- were identified with mutations in GNAO1, a growing number of clinical cases of patients presenting with epilepsy and movement disorders have been found to exhibit de novo mutations in the gene encoding the protein Gao (GNAO1). To date there are over 70 clinical cases of children with mutations in GNAO1 presenting with early infantile epileptic encephalopathy (EIEE17; OMIM 615473) and/or neurodevelopmental disorder with involuntary movements (NEDIM; OMIM 617493)[13-18, 23, 24, 33, 42, 43, 61-66, 87-101] There have been forty-three pathological variants of GNAO1 reported. Our lab has previously classified these variants by the ability of the mutated Gao proteins to support inhibition of cAMP production [19]. Gao proteins with functioning mutations, which inhibit cAMP normally or even more efficiently, are associated with movement disorder patients. Non- functioning mutants, those that showed less cAMP inhibition, are associated with epilepsy patients [19]. Recently our lab created a mouse model with a GNAO1 GOF mutation, G203R, that was identified in patients who showed both epilepsy and movement disorders. As predicted, the mice exhibited with motor coordination and gait abnormalities as well as enhanced seizure susceptibility in pentylenetrazol (PTZ) kindling studies. While this model’s predictivity is a useful 51 tool in understanding the phenotypic spectrum of GNAO1 disorders, there are other more commonly seen variants being identified. The R209H mutations is one of the most commonly seen mutations in the clinical cases [24]. Patients with de novo R209H mutations display severe choreoathetosis and dystonia but not a seizure phenotype [13, 18, 23, 62, 91, 93]. Interestingly, the R209H mutation was classified as having normal function in cAMP inhibition, however, it still causes a severe form of movement disorder in patients, often requiring intensive care unit admission [23, 91]. The fact that our initial analysis classified R209H as a normal functioning mutation while it is pathological clinically implies our initial in vitro functional readout was not sufficient to fully predict the clinical outcome. Therefore, this specific mutation R209H is a good choice for us to expand our initial study. Heterozygous mutant mice (Gnao1+/R209H) were created on C57BL/6J background with CRISPR/Cas9. Using a battery of behavioral tests, we analyzed the mice to determine the phenotypic correlation between our mouse model and the human patients. There is a wide heterogeneity of movement disorders patterns of patients [5] therefore we use a battery of tests to measure motor skills. Our previous model, Gnao1+/G203R displayed motor abnormalities on a few behavioral tests but they did not show significant differences on the open field test. Here we show that Gnao1+/R209H mice of either sex displayed significant hyperactivity during the open field assessment. This difference in model phenotype may account for differences in specific motor disorders of patients. Gnao1+/R209H mice also did not show enhanced seizure susceptibility to PTZ kindling studies. While expected, these findings are promising as patients with that same mutation display hyperactive movement disorders but do not express a seizure phenotype. 52 Having a model that displays hyperactivity while lacking seizure susceptibility expands on our previous model and shows similarities to patients with the same mutation who also lack seizure disorders while exhibits hyperactive movement disorders. Having this new model allowed us to begin allele-specific preclinical drug testing. The neuroleptic risperidone was reportedly beneficial in a patient with R209H [23]. Here we show that risperidone also attenuates the hyperactivity of our animal model. This implies that risperidone treatment may be beneficial for other GNAO1 patients with the R209H mutation with hyperkinetic movement disorders. 53 Materials and Methods Animals Gnao1 +/R209H mice on a C57BL/6J background were generated in the MSU transgenic core. Mice (8-12 weeks old) were housed on a 12-hour light/dark cycle, with ad libitum access to food and water. All experiments were performed in accordance with NIH guidelines and protocols were approved by the Michigan State University Institutional Animal Care and Use Committee. Generation of Gnao1 R209H edited mice Mutant Gnao1 +/R209H mice were generated using CRISPR/Cas9 genome editing on a C57BL/6J genomic background. CRISPR gRNA selection and locus analysis was 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. Single stranded oligodeoxynucleotide (ssODN) carrying the R209H mutation CGC > CAC with short homology arms was used as a repair template (Figure 3-1 and Table 3-1). Ribonucleoprotein (RNP) complexes consisting of crRNA/tracrRNA and Alt-R® S.p. Cas9 Nuclease V3 (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 2017, 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. 54 Table 3-1. Location and sequence of gRNA and ssODN template for CRISPR-Cas targeting Gnao1 locus; primers and genotyping method for Gnao1+/R209H mice Gnao1 R209H Chr 8: 93,950,334 5’ AGCGATCTGAACGCAAGAAG TGG 3’ GTTTCGTCCTCGTGGAGCACCTGGTCATAGCCGCTGAGTGCGAC ACAGAAGATGATGGCCGTGACATCCTCAAAGCAGTGGATCCACTTCTTG tGTTCAGATCGCTGGCCCCCGACGTCAAACAGCCTGCAGGGAGTCAGGG AAAGCTGTGAGGGCGGGGACGCCTA O586 FWD: 5' GGACAGGTGTCACAGGGGAT 3’ O587 REV: 5' ACTGGCCTCCCTTGGCAATA 3' By Sanger Sequencing Location gRNA target 5’ N20-PAM -3’ ssODN template (reverse complement) PCR primers Genotyping Figure 3-1. 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 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 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. 55 Genotyping and Breeding Studies were done on N1 R209H heterozygotes with comparisons to littermate controls. To generate Gnao1 +/R209H heterozygotes N1 backcrosses, 2 founder Gnao1 +/R209H mice, 1 male and 1 female, were crossed with C57BL/6J mice. DNA was extracted by an alkaline method (26) ear clips done before weaning. PCR products were generated with primers flanking the mutation site (Fwd 5' GGACAGGTGTCACAGGGGAT 3’; 5' ACTGGCCTCCCTTGGCAATA 3'). To produce a X base pair (bp) product reaction conditions were: 0.8 μ l template, 4 μ l 5x Promega PCR buffer, 0.4 μ l 10mM dNTPs, 1 μ l 10 μ M Forward Primer, 1 μ l 10 μ M Reverse Primer, 0.2 μ l Promega GoTaq and 12.6 μ l DNase free water (Promega catalog # M3005, Madison WI). Samples were denatured for 4 minutes at 95C 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). Developmental Milestone Assessment: All tests described below were performed on Gnao1 +/R209H and Gnao1 +/+ littermates of either sex on 5-12 days of age (P5,7,10,12). Tests were done in groups so all mice finished one test before the next was performed. Protocols were established based on Feather-Schussler et al [102]. These studies were performed by a male and female researcher. Ambulation: Mice are placed in a clear enclosure where mice are visible from the top as well as the side. For one minute the mice were evaluated and given a score of 0-3. Score of 0 56 was given if no movement was observed, a score of 1 or 2 was given if slow walking was observed with asymmetric or symmetrical movements respectively. A score of 3 represented fast crawling or walking. Gentle prodding by touching the pup's tail was used motivate the pup to walk. Righting: Mice were placed on their backs and held in position for 5 seconds. After releasing pups, the time taken return to prone position was recorded, with a maximum time of 1 minute. Three trials were done, and the average was taken for analysis. Negative Geotaxis: Mice are placed pointed downward on a 45° incline and held for 5 seconds. The time taken for the mice to face upwards was recorded. The maximum testing time was 2 minutes. Three trials were done, and the average was taken for analysis. Mice that fell down the incline or failed to turn were re-tested two additional times. Failure to turn resulted in a score of 2 minutes. Grip strength/Hang time: Mice are placed on a piece of mesh on top of a flat adjustable surface. The Mesh screen is slowly inverted and the approximate angle of the screen when the pup falls off is recorded. If the mouse holds on to the mesh screen until fully inverted, latency to fall is recorded. Three trials were done, and the average was taken for analysis. Cliff Aversion: Mice are placed on the top edge of a box so that their forepaws, digits and snout are over the edge. The time that it takes mice to move from the edge is recorded. This test is repeated 3 times for a maximum of 30 seconds a trial. If the pup does not move away from the edge within 30 sec, a score of 30 seconds is recorded. If the pup falls off the edge, a single additional trial can be performed. 57 Adult Behavioral Assessment Between 8-12 weeks of age male and female Gnao1 +/R209H mice and their Gnao1 +/+ littermates underwent a battery of behavioral testing to assess motor phenotype as described previously [103]. Before each experiment, mice were acclimated for 10 minutes to the testing room. Experiments were performed by two female researchers. Open Field The open field test is frequently used to assess locomotion, exploration and anxiety [68, 104]. The test was conducted in the Fusion VersaMax clear 42 cm x 42cm x 30cm arenas (Omnitech Electronics, Inc. Columbus, Ohio). Gnao1 +/R209H mice of either sex and littermates were placed in the arena for 30 minutes. Using the Fusion Software, we evaluated distance traveled (cm) in terms of novelty, sustained, and total movement corresponding to the first 10 minutes, 10-20 minutes and total of 30 minutes, respectively. As a potential measure of anxiety, the fraction of time spent in the center was assessed. The center area was defined as the 20.32cm x20.32cm area within the middle of the arena. Rotarod To assess motor skills in Gnao1 +/R209H mice we used the Economex accelerating rotaRod (Columbus Instruments OH). The entire protocol occurred over a two-day period. Day 1 mice were trained on across three-2 min training sessions, with 10 minutes between each training trial. The first two sessions the rotarod maintained a constant rotational speed of 5 rpm, while the third training trial started at 5 rpm and accelerated 0.1rpm/sec throughout the 2 minutes. 58 The following day mice ran three more trials, two 2 min trials and a final 5 min trial, with a 10 min break in between. Each of these tests started at 5rpm with constant acceleration of 0.1 rpm/sec. For all training and test trials latency to fall off the spindle was recorded. Grip Strength To assess mouse grip strength, we used seven home-made weights (10, 18, 26, 34, 42, 49, 57 grams). The mouse was held by the middle/base of the tail and lowered to the weight once the mouse grasped the weighted ring with its forepaws the mouse was lifted until weights cleared the bench. For each weight a mouse was given up to three trials to suspend the weight for 3 seconds. If cleared the next heaviest weight was tried, otherwise total time and maximum weight lifted was recorded. Protocol and calculated score was adapted from [51], and normalized to mouse body weight which was measured the day of the test. DigiGait Mouse gait analysis was performed on the DigiGait apparatus (Mouse Specifics, Inc, Framingham, MA). After acclimation, each mouse was subject to run at speed 18, 20, 22, 25, 28, 32, 36 cm/s on DigiGait for 10 sec with a video camera located at the bottom of the belt. There was a 5 min rest between each speed. Then all the recordings were analyzed with DigiGait analysis program. 59 PTZ Kindling Study A PTZ kindling protocol was performed as described [11, 103] to assess mouse susceptibility to epilepsy. Mice were injected with 40mg/kg PTZ (i.p.) every other day and observed for 30 minutes and scored for 24 days. Kindling, which was defined as tonic-clonic seizures on two consecutive injection days or death, marked the end of the study for each animal. Tests of Risperidone on motor behavior Naïve 8-12 week old Gnao1 +/R209H and Gnao1 +/+ littermates of either sex were tested for effects of risperidone on their hyperactivity. The entire test occurred over 5 days; 3 days of testing with a break in between each testing day. On Monday mice underwent the open field protocol as described above to establish baseline. Wednesday mice were habituated in the experimental room for 10 min then given a singly i.p. dose of either 2mg/kg, 0.5mg/kg risperidone (Cayman Chemical) or vehicle control. Risperidone was prepared by dissolving in DMSO at a concentration of 5mg/ml, Further dilutions were done in DI water. 30 minutes following injection mice were placed in the open field arena for a 30-minute testing time. On Friday mice underwent the same open field protocol as Monday, this was done to assess locomotor activity following risperidone depletion. SDS-PAGE and Western Blots Mice between 6 to 8 weeks old were sacrifice and their brains were dissected into different regions and flash-frozen in liquid nitrogen. For western blots analysis, tissues were thawed on ice and homogenized with 0.5mm zirconium beads in the Bullet Blender (Next 60 Advance; Troy, NY) in radioimmunoprecipitation assay buffer (RIPA buffer) with protease inhibitor. Sample homogenates were centrifuged for 5 min at 4°C at 13,000 G. Tissue lysates were then moved to a new tube and protein concentrations were 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) as a reducing agent. For all samples and tissue types, 30 µg of protein was loaded onto a 12% Bis- Tris gradient gel, and samples were separated by running the gel for 1.5 hrs at 160V. Samples were then transferred to an Immobilon-FL PVDF membrane (Millipore, Billerica, MA) for 2 h at 100 V, 400 mA or overnight at 30V, 50mA on ice and subjected to Quantitive Infrared Western immunoblot analysis. Immediately after transfer, PDVF membranes were washed and blocked in Odyssey PBS blocking buffer (Li-Cor) 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) 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 final rinse in PBS for 5 min. The membrane was kept in the dark and the infrared signals at 680 and 800nm 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). 61 Statistical Analysis This data was analyzed with the unpaired Students t-test, Mantel-Cox, two-way ANOVA with Bonferroni corrections. All analysis was done using Graphpad Prism 7.0 (GraphPad; La Jolla, CA). Multiple comparison correction of the dataset from DigiGait was performed as described before [103]. A p < 0.05 was considered critical value for significant throughout the entire study. Detailed discussion can be found within figure legends. 62 Results Gnao1 +/R209H mice have expected frequency and normal viability Out of 98 offspring, 51 heterozygotes and 47 WT were observed between a cross of Gnao1+/R209H with WT mice. Gnao1+/R209H mice exhibit no overt postural abnormalities at basal conditions. Weights between adult mice showed no statistically significant differences between WT and Het of either sex. Gnao1 +/R209H Mice display delayed development of milestones on assessments of negative geotaxis As children with GNAO1 exhibit motor developmental delay and intellectual delay [18, 23, 62, 93], we assessed mice pups between P4 to P12 for neonatal motor deficits. Gnao1 +/R209H mice exhibited a reduced time to prone position at P7 during the negative geotaxis test compared to their wildtype littermates (Figure 3-2C). This might suggest a motor coordination delay which is consistent with patient observations. The model showed no significant differences in ambulation, righting reflex, cliff avoidance, and hang time (Figure 3-2A, 3-2B, 3-2D). 63 Figure 3-2. Gnao1+/R209H exhibited a developmental delay during the negative geotaxis assessment (A-C) Gnao1+/R209H mice do not exhibit significant delays in the cliff aversion, righting or hang time tests. (C) Gnao1+/R209H exhibit a delay on P7 compared to wildtype littermates (2- way ANOVA with Bonferroni multiple comparison post-test). 64 Gnao1 +/R209H of either sex have hyperactive phenotype in the Open Field arena Patients with R209H mutation present with hyperkinetic movement disorders[18, 23, 62, 93]. In order to see if our mouse model phenocopied patients of the same mutation, Gnao1 +/R209H mice were subject to a battery of behavioral tests. Open field arena was used to test overall locomotion activity. It was reported that novel environments may overshadow potential behavioral impairments [69]. To account for this, we divided the test into two sections, novelty as the first 10 minutes then minutes 10-30 as sustained time. Gnao1 +/R209H mice of both sexes showed significantly increased activity in the novel period compared to their wildtype littermates. However, both male and female Gnao1+/R209H displayed significant hyperactivity in the sustained period of the open field test as well (Figure 3-3B). This suggests the observed hyperkinetic movements are due to strain differences but not environmental salience. Additionally, the open field test may be used to assess anxiety-like behaviors. In a measure for anxiety-like behavior male and female Gnao1+/R209H mice also displayed reduced time in center (Figure 3-3B). An accelerating rotarod was used to asses motor coordination and balance. Neither male nor female Gnao1+/R209H mice display an impaired performance (Figure 3-3C). Grip strength, which is used to assess for differences in neuromuscular tone showed no differences between Gnao1 +/R209H and wildtype littermates in either sexes (Figure 3-3D). 65 Figure 3-3. Gnao1 +/R209H shows significant hyperactivity and reduced time in center in the open field arena (A) Representative heat maps show Gnao1 +/R209H comparing time Gnao1 +/+ and Gnao1+/R109H (B) Time spent in the open field arena was separated by time of 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 increase in distance traveled (cm) (2- way ANOVA; ****p < 0.0001, ***p < 0.001, * p < 0.05). Gnao1 +/R209H Mice of both sex spend less time in center areas of the open field arena compared to wildtype littermates. (C) Male nor female Gnao1 +/R209H mice show significant differences on the rotarod. (D) There is no significant difference between grip strength between wildtype and Gnao1+/R209H mice. Data are shown as mean ± SEM. 66 Male Gnao1+/R209H mice display reduced stride length Gait patterns were assessed using DigiGait analysis. Male Gnao1+/R209H mice showed reduced stride length compared to wildtype littermates (P<0.001, 2-way ANOVA). Female Gnao1+/R209H mice do not show significant differences from WT (Figure 3-4C & 3-4D). However, the female Gnao1+/R209H showed a significantly reduced maximum speed to run on the treadmill (Figure 3-4E). 67 Figure 3-4. 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 remain normal. (C & D) Neither male or female Gnao1 +/R209H exhibited significant differences in stride length compared to wildtype littermates. (E) at speeds greater than 25 cm/s Female Gnao1 +/R209H shows reduced ability to run on a treadmill 68 Gnao1 +/R209H mice are not sensitive to PTZ kindling Repeated application of a sub-threshold convulsive stimulus, leads to the generation of full-blown convulsions [72]. GNAO1 variants differ in their ability to cause epileptic seizure, GNAO1+/R209H patients do not exhibit seizure disorders [18, 23, 62, 93]. In accordance with the patients’ symptoms, Gnao1+/R209H mice of neither sex showed increased susceptibility to epileptic seizures (Figure 3-5 A&B). Figure 3-5. Gnao1 +/R209H mice do not have an enhanced Pentylenetetrazol (PTZ) kindling response (A&B) Neither Male or female Gnao1 +/R209H mice showed significant differences in sensitivity to PTZ injection compared to wildtype littermates. Mantel-cox test ns 69 Risperidone treatment attenuated the hyperactivity of Gnao1+/R209H mice Patients with GNAO1 mutations were tested with multiple treatments to alleviate motor symptoms, (Table 3-2). Risperidone, an atypical antipsychotic drug showed effects in one of the patients. In the literature, risperidone has also been shown to control drug-induced dyskinesia [105]. We show that Gnao1+/R209H mice exhibit complete abrogation of movement at 2mg/kg risperidone, which recovers after 2 days (Figure 3-6A&C). WT mice also show a significant decrease in locomotion after 2mg/kg risperidone treatment (Figure 3-6A). After a single 0.5mg/kg dose of risperidone both WT and Gnao1+/R209H mice exhibit a decrease in locomotion (Figure 3- 6B). As expected, hyperactivity of mutant mice was observed during baseline testing on day 1. The hyperactivity returned following a 2-day washout period (Figure 3-6C). Neither 2.0 mg/kg nor 0.5 mg/kg selectively affected Gnao1+/R209H as assessed by percent suppression (Supplement Figure 3-1). 70 Table 3-2. GNAO1 R209H patient classification Patient No. Sex Amino Acid Change Age of Onset Presence of Epilepsy Movement Disorder Treatment Motor Developmental Delay(MDD)/Intellectual Delay(ID) M R209H 17 mo M R209H M R209H M R209H 2 y 3 y 1 y M R209H 10 mo M R209H 15 mo - - - - - Chorea DBS MDD Chorea DBS MDD Chorea Risperidone, BZD MDD/ID Chorea NA MDD/ID Chorea Dystonia TBZ, THP MDD/NA Chorea, Dystonia DBS F R209H 6 mo - Dystonia NA F R209C NA NA Chorea NA MDD/ID MDD/MID MDD/ID F R209G 3 y M R209L 2 y - - Chorea None MDD/ID Chorea NA MDD/ID 71 1 2 3 4 5 6 7 - - - Reference Kulkarni et. al (2016) Kulkarni et. al (2016) Anath et. al (2016) Menke et al (2016) Dhamija et al (2016) Marecos et al (2018) Kelly et al (2018 Saitsu et al (2016) Anath et al (2016) Menke et al (2016) Figure 3-6. Risperidone treatments decreases hyperkinetic movements in Gnao1 +/R209H (A) Gnao1+/R209H mice show complete abrogation of movement compared to vehicle treated Gnao1+/R209H following a 2.0 mg/kg dose of risperidone. Students unpaired T-test (B) At 0.5 mg/kg both WT and Gnao1+/R209H exhibit a significant decrease in locomotion. Students unpaired T-test. Wildtype mice also show a decrease in locomotion after 0.5 mg/kg risperidone treatment, (C) Comparison of 2.0 mg/kg and 0.5 mg/kg treatment in WT and Gnao1+/R209H mice. Hyperactivity of Gnao1+/R209H mice was observed during baseline testing and recovered following the 2-day risperidone washout. 72 Gnao1+/R209H mice did not show any abnormity in Gαo protein expression Cortex, hippocampus, striatum, cerebellum, brain stem and olfactory bulb were harvested and homogenized to measure mutation R209H’s effect on Gαo protein expression level. Western blots showed no difference in the above brain regions of Gαo protein expression between WT and Gnao1+/R209H (Figure 3-7), which is consistent with our tested protein expression level in HEK293T cells with transiently transfected R209H plasmid [19] Figure 3-7. Western blot shows no statistical difference in Gαo protein between Gnao1 +/R209H and WT mice Brain regions (cortex, hippocampus, striatum, cerebellum, brain stem and olfactory bulb homogenates) from WT and Gnao1+/R209H mice were quantified for levels of Gαo protein. There was no significant difference in any of the regions between WT and mutant mice. 73 Discussion Here we show that the Gnao1 R209H mutant mice display both motor and developmental abnormalities which is consistent with R209H patients, who present with psychomotor delay with the presence of involuntary movements (NEDIM). The mouse developmental milestones showed a significant delay in onset of negative geotaxis. Differences in this test likely correlate with a delay in motor coordination [102]. The Gnao1 +/R209H mice displayed a significant hyperkinetic phenotype in the open field arena and shorter stride length on digigait analysis. Moreover, we also showed that R209H mice are also not sensitive to PTZ-kindling. All the above results are consistent with human patients with the R209H mutation. Interestingly, Gnao1 +/R209H mice did not display any significant differences in the RotaRod test, which is traditionally used to assess motor coordination of rodents [51]. In addition, there were no significant differences observed from the grip strength assessment. This result was unexpected as our previous GNAO1 Mouse model, Gnao1+/G203R, showed significant motor impairments in the both tests [103]. Patients with either the G203R or R209H mutations both display movement disorder; however, patients with the G203R variant have been reported with chorea, dystonia and/or dyskinesias [4] while patients with the R209H variant most commonly are diagnosed with chorea[3]. One likely explanation for the motor differences between the mutant mouse models might be behind the heterogeneity of movement disorders between patients. As hyperkinetic movements in patients have been shown to be exacerbated under stress, illness or high temperature [23, 61], it would be interesting to see if we could induce abnormalities through physical or pharmacological induction. 74 It is important to find an effective treatment as patients with the R209H mutation experience multiple incidents of hospitalizations [23, 24, 91]. Deep brain stimulation in the Globus pallidus has proven effective in GNAO1 patients in attenuating MD [18, 63, 65, 92]. However, the invasive treatment is reserved for refractory patients. Risperidone is one of the oral treatments that has proven to be beneficial, specifically in a patient with the R209H mutation [23]. Risperidone is an atypical neuroleptic, antagonizing D2 and 5-HT receptors. Gao couples to a myriad of G-protein coupled receptors including the dopamine D2 receptor which is involved in movement control [106]. In our experiment, risperidone was able to significantly decrease the hyperlocomotion seen in our heterozygous mutant. This is a promising finding which might allow physicians to narrow down which drugs they try first in the patients. At both the 0.5 mg/kg and 2.0 mg/kg dose of risperidone, hyperactivity was attenuated in our R209H mouse model. However, this response was not selective to our Gnao1+/R209H model as the WT mice also displayed a significant decrease in locomotion. This outcome suggests that risperidone treatment may be effective in repressing global movement, while not specifically targeting a R209H mechanism. While Gao regulates cAMP production through AC, studies have shown that Gao also negatively regulates N-type calcium channels in the presynaptic nerve terminal, reducing release of neurotransmitters [37]. Mutations in other genes known to regulate neurotransmitter release have also been found in individuals with motor impairments [107, 108]. To look into this, future studies should be done to measure concentration of released dopamine and 5-HT within the brains of the Gnao1+/R209H mice. 75 In our previous work, we uncovered a mutation pattern where GOF mutations for to cAMP inhibition are associated with movement disorders and LOF mutations correlate to EIEE17[19]. However, in that model the R209H mutation was classified as having normal- function. This possibly implies that the mechanism is more complex than cAMP inhibition. Since the canonical pathways of Gαo also include Gβγ-mediated inhibition of N-type calcium channels and activation of GIRK channels, it is possible that the R209H mutation could affect other mechanistic pathways of GNAO1. 76 CHAPTER 4: CONCLUSIONS AND FUTURE DIRECTIONS 77 The goals of my thesis work were to: 1) define a phenotype in various GNAO1 mutant mouse models, 2) assess those models’ response to pharmacological treatments and 3) begin to elucidate molecular differences within the models. In Chapters 1 and 2, we defined 3 animal models with gain-of-function associated mutations; Gnao1+/G184S, Gnao1+/G203R, and Gnao1+/R209H. Aside from the G184S model which has not been found in patients, each of these models displayed behavioral abnormalities consistent with patients presenting with the same variant. We were then able to use these models in studying response to treatment in an allele-specific personalized medicine approach. While both trihexyphenidyl and risperidone alleviated MD symptoms in certain patients, only risperidone treated mice showed suppression of the mutant phenotype. Biologically, risperidone works by inhibiting dopamine and 5-HT. As it was effective in reducing the hyperactive phenotype it is possible that this is a mechanism by which R209H variants, alter movement in the patients; however, as risperidone did not seem to exert a more significant effects on the Gnao1+/R209H mice compared to wildtype mice, this needs to be further studied. Gαo has been shown to decrease release of neurotransmitters through modulation of potassium and calcium channels [5, 6] as well as by direct effects on the vesicle release machinery [109]. Surprisingly in our Gnao1+/G203R model we showed no statistically significant decrease within any neurotransmitters we measured by HPLC. In fact, we saw an increase in the amount of norepinephrine within the brain hemisphere samples of the Gnao1+/G203R mice. This result may be less surprising as norepinephrine serves as both pro and anti-convulsant in models of epilepsy [85]. Therefore, our result showing increased amounts of epinephrine in a model positive for PTZ sensitivity, may serve as an indicator that in patients with EIEE, norepinephrine may be playing a 78 proconvulsant role. Follow-up studies looking at the effects depletion of norepinephrine has on our PTZ kindling test might be useful in indicating a treatment option for GNAO1 patients presenting with epilepsy. We analyzed neurotransmitters only for our G203R mice; future studies should also be done to look at our other model Gnao1+/R209H as well. Additionally, our first analysis used an entire brain hemisphere to look at neurotransmitters. It is widely known that movement is controlled within specific regions of the brain including the striatum and the cerebellum. It is also known that our protein of interest Gαo is respectively more abundant within the cerebellum, striatum and hippocampus [1]. By choosing to first look at the whole brain, we may have missed subtle regional differences that other areas compensate for. Additionally, the lack of significant difference between WT and mutant might be due to the differences between total levels of neurotransmitters and specific amount of neurotransmitters released. Future work should be done in analyzing regions specific to both movement and the Gαo protein targeting amount of released neurotransmitters. We saw that the nonselective cholinergic agonist, oxotremorine brought about motor and postural abnormalities in the Gnao1+/G203R mice that were not as pronounced in WT mice. Interestingly, pilot studies using trihexyphenidyl, a cholinergic antagonist showed no reprieve of motor abnormalities. As such it may be valuable to test the response of Gnao1+/G203R mice to a nonselective cholinergic antagonist or an M2 selective antagonist such as benztropine. This work assessed on 2 out of the 35 GNAO1 variants that are currently known. However, with the increasing availability of genomic sequencing, more patients and more variants, of GNAO1 will likely be identified. It would be beneficial to try and create different variant models. 79 The ultimate goal of creating predictive animal models of human GNAO1 mutations is to apply what we learn about the mechanisms of Gαo mutations from those models to clinical applications. Overall from work described here in my thesis, I have identified 2 comparative models that will continue to be useful in researching the GNAO1-associated disorders of NEDIM and EIEE17. The Gnao1+/R209H and Gnao1+/G203R mouse models should be able to guide drug repurposing efforts. 80 APPENDICES 81 APPENDIX A Chapter 2 82 Supplemental Figure 2-1. 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). 83 Supplemental Figure 2-2. 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. 84 Supplemental Figure 2-3. 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 sexes 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. 85 Supplemental Figure 2-4. False discovery rate (FDR) calculation probed through all the parameters given by DigiGait in Gnao1+G184S mice All parameters 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. 86 Supplemental Figure 2-5. False discovery rate (FDR) calculation probed through all the parameters given by DigiGait in Gnao1+/G203R mice All parameters that showed significance are plotted here. A&B) Female Gnao1+/G203R and their littermate controls showed 9 parameters with significance detected by the FDR analysis. C&D) 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. 87 Supplemental Table 2-1. Gait analysis parameters Male Gnao1 G203R mutants 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 88 Supplemental Table 2-2. Gait analysis parameters Female Gnao1 G203R mutants Feng et al Table S2 Gait analysis parameters Female Gnao1 G203R mutants 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 0.143574 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 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 49.1166667 8.051334 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 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 0.47349558 0.90159292 -1.96256637 -0.00261062 1 0.2414856 0.3272514 0.3777178 0.7706896 0 89 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 Supplemental Table 2-3. Gait analysis parameters Male Gnao1 G184S mutants Feng et al Table S3 Gait analysis parameters Male Gnao1 G184S mutants 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 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 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 0.51178571 0.91821429 -2.76369048 0.01333333 1 0.1905993 0.3516473 0.3417171 0.8200578 0 90 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 0 0 0 1 1 1 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 Supplemental Table 2-4. Gait analysis parameters Female Gnao1 G184S mutants Feng et al Table S4 Gait analysis parameters Female Gnao1 G184S mutants 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 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 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 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 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 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 91 Supplemental Table 2-6. Benchling off-target list for Gnao1 G203 gRNA Feng%at%al%Table&S5& &Benchling)off,target)list)for)Gnao1)G203)gRNA)) & PAM& Score& Chromosome& Mismatches& 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) ) chr1) 0.3804) 0.3169) ENSMUSG00000032497) chr9) ) chr3) 0.2931) 0.1953) ) chr1) 0.1929) ) chr13) 0.1923) ) chr1) 0.1710) ) chr1) 0.1556) ENSMUSG00000086805) chr8) 0.1543) ENSMUSG00000057614) chr5) 0.1515) ) chrX) ) chr4) 0.1480) 0.1450) ) chr17) 0.1403) ) chr18) 0.1343) ENSMUSG00000026413) chr1) ) chrX) 0.1262) 0.1144) ) chr11) 0.1127) ) chr5) 0.1127) ) chr1) ) chr10) 0.1004) 0.1002) ) chr11) 0.0954) ) chrX) 0.0933) ) chr3) 0.0930) ENSMUSG00000020015) chr10) 0.0865) ) chrX) 0.0862) ) chr2) 0.0836) ) chr11) 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) 92 Supplemental Figure 2-6. GABA and glutamate show no significant differences between Gnao1+/R209H and wildtype littermates HPLC analysis of a second cohort of Gnao1+/G203R mice, there was no significant difference in GABA or glutamate within the left hemisphere. Students unpaired T-test: nonsignificant. 93 APPENDIX B Chapter 3 94 Supplemental Figure 3-1. WT and Gnao1+/R209H mice show no difference in percent suppression of locomotion after oxotremorine treatment Gnao1+/R209H mice show similar sensitivity to risperidone treatment at 2.0 mg/kg or 0.5 mg/kg compared to WT treated mice, Student t-test. 95 REFERENCES 96 REFERENCES from bovine cerebral cortex. Huff RM, Axton JM, Neer EJ. Physical and immunological characterization of a guanine J Biol Chem. Worley PF, Baraban JM, Van Dop C, Neer EJ, Snyder SH. Go, a guanine nucleotide-binding 1. protein: immunohistochemical localization in rat brain resembles distribution of second messenger systems. Proc Natl Acad Sci U S A. 1986;83(12):4561-5. PubMed PMID: 3086888; PubMed Central PMCID: PMCPMC323774. 2. Sternweis PC, Robishaw JD. Isolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain. J Biol Chem. 1984;259(22):13806-13. PubMed PMID: 6438083. 3. Neer EJ, Lok JM, Wolf LG. Purification and properties of the inhibitory guanine nucleotide regulatory unit of brain adenylate cyclase. J Biol Chem. 1984;259(22):14222-9. PubMed PMID: 6150041. 4. nucleotide-binding protein purified 1985;260(19):10864-71. PubMed PMID: 3928624. Jiang M, Bajpayee NS. Molecular mechanisms of go signaling. Neurosignals. 5. 2009;17(1):23-41. Epub 2009/02/12. doi: 10.1159/000186688. PubMed PMID: 19212138; PubMed Central PMCID: PMCPMC2836949. 6. Colecraft HM, Brody DL, Yue DT. G-protein inhibition of N- and P/Q-type calcium channels: distinctive elementary mechanisms and their functional impact. J Neurosci. 2001;21(4):1137-47. PubMed PMID: 11160384. 7. 2012;3:95. doi: 10.3389/fphys.2012.00095. PubMed PMID: article. 8. Fu Y, Zhong H, Nanamori M, Mortensen RM, Huang X, Lan K, et al. RGS-insensitive G- protein mutations to study the role of endogenous RGS proteins. Methods Enzymol. 2004;389:229-43. doi: 10.1016/S0076-6879(04)89014-1. PubMed PMID: 15313569. 9. Goldenstein BL, Nelson BW, Xu K, Luger EJ, Pribula JA, Wald JM, et al. Regulator of G protein signaling protein suppression of Galphao protein-mediated alpha2A adrenergic receptor inhibition of mouse hippocampal CA3 epileptiform activity. Mol Pharmacol. 2009;75(5):1222-30. Epub 2009/02/18. doi: 10.1124/mol.108.054296. PubMed PMID: 19225179; PubMed Central PMCID: PMCPMC2672807. 10. Nakamura K, Kodera H, Akita T, Shiina M, Kato M, Hoshino H, et al. De Novo mutations in GNAO1, encoding a Gαo subunit of heterotrimeric G proteins, cause epileptic encephalopathy. Am J Hum Genet. 2013;93(3):496-505. Epub 2013/08/29. doi: 10.1016/j.ajhg.2013.07.014. Adele S, Jie H, Rory F. RGS Proteins in heart: Brakes on the Vagus. Frontiers in physiology. 97 PubMed PMID: 23993195; PubMed Central PMCID: PMCPMC3769919. 11. M. KJ, Sahaya K, Dalton HM, Charbeneau RA, Kohut KT, Gilbert K, et al. Gain-of-function mutation in Gnao1: a murine model of epileptiform encephalopathy (EIEE17)? Mamm Genome. 2014;25(5-6):202-10. Epub 2014/04/05. doi: 10.1007/s00335-014-9509-z. PubMed PMID: 24700286; PubMed Central PMCID: PMCPMC4042023. 12. Shimada T, Yamagata K. Pentylenetetrazole-Induced Kindling Mouse Model. J Vis Exp. 2018;(136). Epub 2018/06/12. doi: 10.3791/56573. PubMed PMID: 29985308; PubMed Central PMCID: PMCPMC6101698. 13. Meredith K, Meredith P, Ivana M, Anne R, Marie G, Eduardo P-P, et al. Spectrum of neurodevelopmental disease associated with the GNAO1 guanosine triphosphate-binding region. Epilepsia. 2019. doi: 10.1111/epi.14653. PubMed PMID: kelly_2019. 14. Gawlinski P, Renata P, Tomasz G, Danuta S, Monika C, Beata N, et al. PEHO Syndrome May Represent Phenotypic Expansion at the Severe End of the Early-Onset Encephalopathies. Pediatr Neurol. 2016;60:83-7. doi: 10.1016/j.pediatrneurol.2016.03.011. PubMed PMID: gawlinski_2016. 15. Law C-Y, Tzu-Lun CS, Young CS, Kin-Cheong YE, Sui-Fun NG, Nai-Chung F, et al. Clinical whole-exome sequencing reveals a novel missense pathogenic variant of GNAO1 in a patient with infantile-onset epilepsy. Clin Chim Acta. 2015;451(Pt B):292-6. doi: 10.1016/j.cca.2015.10.011. PubMed PMID: law_2015. 16. Marcé-Grau A, James D, Javier L-P, Concepción G-JM, Lorena M-G, Ester C-L, et al. GNAO1 encephalopathy: further delineation of a severe neurodevelopmental syndrome affecting females. Orphanet J Rare Dis. 2016;11:38. doi: 10.1186/s13023-016-0416-0. PubMed PMID: marcgrau_2016. 17. Saitsu H, Ryoko F, Bruria B-Z, Yasunari S, Masakazu M, Nobuhiko O, et al. Phenotypic spectrum of GNAO1 variants: epileptic encephalopathy to involuntary movements with severe developmental delay. Eur J Hum Genet. 2016;24(1):129-34. doi: 10.1038/ejhg.2015.92. PubMed PMID: saitsu_2016. 18. Kulkarni N, Sha T, Ratan B, Saunder B, A. GT. Progressive movement disorder in brothers carrying a GNAO1 mutation responsive to deep brain stimulation. J Child Neurol. 2016;31(2):211- 4. doi: 10.1177/0883073815587945. PubMed PMID: kulkarni_2016. 19. Feng H, Sjögren B, Karaj B, Shaw V, Gezer A, Neubig RR. Movement disorder in. Neurology. 2017;89(8):762-70. Epub 2017/07/26. doi: 10.1212/WNL.0000000000004262. PubMed PMID: 28747448; PubMed Central PMCID: PMCPMC5580866. 20. Mortal Wkly Rep. 2012;61(45):909-13. PubMed PMID: 23151949. (CDC) CfDCaP. Epilepsy in adults and access to care--United States, 2010. MMWR Morb 98 21. McTague A, Howell KB, Cross JH, Kurian MA, Scheffer IE. The genetic landscape of the epileptic encephalopathies of infancy and childhood. Lancet Neurol. 2016;15(3):304-16. Epub 2015/11/17. doi: 10.1016/S1474-4422(15)00250-1. PubMed PMID: 26597089. 22. Subramony SH, Schott K, Raike RS, Callahan J, Langford LR, Christova PS, et al. Novel CACNA1A mutation causes febrile episodic ataxia with interictal cerebellar deficits. Ann Neurol. 2003;54(6):725-31. doi: 10.1002/ana.10756. PubMed PMID: 14681882. 23. Ananth AL, Robichaux-Viehoever, Young-Min A, Young-Min K, Andrea H-K, Rachel C, et al. Clinical Course of Six Children With GNAO1 Mutations Causing a Severe and Distinctive Movement Disorder. Pediatr Neurol. 2016;59:81-4. doi: 10.1016/j.pediatrneurol.2016.02.018. PubMed PMID: ananth_2016. Schirinzi T, Giacomo G, Lorena T, Gessica V, Serena G, Loreto R, et al. Phenomenology and 24. clinical course of movement disorder in GNAO1 variants: Results from an analytical review. Parkinsonism Relat Disord. 2018. doi: 10.1016/j.parkreldis.2018.11.019. PubMed PMID: schirinzi_2018. 25. Chen YZ, Matsushita MM, Robertson P, Rieder M, Girirajan S, Antonacci F, et al. Autosomal dominant familial dyskinesia and facial myokymia: single exome sequencing identifies a mutation in adenylyl cyclase 5. Arch Neurol. 2012;69(5):630-5. doi: 10.1001/archneurol.2012.54. PubMed PMID: 22782511; PubMed Central PMCID: PMCPMC3508680. 26. Carecchio, Miryam, E. MN. Emerging monogenic complex hyperkinetic disorders. Curr Neurol Neurosci Rep. 2017;17(12):97. doi: 10.1007/s11910-017-0806-2. PubMed PMID: carecchio_2017. 27. local GC 10.1093/molbev/msi043. PubMed PMID: 15537806. Jang HS, Shin WJ, Lee JE, Do JT. CpG and Non-CpG Methylation in Epigenetic Gene 28. Regulation and Brain Function. Genes (Basel). 2017;8(6). Epub 2017/05/23. doi: 10.3390/genes8060148. PubMed PMID: 28545252; PubMed Central PMCID: PMCPMC5485512. 29. Messé LA, Aronoff J, Wilson JP. Motivation as a mediator of the mechanisms underlying role assignments in small groups. J Pers Soc Psychol. 1972;24(1):84-90. PubMed PMID: 5079558. 30. Kong A, Frigge ML, Masson G, Besenbacher S, Sulem P, Magnusson G, et al. Rate of de novo mutations and the importance of father's age to disease risk. Nature. 2012;488(7412):471- 5. doi: 10.1038/nature11396. PubMed PMID: 22914163; PubMed Central PMCID: PMCPMC3548427. 31. Fryxell KJ, Moon WJ. CpG mutation rates in the human genome are highly dependent on doi: Panchin AY, Makeev VJ, Medvedeva YA. Preservation of methylated CpG dinucleotides in content. Mol Biol Evol. 2005;22(3):650-8. Epub 2004/11/10. 99 human CpG islands. Biol Direct. 2016;11(1):11. Epub 2016/03/22. doi: 10.1186/s13062-016- 0113-x. PubMed PMID: 27005429; PubMed Central PMCID: PMCPMC4804638. Urdinguio RG, Sanchez-Mut JV, Esteller M. Epigenetic mechanisms in neurological 32. diseases: genes, syndromes, and therapies. Lancet Neurol. 2009;8(11):1056-72. doi: 10.1016/S1474-4422(09)70262-5. PubMed PMID: 19833297. 33. Schorling DC, Tobias D, Christina E, Katrin H, Rudolf K, Daniel E, et al. Expanding phenotype of de novo mutations in GNAO1 : four new cases and review of literature. Neuropediatrics. 2017;48(5):371-7. doi: 10.1055/s-0037-1603977. PubMed PMID: schorling_2017. 34. Feng H, Suad K, R. NR, Christos S. A mechanistic review on GNAO1 -associated movement disorder. Neurobiol Dis. 2018;116:131-41. doi: 10.1016/j.nbd.2018.05.005. PubMed PMID: feng_2018. 35. Gazi L, Nickolls SA, Strange PG. Functional coupling of the human dopamine D2 receptor with G alpha i1, G alpha i2, G alpha i3 and G alpha o G proteins: evidence for agonist regulation of G protein selectivity. Br J Pharmacol. 2003;138(5):775-86. doi: 10.1038/sj.bjp.0705116. PubMed PMID: 12642378; PubMed Central PMCID: PMCPMC1573727. 36. Zhang Q, Pacheco MA, Doupnik CA. Gating properties of GIRK channels activated by Galpha(o)- and Galpha(i)-coupled muscarinic m2 receptors in Xenopus oocytes: the role of receptor precoupling in RGS modulation. J Physiol. 2002;545(2):355-73. PubMed PMID: 12456817; PubMed Central PMCID: PMCPMC2290703. 37. Li Q, Lau A, Morris TJ, Guo L, Fordyce CB, Stanley EF. A syntaxin 1, Galpha(o), and N-type calcium channel complex at a presynaptic nerve terminal: analysis by quantitative immunocolocalization. J Neurosci. 2004;24(16):4070-81. doi: 10.1523/JNEUROSCI.0346-04.2004. PubMed PMID: 15102922. 38. Strittmatter SM, Fishman MC, Zhu XP. Activated mutants of the alpha subunit of G(o) promote an increased number of neurites per cell. J Neurosci. 1994;14(4):2327-38. PubMed PMID: 8158271. 39. Bromberg KD, Iyengar R, He JC. Regulation of neurite outgrowth by G(i/o) signaling pathways. Front Biosci. 2008;13:4544-57. Epub 2008/05/01. PubMed PMID: 18508528; PubMed Central PMCID: PMCPMC3068557. Lan KL, Sarvazyan NA, Taussig R, Mackenzie RG, DiBello PR, Dohlman HG, et al. A point 40. mutation in Galphao and Galphai1 blocks interaction with regulator of G protein signaling proteins. J Biol Chem. 1998;273(21):12794-7. PubMed PMID: 9582306. 41. Kehrl JM, Sahaya K, Dalton HM, Charbeneau RA, Kohut KT, Gilbert K, et al. Gain-of-function mutation in Gnao1: a murine model of epileptiform encephalopathy (EIEE17)? Mamm Genome. 2014;25(5-6):202-10. Epub 2014/04/05. doi: 10.1007/s00335-014-9509-z. PubMed PMID: 100 24700286; PubMed Central PMCID: PMCPMC4042023. 42. Arya R, Christine S, L. GD, L. LJ, D. HK. GNAO1 -associated epileptic encephalopathy and movement disorders: c. 607G\textgreaterA variant represents a probable mutation hotspot with a distinct phenotype. Epileptic Disord. 2017;19(1):67-75. doi: 10.1684/epd.2017.0888. PubMed PMID: arya_2017. 43. Xiong J, Jing P, Hao-Lin D, Chen C, Xiao-Le W, Shi-Meng C, et al. Recurrent convulsion and pulmonary infection complicated by psychomotor retardation in an infant . Zhongguo Dang Dai Er Ke Za Zhi. 2018;20(2):154-7. doi: 10.7499/j.issn.1008-8830.2018.02.014. PubMed PMID: xiong_2018. Qin W, Dion SL, Kutny PM, Zhang Y, Cheng AW, Jillette NL, et al. Efficient CRISPR/Cas9- 44. in Mice by Zygote Electroporation of Nuclease. Genetics. Mediated Genome Editing 2015;200(2):423-30. Epub 2015/03/27. doi: 10.1534/genetics.115.176594. PubMed PMID: 25819794; PubMed Central PMCID: PMCPMC4492369. 45. Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016;34(2):184-91. Epub 2016/01/18. doi: 10.1038/nbt.3437. PubMed PMID: 26780180; PubMed Central PMCID: PMCPMC4744125. 46. Haeussler M, Schönig K, Eckert H, Eschstruth A, Mianné J, Renaud JB, et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 2016;17(1):148. Epub 2016/07/05. doi: 10.1186/s13059-016-1012-2. PubMed PMID: 27380939; PubMed Central PMCID: PMCPMC4934014. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, et al. DNA targeting 47. specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31(9):827-32. Epub 2013/07/21. doi: 10.1038/nbt.2647. PubMed PMID: 23873081; PubMed Central PMCID: PMCPMC3969858. Iyer V, Boroviak K, Thomas M, Doe B, Riva L, Ryder E, et al. No unexpected CRISPR-Cas9 48. off-target activity revealed by trio sequencing of gene-edited mice. PLoS Genet. 2018;14(7):e1007503. Epub 2018/07/09. doi: 10.1371/journal.pgen.1007503. PubMed PMID: 29985941; PubMed Central PMCID: PMCPMC6057650. 49. Truett GE, Heeger P, Mynatt RL, Truett AA, Walker JA, Warman ML. Preparation of PCR- quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). Biotechniques. 2000;29(1):52, 4. doi: 10.2144/00291bm09. PubMed PMID: 10907076. 50. Hirata H, Takahashi A, Shimoda Y, Koide T. Caspr3-Deficient Mice Exhibit Low Motor Learning during the Early Phase of the Accelerated Rotarod Task. PLoS One. 2016;11(1):e0147887. Epub 2016/01/25. doi: 10.1371/journal.pone.0147887. PubMed PMID: 26807827; PubMed Central PMCID: PMCPMC4726695. 101 Int J Drug Policy. Epub 2016/06/11. 2016;36:104-11. 51. Deacon RM, Nielsen S, Leung S, Rivas G, Cubitt T, Monds LA, et al. Alprazolam use and related harm among opioid substitution treatment clients - 12 months follow up after regulatory rescheduling. doi: 10.1016/j.drugpo.2016.06.006. PubMed PMID: 27453147. 52. Hansen ST, Pulst SM. Response to ethanol induced ataxia between C57BL/6J and 129X1/SvJ mouse strains using a treadmill based assay. Pharmacol Biochem Behav. 2013;103(3):582-8. Epub 2012/10/24. doi: 10.1016/j.pbb.2012.10.010. PubMed PMID: 23103202; PubMed Central PMCID: PMCPMC4900535. Franco-Pons N, Torrente M, Colomina MT, Vilella E. Behavioral deficits in the cuprizone- 53. induced murine model of demyelination/remyelination. Toxicol Lett. 2007;169(3):205-13. Epub 2007/02/02. doi: 10.1016/j.toxlet.2007.01.010. PubMed PMID: 17317045. 54. DiBello PR, Garrison TR, Apanovitch DM, Hoffman G, Shuey DJ, Mason K, et al. Selective uncoupling of RGS action by a single point mutation in the G protein alpha-subunit. J Biol Chem. 1998;273(10):5780-4. PubMed PMID: 9488712. 55. Grecksch G, Becker A, Schroeder H, Kraus J, Loh H, Höllt V. Accelerated kindling development in mu-opioid receptor deficient mice. Naunyn Schmiedebergs Arch Pharmacol. 2004;369(3):287-93. Epub 2004/02/12. doi: 10.1007/s00210-004-0870-4. PubMed PMID: 14963640. 56. Wilczynski GM, Konopacki FA, Wilczek E, Lasiecka Z, Gorlewicz A, Michaluk P, et al. Important role of matrix metalloproteinase 9 in epileptogenesis. J Cell Biol. 2008;180(5):1021-35. doi: 10.1083/jcb.200708213. PubMed PMID: 18332222; PubMed Central PMCID: PMCPMC2265409. 57. Ahmad MF, Ferland D, Ayala-Lopez N, Contreras GA, Darios E, Thompson J, et al. Perivascular Adipocytes Store Norepinephrine by Vesicular Transport. Arterioscler Thromb Vasc Biol. 2019;39(2):188-99. doi: 10.1161/ATVBAHA.118.311720. PubMed PMID: 30567483; PubMed Central PMCID: PMCPMC6344267. 58. Donzanti BA, Yamamoto BK. An improved and rapid HPLC-EC method for the isocratic separation of amino acid neurotransmitters from brain tissue and microdialysis perfusates. Life Sci. 1988;43(11):913-22. PubMed PMID: 2901021. Gillies GE, Murray HE, Dexter D, McArthur S. Sex dimorphisms in the neuroprotective 59. effects of estrogen in an animal model of Parkinson's disease. Pharmacol Biochem Behav. 2004;78(3):513-22. doi: 10.1016/j.pbb.2004.04.022. PubMed PMID: 15251260. 60. J. BTU, DesRoches CL, Wilson D, Chau V, Nakagawa T, Yamasaki M, et al. Prospective cohort study for identification of underlying genetic causes in neonatal encephalopathy using whole- exome doi: sequencing. Genet Med. 2018;20(5):486-94. Epub 2017/08/17. 102 10.1038/gim.2017.129. PubMed PMID: 28817111. 61. Danti FR, Serena G, Marta R, Martino M, J. CK, Lucy RF, et al. GNAO1 encephalopathy: Broadening the phenotype and evaluating treatment and outcome. Neurol Genet. 2017;3(2):e143. doi: 10.1212/ NXG .0000000000000143. PubMed PMID: danti_2017. 62. Menke LA, Marc E, Mariel A, J. OVJ, Frank B, M. CJ. Recurrent GNAO1 mutations associated with developmental delay and a movement disorder. J Child Neurol. 2016;31(14):1598-601. doi: 10.1177/0883073816666474. PubMed PMID: menke_2016. Honey CM, K. MA, Maja T-G, M. vKCD, Gabriella H, Adi S. GNAO1 Mutation-Induced 63. Pediatric Dystonic Storm Rescue With Pallidal Deep Brain Stimulation. J Child Neurol. 2018;33(6):413-6. doi: 10.1177/0883073818756134. PubMed PMID: honey_2018. 64. Waak M, S. MS, David C, Kate S, Lisa C, Peter S, et al. GNAO1 -related movement disorder with life-threatening exacerbations: movement phenomenology and response to DBS . J Neurol Neurosurg Psychiatr. 2018;89(2):221-2. doi: 10.1136/jnnp-2017-315653. PubMed PMID: waak_2018. 65. Sanem Y, Tuncer T, Serdar C, Sarenur G, Hasan T, Gul S. Excellent response to deep brain stimulation in a young girl with GNAO1 -related progressive choreoathetosis. Childs Nerv Syst. 2016;32(9):1567-8. doi: 10.1007/s00381-016-3139-6. PubMed PMID: yilmaz_2016. 66. Consortium E-R, Project EPG, Consortium EK. De novo mutations in synaptic transmission genes including DNM1 cause epileptic encephalopathies. Am J Hum Genet. 2014;95(4):360-70. doi: 10.1016/j.ajhg.2014.08.013. PubMed PMID: euroepinomicsresconsortium_2014. Zhu X, Petrovski S, Xie P, Ruzzo EK, Lu YF, McSweeney KM, et al. Whole-exome sequencing 67. in undiagnosed genetic diseases: interpreting 119 trios. Genet Med. 2015;17(10):774-81. Epub 2015/01/15. doi: 10.1038/gim.2014.191. PubMed PMID: 25590979; PubMed Central PMCID: PMCPMC4791490. 68. Tatem KS, Quinn JL, Phadke A, Yu Q, Gordish-Dressman H, Nagaraju K. Behavioral and locomotor measurements using an open field activity monitoring system for skeletal muscle diseases. J Vis Exp. 2014;(91):51785. Epub 2014/09/29. doi: 10.3791/51785. PubMed PMID: 25286313; PubMed Central PMCID: PMCPMC4672952. Parr T, Friston KJ. Working memory, attention, and salience in active inference. Sci Rep. 69. 2017;7(1):14678. Epub 2017/11/07. doi: 10.1038/s41598-017-15249-0. PubMed PMID: 29116142; PubMed Central PMCID: PMCPMC5676961. 70. Stroobants S, Gantois I, Pooters T, D'Hooge R. Increased gait variability in mice with small cerebellar cortex lesions and normal rotarod performance. Behav Brain Res. 2013;241:32-7. Epub 2012/12/03. doi: 10.1016/j.bbr.2012.11.034. PubMed PMID: 23219967. 103 Dhir A. Pentylenetetrazol (PTZ) kindling model of epilepsy. Curr Protoc Neurosci. Korchounov AM. Role of D1 and D2 receptors in the regulation of voluntary movements. 71. Song CH, Fan X, Exeter CJ, Hess EJ, Jinnah HA. Functional analysis of dopaminergic systems in a DYT1 knock-in mouse model of dystonia. Neurobiol Dis. 2012;48(1):66-78. Epub 2012/05/31. doi: 10.1016/j.nbd.2012.05.009. PubMed PMID: 22659308; PubMed Central PMCID: PMCPMC3498628. 72. 2012;Chapter 9:Unit9.37. doi: 10.1002/0471142301.ns0937s58. PubMed PMID: 23042503. 73. Pelosi A, Menardy F, Popa D, Girault JA, Hervé D. Heterozygous Gnal Mice Are a Novel Animal Model with Which to Study Dystonia Pathophysiology. J Neurosci. 2017;37(26):6253-67. Epub 2017/05/25. doi: 10.1523/JNEUROSCI.1529-16.2017. PubMed PMID: 28546310. 74. Bull Exp Biol Med. 2008;146(1):14-7. PubMed PMID: 19145338. 75. Raike RS, Jinnah HA, Hess EJ. Animal models of generalized dystonia. NeuroRx. 2005;2(3):504-12. doi: 10.1602/neurorx.2.3.504. PubMed PMID: 16389314; PubMed Central PMCID: PMCPMC1144494. 76. Oleas J, Yokoi F, DeAndrade MP, Pisani A, Li Y. Engineering animal models of dystonia. Mov Disord. 2013;28(7):990-1000. doi: 10.1002/mds.25583. PubMed PMID: 23893455; PubMed Central PMCID: PMCPMC3800691. 77. Wettschureck N, Offermanns S. Mammalian G proteins and their cell type specific functions. Physiol Rev. 2005;85(4):1159-204. doi: 10.1152/physrev.00003.2005. PubMed PMID: 16183910. 78. 10.18632/oncotarget.22067. PMCPMC5963625. 79. Smith KM, Dahodwala N. Sex differences in Parkinson's disease and other movement disorders. Exp Neurol. 2014;259:44-56. Epub 2014/03/28. doi: 10.1016/j.expneurol.2014.03.010. PubMed PMID: 24681088. 80. Revankar CM, Cimino DF, Sklar LA, Arterburn JB, Prossnitz ER. A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science. 2005;307(5715):1625-30. Epub 2005/02/10. doi: 10.1126/science.1106943. PubMed PMID: 15705806. 81. Wooten GF, Currie LJ, Bovbjerg VE, Lee JK, Patrie J. Are men at greater risk for Parkinson's disease than women? J Neurol Neurosurg Psychiatry. 2004;75(4):637-9. PubMed PMID: 15026515; PubMed Central PMCID: PMCPMC1739032. 82. Solis GP, Katanaev VL. Gαo (Oncotarget. 2018;9(35):23846-7. Epub 2017/10/25. doi: PMCID: Kompoliti K. Estrogen and movement disorders. Clin Neuropharmacol. 1999;22(6):318-26. PubMed PMID: 29844856; PubMed Central 104 Schutsky K, Ouyang M, Thomas SA. Xamoterol Consortium G. The Genotype-Tissue Expression PubMed PMID: 10626091. 83. Madalan A, Yang X, Ferris J, Zhang S, Roman G. G(o) activation is required for both appetitive and aversive memory acquisition in Drosophila. Learn Mem. 2012;19(1):26-34. Epub 2011/12/21. doi: 10.1101/lm.024802.111. PubMed PMID: 22190729. 84. impairs hippocampus-dependent emotional memory retrieval via Gi/o-coupled β2-adrenergic signaling. Learn Mem. 2011;18(9):598-604. Epub 2011/08/30. doi: 10.1101/lm.2302811. PubMed PMID: 21878527; PubMed Central PMCID: PMCPMC3166789. 85. Fitzgerald PJ. Is elevated norepinephrine an etiological factor in some cases of epilepsy? Seizure. 2010;19(6):311-8. Epub 2010/05/20. doi: 10.1016/j.seizure.2010.04.011. PubMed PMID: 20493725. 86. (GTEx) project. Nat Genet. 2013;45(6):580-5. doi: 10.1038/ng.2653. PubMed PMID: 23715323; PubMed Central PMCID: PMCPMC4010069. 87. Nakamura K, Hirofumi K, Tenpei A, Masaaki S, Mitsuhiro K, Hideki H, et al. De Novo mutations in GNAO1 , encoding a G\alphao subunit of heterotrimeric G proteins, cause epileptic encephalopathy. Am J Hum Genet. 2013;93(3):496-505. doi: 10.1016/j.ajhg.2013.07.014. PubMed PMID: nakamura_2013. 88. Blumkin L, Tally L-S, Ana W, Hilla B-P, Ayelet Z, Keren Y, et al. Multiple Causes of Pediatric Early Onset Chorea-Clinical and Genetic Approach. Neuropediatrics. 2018;49(4):246-55. doi: 10.1055/s-0038-1645884. PubMed PMID: blumkin_2018. 89. Sakamoto S, Yukifumi M, Ryoko F, Noriko M, Hiroshi S, Akihiko M, et al. A case of severe movement disorder with GNAO1 mutation responsive to topiramate. Brain Dev. 2017;39(5):439- 43. doi: 10.1016/j.braindev.2016.11.009. PubMed PMID: sakamoto_2017. 90. Rim JH, Hee KS, Sik HI, Sung KS, Jieun K, Woo KH, et al. Efficient strategy for the molecular diagnosis of intractable early-onset epilepsy using targeted gene sequencing. BMC Med Genomics. 2018;11(1):6. doi: 10.1186/s12920-018-0320-7. PubMed PMID: rim_2018. 91. Marecos C, S. D, I. A, E. C, A. M. GNAO1 : a new gene to consider on early-onset childhood dystonia . Rev Neurol. 2018;66(9):321-2. PubMed PMID: marecos_2018. 92. Koy A, Sebahattin C, Victoria G, Kerstin B, Thomas R, Christophe M, et al. Deep brain stimulation is effective in pediatric patients with GNAO1 associated severe hyperkinesia. J Neurol Sci. 2018;391:31-9. doi: 10.1016/j.jns.2018.05.018. PubMed PMID: koy_2018. 93. Dhamija R, W. MJ, B. SB, P. GH. GNAO1 -Associated Movement Disorder. Mov Disord Clin 105 doi: PubMed case report and review of literature. 10.1016/j.ajhg.2014.08.013. Pract. 2016;3(6):615-7. doi: 10.1002/mdc3.12344. PubMed PMID: dhamija_2016. 94. Consortium. De novo mutations in SLC1A2 and CACNA1A are important causes of epileptic encephalopathies. Am J Hum Genet. 2016;99(2):287-98. doi: 10.1016/j.ajhg.2016.06.003. PubMed PMID: epi4kconsortium_2016. 95. Talvik I, S. MoR, Merilin V, Ulvi V, Hg LL, A. DH, et al. Clinical phenotype of de novo GNAO1 mutation: Child Neurology Open. 2015;2(2):2329048X15583717. doi: 10.1177/ 2329048X15583717. PubMed PMID: talvik_2015. Consortium, Epilepsy Phenome/Genome Project and C. De novo mutations in synaptic 96. transmission genes including DNM1 cause epileptic encephalopathies. Am J Hum Genet. 2014;95(4):360-70. PMID: euroepinomicsresconsortium_2014. 97. Gerald B, Keri R, Newell B, Szabolcs S, L. SA, Chris B, et al. Neonatal epileptic encephalopathy caused by de novo GNAO1 mutation misdiagnosed as atypical Rett syndrome: Cautions in interpretation of genomic test results. Semin Pediatr Neurol. 2018;26:28-32. doi: 10.1016/j.spen.2017.08.008. PubMed PMID: gerald_2018. Bruun TUJa. Prospective cohort study for identification of underlying genetic causes in 98. neonatal encephalopathy using whole-exome sequencing. Genet Med. 2018;20(5):486-94. doi: 10.1038/gim.2017.129. PubMed PMID: bruun_2018. 99. Takezawa Y, Atsuo K, Kazuhiro H, Tetsuya N, Yurika N-U, Takehiko I, et al. Genomic analysis identifies masqueraders of full-term cerebral palsy. Ann Clin Transl Neurol. 2018;5(5):538-51. doi: 10.1002/acn3.551. PubMed PMID: takezawa_2018. 100. Okumura A, Koichi M, Mami S, Hirokazu K, Atsushi I, Shingo N, et al. A patient with a GNAO1 mutation with decreased spontaneous movements, hypotonia, and dystonic features. Brain Dev. 2018;40(10):926-30. doi: 10.1016/j.braindev.2018.06.005. PubMed PMID: okumura_2018. 101. Consortium EK. De novo mutations in SLC1A2 and CACNA1A are important causes of epileptic doi: 10.1016/j.ajhg.2016.06.003. PubMed PMID: epi4kconsortium_2016. Feather-Schussler DN, Ferguson TS. A Battery of Motor Tests in a Neonatal Mouse Model 102. of Cerebral Palsy. J Vis Exp. 2016;(117). Epub 2016/11/03. doi: 10.3791/53569. PubMed PMID: 27842358; PubMed Central PMCID: PMCPMC5226120. 103. movement disorder: Allele- and sex-specific differences 2019;14(1):e0211066. doi: 10.1371/journal.pone.0211066. PubMed PMID: feng_2019. Feng H, L. LC, Y. DE, Huirong X, R. LJ, R. NR. Mouse models of GNAO1 -associated in phenotypes. PLoS ONE. 2016;99(2):287-98. encephalopathies. Am J Hum Genet. 106 104. Seibenhener ML, Wooten MC. Use of the Open Field Maze to measure locomotor and anxiety-like behavior in mice. J Vis Exp. 2015;(96):e52434. Epub 2015/02/06. doi: 10.3791/52434. PubMed PMID: 25742564; PubMed Central PMCID: PMCPMC4354627. 105. Carvalho RC, Silva RH, Abílio VC, Barbosa PN, Frussa-Filho R. Antidyskinetic effects of risperidone on animal models of tardive dyskinesia in mice. Brain Res Bull. 2003;60(1-2):115-24. PubMed PMID: 12725899. 106. Neve KA, Seamans JK, Trantham-Davidson H. Dopamine receptor signaling. J Recept Signal Transduct Res. 2004;24(3):165-205. PubMed PMID: 15521361. 107. Alkufri F, Shaag A, Abu-Libdeh B, Elpeleg O. Deleterious mutation in GPR88 is associated with chorea, speech delay, and learning disabilities. Neurol Genet. 2016;2(3):e64. Epub 2016/03/09. doi: 10.1212/NXG.0000000000000064. PubMed PMID: 27123486; PubMed Central PMCID: PMCPMC4830197. 108. splicing 10.1212/01.wnl.0000169020.82223.dd. PubMed PMID: 16043807. 109. Betke KM, Wells CA, Hamm HE. GPCR mediated regulation of synaptic transmission. Prog Neurobiol. 2012;96(3):304-21. Epub 2012/01/28. doi: 10.1016/j.pneurobio.2012.01.009. PubMed PMID: 22307060; PubMed Central PMCID: PMCPMC3319362. Eunson LH, Graves TD, Hanna MG. New calcium channel mutations predict aberrant RNA doi: 2005;65(2):308-10. ataxia. Neurology. in episodic 107