INVESTIGATING NOVEL THERAPEUTIC TARGETS FOR THE TREATMENT OF
FRAGILE X SYNDROME
By
Ferzin Sethna

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Genetics—Doctor of Philosophy
2014

	
  
ABSTRACT
INVESTIGATING NOVEL THERAPEUTIC TARGETS FOR THE TREATMENT OF
FRAGILE X SYNDROME
By
Ferzin Sethna
Fragile X syndrome (FXS) is the most common form of inherited mental retardation and
a leading cause of autism. Most cases of FXS result from CGG repeat expansion in the
5’ UTR of the FMR1 gene, which results in lack of expression of its protein product,
FMRP. FMRP is an mRNA binding protein, which predominantly suppresses the
translation of its targets. The absence of FMRP thus leads to excessive protein
synthesis and altered synaptic signaling, which are believed to underlie the
pathophysiology of FXS. There is currently no cure for FXS and often multiple drugs are
administered to manage the symptoms. This highlights the importance of understanding
the molecular mechanisms that are altered in FXS brains, in order to develop better
therapeutic targets. My study identifies that translation of the brain-specific type-1
adenylyl cyclase (AC1) mRNA is controlled by FMRP and that AC1 protein is
overexpressed in the absence of FMRP. Using genetic knockdown and pharmacological
inhibition of AC1, I show that reducing AC1 activity can rescue several cellular and
behavioral phenotypes in the Fmr1 knockout mouse model. My research also reveals

	
  

calmodulin inhibitor can rectify several cellular and behavioral phenotypes in Fmr1 KO
mice. In summary this thesis describes the identification and validation of a novel
therapeutic target and an FDA approved drug for treatment of FXS using the Fmr1 KO
mouse model.

ACKNOWLEDGEMENTS

I would like to thank my advisor Dr. Hongbing Wang for allowing me to work on this
wonderful project. Thank you for all the ideas and discussions that helped shape my
research. I would also like to thank my committee members, Dr. David Arnosti, Dr.
Laura McCabe and Dr. Tony Nunez for the helpful discussions, ideas and support they
provided during my PhD. The Genetics Program, Dept. of Physiology, Neuroscience
Program, for funding and support extended throughout my PhD. career. I would like to
thank: Wang lab members especially Dr. Fei Zhang who patiently taught me most of the
techniques; Dr. Ming Zhang and our collaborators Dr. Eric Klann and Dr. Hanoc
Kaphzan for providing electrophysiology data; Dr. Yue Feng and Dr. Wei Feng for
performing the ribosome density gradient centrifugation experiment; Dr. Cara Westmark
and Dr. James Malter for providing Fmr1 knockout mice; Dr. Melinda Frame for help
with microscopy. I sincerely thank Dr. A. J. Robison and Dr. Kenneth Moon for helping
me set up the software for dendritic spine measurements. Thank you Dr. Xiao, Cindy,
Mark and Feiye for always accommodating me to use various lab equipment. All staff
members of the University Laboratory Animal Resources (ULAR), Diagnostic center for
Population and Animal Health and Pharmacokinetics core (University of Michigan). I
would like to thank the FRAXA research foundation for not only providing us a startup

	
  

iv	
  

grant but also providing me the opportunity to get to interact with very helpful colleagues
working on Fragile X syndrome. These interactions were crucial to the development of
my project ideas and experimental design, since our lab was just beginning research on
Fragile X. I would also like to thank NIH (R01MH093445) for funding our research.
Thank you, Mrs. Kelley Peel for introducing me to two lovely children with Fragile
X and giving me the opportunity to talk to their parents. This experience really put my
research in the lab into real-life perspective. Thanks to my family and friends especially
Mom and Beans for their unconditional love and support. Thank you Sudarshan- for
everything, I don’t know where to begin!

	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  

	
  

v	
  

TABLE OF CONTENTS

LIST OF TABLES…………………………………………………………………………….... ix
LIST OF FIGURES………………………………………………………………………………x
KEY TO SYMBOLS OR ABBREVIATIONS………………………………………………....xii
CHAPTER 1: INTRODUCTION………………………………………………………………..1
1.1 Fragile X Syndrome: A genetic disorder………………………………………….1
1.2 Animal models of FXS……………………………………………………………...2
1.3 Role of FMRP in mRNA metabolism……………………………………………...7
1.3.1 RNA binding activity of FMRP…………………………………………........7
1.3.2 RNA targets of FMRP............................................................................10
1.3.3 FMRP regulates mRNA metabolism......................................................12
1.4 Role of FMRP in regulating synaptic function……………………………….....14
1.4.1 Exaggerated mGluR1/5-LTD in Fmr1 KO mice.....................................14
1.4.2 Effects of mGluR inhibition on FXS.......................................................16
1.4.3 mGluR-Independent mechanisms.........................................................17
1.5 Effects of manipulating FMRP targets on FXS…………………………………18
1.6 Connecting the mGluR theory and FMRP targets……………………………..24
1.7 Concluding remarks……………………………………………………………….25
APPENDIX......................................................................................................................27
RERERENCES………………………………………………………………………………...37
CHAPTER 2: TYPE I ADENYLYL CYCLASE AS A POTENTIAL THERAPEUTIC
TARGET FOR FRAGILE X SYNDROME…………………….....................55
2.1 ABSTRACT....................................................................................................55
2.2 Introduction……………………………………………………………..................56
2.3 Materials and Methods………………………………………….........................57
2.3.1 Animals..................................................................................................57
2.3.2 Biochemical analyses............................................................................57
2.3.3 Drugs and treatments............................................................................59
2.3.4 Behavioral testing..................................................................................60
2.3.5 Dendritic spine analysis.........................................................................62
2.3.6 NB001 toxicology studies......................................................................63
2.3.7 NB001 pharmacokinetic studies............................................................63

	
  

iv	
  

2.3.8 Statistical analysis.................................................................................64
2.4 Results………………………………………………………...............................65
2.4.1 AC1 mRNA translation is elevated in the Fmr1 KO hippocampus........65
2.4.2 FMRP regulates AC1 translation by microRNA mediated
mechanisms...........................................................................................66
2.4.3 Rescue of several FXS phenotypes in DKO mice.................................67
2.4.4 Pharmacological rescue of FXS phenotypes by an AC1 inhibitor.........70
2.4.5 NB001 toxicity and pharmacokinetics....................................................72
2.5 Discussion……………………………………………………………...................73
APPENDIX......................................................................................................................76
REFERENCES………………………………………………………………………………..102
CHAPTER 3: TRIFLUOPERAZINE INHIBITS GQ-COUPLED RECEPTOR SIGNALING
AND RESCUES CELLULAR AND BEHAVIORAL ABNORMALITIES IN A
MOUSE MODEL OF FRAGILE X SYNDROME……………....................107
3.1 ABSTRACT..................................................................................................107
3.2 Introduction…………………………………………………..............................109
3.3 Materials and Methods…………………………………………………............112
3.3.1 Animals................................................................................................112
3.3.2 Neuronal cell culture and Western blotting..........................................112
3.3.3 Electrophysiology................................................................................113
3.3.4 Behavioral tests...................................................................................114
3.3.5 Administration of Trifluoperazine.........................................................116
3.3.6 Measurements of protein synthesis in cultured neurons.....................116
3.3.7 Statistical analysis...............................................................................117
3.4 Results……………………………………………………………......................118
3.4.1 Calmodulin activity is required for mGluR1/5-mediated intracellular
signaling...............................................................................................118
3.4.2 CaM activity is required for mGluR1/5-mediated LTD.........................119
3.4.3 ERK1/2 activity is required for mGluR1/5-mediated upregulation of
Arc expression.....................................................................................120
3.4.4 Trifluoperazine attenuates audiogenic seizures and corrects
hyperactivity, repetitive behavior, and defective passive avoidance
memory in Fmr1 KO mice....................................................................121
3.4.5 Trifluoperazine blocks intracellular signaling triggered by the activation
of Gq-coupled receptors.......................................................................124
3.4.6 Trifluoperazine corrects the enhanced protein synthesis in Fmr1 KO
neurons................................................................................................125
3.5 Discussion………………………………………………………………………...126
APPENDIX....................................................................................................................132
	
  

v	
  

REFERENCES………………………………………………………………………………..150
CHAPTER 4: CONCLUDING REMARKS AND FUTURE DIRECTIONS……………....154

	
  

vi	
  

LIST OF TABLES

Table 1

Human FXS traits that are recapitulated in Fmr1 knockout mice…..........28

Table 2

FMRP targets that may be functionally involved in FMRP-regulated
translation and FXS-related phenotypes………........................................29

Table 3

Rescue of audiogenic seizure in DKO mice………………........................84

Table 4

NB001 rescues audiogenic seizure in Fmr1 KO mice…………….............91

Table 5

Chronic high dose NB001 administration does not alter blood chemistry in
mice…………………………………………………….................................101

Table 6

TFP rescues audiogenic seizure in Fmr1 KO mice……………...............140

	
  

vii	
  

LIST OF FIGURES

Figure 1

FMRP mRNA targets whose proteins are activated/translated upon
mGluR5 stimulation………………………………………….........................36

Figure 2

FMRP represses AC1 translation…………………………………...............77

Figure 3

Rapid translation of AC1 upon mGluR5 activation in the presence of
FMRP…………………………….................................................................79

Figure 4

FMRP controls AC1 translation by microRNA-mediated mechanisms......81

Figure 5

Rescue of hyperactive/repetitive phenotypes in DKO mice…………........85

Figure 6

Rescue of social behavior but not open-field hyperactivity in DKO
mice….......................................................................................................87

Figure 7

Impaired cognitive function in DKO mice mice……………........................89

Figure 8

Rescue of dendritic spine phenotype in DKO mice………………………...90

Figure 9

NB001 rescues hyperactive/repetitive phenotypes in Fmr1 KO mice…...92

Figure 10

NB001 rescues open-field hyperactivity and social deficit in Fmr1 KO
mice….......................................................................................................94

Figure 11

NB001 rescues a cognitive deficit in Fmr1 KO mice.................................96

Figure 12

NB001 normalizes exaggerated protein synthesis in Fmr1 KO
neurons…..................................................................................................97

Figure 13

NB001 toxicity studies………………………………………………………...98

Figure 14

Pharmacokinetics of NB001…………………………………………….......100

	
  

viii	
  

Figure 15

mGluR5 stimulation leads to phosphorylation of ERK1/2 and S6 kinase
and Arc translation………………………………………….........................133

Figure 16

Calmodulin activity is required for mGluR1/5 signaling……....................135

Figure 17

Calmodulin activity is required for mGluR1/5-mediated synaptic LTD….137

Figure 18

Inhibition of ERK1/2 suppresses mGluR1/5 mediated Arc upregulation
and LTD………………..……………….....................................................138

Figure 19

Trifluoperazine attenuates the hyperactivity phenotype in Fmr1 KO
mice.........................................................................................................141

Figure 20

Trifluoperazine rescues repetitive behavior in Fmr1 KO mice…….........143

Figure 21

Trifluoperazine rescues the impaired passive avoidance memory in Fmr1
KO mice………………….........................................................................145

Figure 22

Trifluoperazine suppresses mGluR1/5- and muscarinic receptor-mediated
intracellular signaling……………………………………............................146

Figure 23

Trifluoperazine suppresses enhanced basal protein translation in Fmr1 KO
neurons…………………………………………..........................................148

	
  

ix	
  

KEY TO SYMBOLS OR ABBREVIATIONS

AC1

Adenylyl cyclase 1

Adcy1

Adenylyl cyclase 1

AGS

Audiogenic seizure

Arc

Activity-regulated cytoskeleton-associated protein

CaM

Calmodulin

CCh

Carbamylcholine chloride

DHPG

Dihydroxyphenylglycine

DKO

Double knockout

Fmr1

Fragile X mental retardation 1

FMRP

Fragile X mental retardation 1 protein

FXS

Fragile X syndrome

Gq

Gq protein

KO

Knockout

LTD

Long- term depression

mAchR

Muscarinic acetylcholine receptor

MEK

Mitogen-activated kinase kinase

	
  

x	
  

mGluR1/5

Metabotropic glutamate receptor 1/5

miRNA

microRNA

NB001

5-[[2-(6-Amino-9H-purin-9-yl)ethyl]amino]-1-pentanol

pERK1/2

Phospho extracellular-signal-regulated kinases 1/2

S6K

Ribosomal S6 kinase

tERK1/2

Total extracellular-signal-regulated kinases 1/2

TFP

Trifluoperazine- hydrochloride

U0126

1,4-Diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene

UTR

Untranslated region

W13

N-(4-Aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride

WT

Wild-type

	
  

xi	
  

CHAPTER 1: INTRODUCTION

Adapted from Publication: Sethna, F., Moon, C., Wang, H., 2013. From FMRP Function
to Potential Therapies for Fragile X Syndrome. Neurochemical Research [Epub ahead
of print]

1.1 Fragile X Syndrome: A genetic disorder
Fragile X syndrome (FXS), is the most common form of inherited intellectual
disability (or mental retardation) and a leading cause of autism (Santoro et al., 2012).
The incidence of FXS in males is approximately 1 in 2,500–5,000 and in females is 1 in
4,000–6,000 (Bagni et al., 2012). The cytogenetic discovery of the ‘‘fragile site’’ on the X
chromosome in patients (Krawczun et al., 1985) and the higher incidence in males
strongly suggest that FXS is a genetic disease. Positional cloning definitively
demonstrates the link between mutations in the fragile X mental retardation 1 (FMR1)
gene, whose chromosome locus is at the Xq27.3 fragile site, and FXS (Pixxuti et al.,
1991). Most FXS patients have a significant expansion of CGG trinucleotide repeats in
the 5’ untranslated region (UTR) of the FMR1 gene. While healthy individuals have 5–45
CGG repeats (commonly 29–30 repeats), affected individuals with full mutation normally

	
  

1	
  

have more than 200 repeats (Peprah, 2012). A few cases with missense mutations and
deletions in the FMR1 gene have also been reported (De Boulle K et al., 2010).
Studies show that the high number of the CGG repeats may facilitate
hypermethylation on the cytosine residues in the proximal regions of FMR1, including
the promoter (i.e. about 250 bp to 1 kb upstream of the CGG repeats), leading to
transcriptional silencing and consequently lack of protein expression (Pieretti et al.,
1991; Sutcliffe et al., 1992). Other mechanisms posit that a full mutation in CGG repeats
affects histone modification (including acetylation and methylation) and may in turn
suppress the activity of the FMR1 promoter (Coffee et al., 2002).

1.2 Animal models of FXS
The development of valid animal models has been crucial for understanding FXS
etiology, the function of FMRP and has been invaluable in the conception of potential
therapeutics for FXS. The main animal models of FXS have been generated with mouse
(Dutch-belgian et al., 1994), fruit fly (Morales et al., 2002), and zebrafish (den Broeder
et al., 2009), in which the genetic ortholog of human FMR1 is deleted. In another mouse
model, the wild type Fmr1 allele was mutated to harbor an isoleucine to asparagine
mutation (I304N, corresponding to the I367N mutation in a rare FXS patient) (De Boulle,

	
  

2	
  

et al., 1993; Zang et al., 2009). It is important to note that the mouse model with an
engineered expansion in CGG repeats does not show hypermethylation and lack of
FMRP expression (Brouwer et al., 2007). Thus, animal models with perfect construct
validity are not available. Stem cells from FXS patients show FMR1 silencing due to
DNA hypermethylation upon differentiation (Eiges et al., 2007), and can be used for
drug screening and preliminary examination of gene reactivation therapies (Chiurazzi et
al., 1998, 1999).
Behavioral and physiological examinations have demonstrated that the current
animal models show robust if not complete face validity of FXS. Some of the therapeutic
strategies, which attenuate certain FXS-related symptoms in the animal models, have
now been extended to human clinical trials, indicating reasonable predictive validity.
FXS is characterized by mild to severe intellectual disability, susceptibility to seizures,
hyperactivity, hypersensitivity to sensory stimuli, and autistic traits such as social
anxiety, attention deficit, hand biting or flapping (repetitive behavior), and poor eye
contact. Physical manifestations include long facial features with protruding ears, soft
skin, connective tissue problems, and large testicles (macroorchidism). Many of these
symptoms are recapitulated in the Fmr1 knockout (KO) mouse (Table 1). Fmr1 KO mice
show cognitive deficits when examined by Morris water maze (Dobkin et al., 2000;

	
  

3	
  

Baker et al., 2010)(but also see (Paradee et al., 1999)), passive avoidance (Qin et al.,
2005; Dölen et al., 2007), contextual fear conditioning (Paradee et al., 1999)(but also
see (Van Dam et al., 2000)), and object recognition (Bhattacharya et al., 2012).
Susceptibility to seizures in Fmr1 KO mice is implicated by wild- running and onset of
seizure after receiving a high intensity sound (e.g. 125 dB at 1,800–6,300 Hz)
(Musumeci et al., 2000; Chen and Toth, 2001). In addition to audiogenic seizures
(AGS), Fmr1 KO mice also show enhanced limbic epileptogenesis and mossy fiber
sprouting

following

a

kindling

paradigm

(Qiu

et

al.,

2009).

Furthermore,

electrophysiological studies have identified prolonged epileptiform discharges in the
Fmr1 KO hippocampus (Chuang et al., 2005). Fmr1 KO mice are hyperactive and have
more locomotor movement in the open field test (Qin et al., 2002). They also show more
entries to and spend more time in the center area of the open field arena (Qin et al.,
2002; Yan et al., 2005), indicating less anxiety (in contrast to the human FXS
phenotype). However, in a modified open field chamber surrounded with mirrored walls,
Fmr1 KO mice avoid the center area (Spencer et al., 2005). Interestingly, independent
groups have found that Fmr1 KO mice show more (Bilousova et al., 2009), normal
(Nielsen et al., 2002), or less anxiety (Liu et al., 2011) in the elevated plus maze test.
Hyperarousal and sensorimotor gating phenotypes have been examined by acoustic

	
  

4	
  

startle responses and prepulse inhibition (PPI), respectively. While some studies show
that low intensity white noise (at 80 dB) elicits higher startle responses and high
intensity stimuli (at 120 dB) cause less startle in Fmr1 KO mice (Nielsen et al., 2002),
other studies demonstrate that deletion of the Fmr1 gene in mice causes no change or
lower startle response to different levels of auditory stimuli (Pietropaolo et al., 2011;
Thomas et al., 2011). Reduced PPI (a symptom observed in human FXS patients)
(Frankland et al., 2004) is seen in some investigations using Fmr1 KO mice (de Vrij et
al., 2008; Levenga et al., 2011), while other reports have described increased PPI
(Chen and Toth, 2001; Nielsen et al., 2002; Frankland et al., 2004; Pietropaolo et al.,
2011; Thomas et al., 2011). Autism-related symptoms are also detected in Fmr1 mutant
mice (Pietropaolo et al., 2011). Fmr1 KO mice show less social dominance than wild
type animals in the social dominance tube test (Spencer et al., 2005; Goebel-Goody et
al., 2012). Fmr1 mutants are less interested in social novelty and social interaction
(Mines et al., 2010; Liu et al., 2011; Pietropaolo et al., 2011; Bhattacharya et al., 2012).
Defective communication (tested by ultrasonic vocalization) (Wöhr and Scattoni, 2013)
and repetitive behavior (tested by marble burying) (Thomas et al., 2009) are also
detected in Fmr1 KO mice (Rotschafer et al., 2012; Roy et al., 2012). FXS model mice
harboring the I304N mutation exhibit hyperactivity, decreased acoustic startle response,

	
  

5	
  

repetitive behavior, and audiogenic seizures (Zang et al., 2009). In addition to the
neurological phenotypes, both Fmr1 KO and I304N mutant mice show macroorchidism
(Dutch-belgian et al., 1994; Zang et al., 2009). Furthermore, increased spine density
and immature spines are observed from postmortem FXS brain samples (Irwin et al.,
2001; Grossman et al., 2006), and such cellular abnormalities are detected in different
brain regions of Fmr1 KO mice as well as in cultured mutant hippocampal neurons
(Grossman et al., 2006; Dölen et al., 2007; Hayashi et al., 2007; Bhattacharya et al.,
2012).
In addition to vertebrate models, Drosophila (fruit fly) has been successfully used
to study FXS. Flies with mutations in dfmr1, whose gene product shows similar function
to that of human FMRP (Wan et al., 2000), show altered synaptic structure (Zhang et
al., 2001), altered social interaction, impaired circadian rhythms (Dockendorff et al.,
2002), and defective cognitive function (McBride et al., 2005). Additionally, dfmr1
mutants have been used to validate therapeutic efficacy, understand signaling
dysregulation in FXS, and screen potential pharmacological compounds for FXS therapy
(McBride et al., 2005). Although these animal models do not recapitulate all FXS
symptoms and inconsistent phenotypes have been reported, it is evident that the current
animal models do show reasonable face validity. The inconsistent behavioral

	
  

6	
  

phenotypes observed with Fmr1 KO mice may be due to differences in the experimental
protocol, age (Yun et al., 2006), animal handling (or environmental factors such as
different housing facilities and stress (Qin et al., 2011)), and genetic background
(Paradee et al., 1999; Baker et al., 2010; Pietropaolo et al., 2011). These inconsistent
observations with animal models might reasonably reflect the fact that human FXS
patients do not necessarily display a full spectrum of the symptoms. Importantly,
differences in genetic background and environmental factors possibly contribute to the
fact that FXS patients do not respond equally to behavioral and medical treatment
(Berry-Kravis et al., 2008; Jacquemont et al., 2011).

1.3 Role of FMRP in mRNA metabolism
1.3.1 RNA binding activity of FMRP
FMRP is expressed in many tissues, but is most abundant in the brain and in the
testis. In addition to its expression in the neuronal cell body, FMRP is also detected at
dendrites and synapses (Berwine and Reenough, 1997). Sequence analysis of FMRP
reveals several RNA binding domains, which mediate FMRP-RNA interaction
(Nussbaum and Dreyfuss, 1993; Siomi et al., 1994), implicating its function in regulating
RNA metabolism. Cellular fractionation experiments demonstrate that FMRP co-

	
  

7	
  

sediments with actively translating polyribosomes (Corbin et al., 1997; Feng et al.,
1997a, 1997b), further suggesting its role in regulating mRNA translation. Among the
three canonical RNA binding domains, the two centrally localized hnRNP K-homology
KH domains bind to the ‘‘kissing complex’’ tertiary motifs in RNA. The RGG (arginineglycine-glycine) box is located close to the C terminus and binds to the G-quartet
structures in RNA. The I367N missense mutation discovered in a human patient with
severe FXS symptoms maps to the RNA binding pocket of the KH2 domain (Siomi et
al., 1994). FMRP with the I367N mutation fails to bind to RNA (Siomi et al., 1994) and
polyribosomes (Feng et al., 1997a). Mutant I304N-FMRP also does not show robust
association with polyribosomes. This suggests that the loss of RNA-binding/translationregulating function of FMRP may be causal for the phenotypes in FXS. RNA selection
experiments in vitro have revealed that the KH2 domain of FMRP binds to an RNA
complex called loop–loop pseudoknot or ‘‘kissing complex’’. This binding activity is
abolished in I304N-FMRP (Darnell et al., 2005). Further, RNA containing the ‘‘kissing
complex’’ but not G-quartet decreases the association of FMRP with polyribosomes
(Darnell et al., 2005), suggesting that the ‘‘kissing complex’’ mimics the site that FMRP
uses to regulate translation of its target mRNA. However, it is important to note that the
kissing complex structure has not been yet convincingly identified in endogenous

	
  

8	
  

mRNAs. The C-terminal RGG box has been found to bind to G-quadruplex RNA
secondary structures in vitro (Darnell et al., 2001). Several FMRP target mRNAs (such
as Fmr1, Map1b, and Sema3f) possess predicted G-quadruplex structures, and in vitro
biochemical examinations have confirmed their binding to FMRP (Santoro et al., 2012).
A new structure SoSLIP (Sod1 stem loops) in Sod1 (superoxide dismutase 1) mRNA
may also interact with FMRP via the RGG domain (Bechara et al., 2009). In addition to
the RNA binding domains, FMRP possesses two other regions (i.e. nuclear localization
signal and nuclear export signal) that enable it to shuttle between the cytoplasm and the
nucleus (Eberhart et al., 1996). It is postulated that FMRP may pick up its mRNA targets
in the nucleus and transport them to the dendrites, where local protein synthesis is
regulated in an activity-dependent manner. It is shown that some protein–protein
interaction domains may also exist to mediate FMRP association with proteins involved
in translational regulation (Napoli et al., 2008) and the RNA- induced silencing complex
(RISC) (Jin et al., 2004).

	
  

9	
  

1.3.2 RNA targets of FMRP
It is estimated that FMRP binds to roughly 4 % of the mRNAs in the brain
(Santoro et al., 2012). An earlier genome-wide microarray study identified 432 FMRPinteracting mRNAs (Brown et al., 2001). A recent study using UV cross-linking to
covalently link FMRP to mRNA followed by stringent coimmunoprecipitation and high
throughput sequencing identified 842 mRNA targets of FMRP in postnatal 11–25 day
mouse brain. This study found that many of the targets were involved in synaptic
function, cell signalling, neural development, and autism. Further, most of the FMRP
binding sites were in the coding region but not in the 5’ or 3’ UTR of the mRNA targets
and no specific sequence or structural feature was identified as preferred FMRP-binding
motifs (Darnell et al., 2011). Another recent study used 4-thiouridine (4SU)
photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PARCLIP) in HEK293 cells expressing HA-tagged FMRP. Complementary DNA libraries
were generated and sequenced. By analyzing the resulting reads, the study identified
around 6,000 mRNAs that were bound to FMRP. More than 95 % of the binding sites
were either in the coding region or the 3’ UTR. Unlike the previous study, this study
identified only few more binding sites in the coding region than in the 3’ UTR. Further,
two RNA-recognition elements (RRE), ACUK and WGGA (where K is G or U and W is A

	
  

10	
  

or U), were found to occur in more than 50 % of the binding sites (Ascano et al., 2012).
Consistent with the study by Darnell et al. (Darnell et al., 2011), many of the identified
FMRP targets were also involved in autism spectrum disorder and synaptic signaling. It
remains unclear whether these FMRP targets are functionally regulated by FMRP and
contribute to cellular and behavioral abnormalities in FXS. Among all the identified
FMRP targets, only a handful of them are verified by independent methods and
functional studies (see Table 2). Interestingly, FMRP also interacts with non-coding
microRNAs (miRNAs) (Jin et al., 2004; Edbauer et al., 2010). Among them, miR-125a
and 125b can cooperate with FMRP to regulate the translation of validated FMRP
targets PSD-95 and NR2A, respectively (Edbauer et al., 2010; Muddashetty et al.,
2011). Further, overexpression of miR-125b results in longer and thinner dendritic
spines (Edbauer et al., 2010), which is a cellular phenotype of FXS (Grossman et al.,
2006). A functional significance of FMRP-miRNA interaction is that FMRP may regulate
translation through coordination with miRNAs that bind to the 3’ UTR of FMRP target
mRNAs.

	
  

11	
  

1.3.3 FMRP regulates mRNA metabolism
The existence of RNA binding domains in FMRP suggests its function in
regulating RNA metabolism. Although some studies have demonstrated that FMRP
regulates mRNA transport (Dictenberg et al., 2008) and stability (Zalfa et al., 2007), it is
well accepted that FMRP mainly suppresses the translation of its target mRNAs. In
addition to mRNAs, FMRP also interacts with proteins such as CYFIP1 that regulate
translation (Napoli et al., 2008). FMRP has been found to co-sediment with actively
translating polyribosomes particularly in synaptic preparations (Corbin et al., 1997; Feng
et al., 1997a). In Fmr1 KO neurons, FMRP targets such as MAP1B (Lu et al., 2004) and
PSD-95 (Muddashetty et al., 2011) mRNAs are more enriched in the actively translating
polyribosomes rather than in the translationally quiescent messenger ribonucleoprotein
(mRNP) complexes. Consequently, the expression of many FMRP targets is upregulated in the absence of FMRP (see Table 2). Thus FMRP is believed to control local
protein synthesis at synapses by acting as a translational repressor and the loss of such
translation control leads to many of the defects seen in FXS. In a rare case, FMRP
binding to Sod1 mRNA positively regulates its translation (Bechara et al., 2009). It is
important to point out that the protein levels of many FMRP targets remain unchanged
or even reduced in Fmr1 KO mice (see Table 2). To fully understand how the

	
  

12	
  

expression level of FMRP targets is controlled, investigations on compensatory
mechanisms and secondary effects are needed. Considering that FMRP interacts with
the translation machinery and some of the FMRP targets (such as S6K1, PI3 K, PIKE,
and ERK1/2) can indirectly stimulate translation (Figure 1), it is understandable that the
basal level of global protein synthesis is increased in Fmr1 KO mouse brains and in cell
cultures derived from FXS patients (Dölen et al., 2007; Gross et al., 2010; Osterweil et
al., 2010). In addition to its involvement in the cap-dependent initiation and elongation
steps ( et al., 2011), FMRP may also impede translation through coordination with
miRNAs that bind to both FMRP and the 3’ UTR of FMRP targets. The interaction
between FMRP and components of the RISC (such as AGO1/2 and Dicer) may provide
another layer of control on RNA metabolism. For example, the expression of FMRP
target NR2A is regulated through the coordination of FMRP and miR-125, and
knockdown of AGO1 increases NR2A expression (Edbauer et al., 2010). As the basal
translation is elevated in FXS, activity-dependent upregulation of translation is
dampened in Fmr1 KO neurons. As opposed to wild type neurons, Fmr1 KO neurons do
not show increased translation following the activation of mGluR1/5 (Todd et al., 2003;
Hou et al., 2006; Muddashetty et al., 2011), NMDAR (Lee et al., 2011), and upon
membrane depolarization (Weiler et al., 2004). Consequently, certain aspects of

	
  

13	
  

mGluR1/5- and NMDAR-dependent synaptic plasticity (such as long-term depression
and long-term potentiation) are altered in Fmr1 KO mice (Bear et al., 2004; Lauterborn
et al., 2007).

1.4 Role of FMRP in regulating synaptic function
1.4.1 Exaggerated mGluR1/5-LTD in Fmr1 KO mice
Synaptic protein synthesis and spine development are altered in FXS, thus it is
hypothesized that FMRP regulates synaptic function and plasticity. Huber et al. (Huber
et al., 2002) showed that synaptic long-term depression (LTD) triggered by mGluR1/5
agonist DHPG is enhanced at the CA1 synapses in the hippocampus of Fmr1 KO mice.
This seminal study builds the foundation of the ‘‘mGluR theory’’, which highlights that
mGluR1/5 signaling is exaggerated in FXS, and explains how FMRP function is
connected to mGluR1/5-mediated synaptic responses (Bear et al., 2004). Multiple lines
of evidence support the functional link between FMRP and mGluR1/5-LTD. First,
mGluR1/5-LTD depends on new protein synthesis. The upregulation of protein
translation following mGluR1/5 activation may be related to the dynamic changes in
FMRP due to mGluR1/5 induced signal transduction. The activation of mGluR1/5
triggers rapid increase in FMRP translation (Berwine and Reenough, 1997) but it is

	
  

14	
  

followed by FMRP degradation and de-phosphorylation, which may cause de-repression
on the translation of certain synaptic mRNA (Hou et al., 2006; Nalavadi et al., 2012;
Niere et al., 2012). Indeed, the translation of several FMRP targets is up-regulated
following DHPG stimulation (see Table 2 and Figure 1). Second, the expression of
mGluR1/5-LTD requires AMPA receptor internalization (Waung et al., 2008). DHPGstimulated receptor internalization depends on the translation of certain ‘‘LTD’’ proteins
such as Arc and STEP (Figure 1). In the absence of FMRP, the translation of these
‘‘LTD’’ proteins is not suppressed. The elevated expression of such ‘‘LTD’’ proteins in
Fmr1 KO neurons consequently facilitates AMPAR internalization (Nakamoto et al.,
2007), leading to enhanced synaptic depression (LTD). It is evident that other
mGluR1/5-mediated synaptic functions are also regulated by FMRP. The activation of
mGluR1/5 stimulates spine growth (Vanderklish and Edelman, 2002) and there are
more immature spines in FXS neurons (Irwin et al., 2001). Collectively, mGluR1/5mediated synaptic function and cellular changes are regulated by FMRP and are
exaggerated in FXS.

	
  

15	
  

1.4.2 Effects of mGluR inhibition on FXS
Based on the mGluR theory, it is postulated that dampening mGluR1/5 activity
may be therapeutic for FXS. Indeed, administration of mGluR5 antagonists to animal
models of FXS has shown promising therapeutic effects. Specifically, administration of
mGluR5 antagonist MPEP attenuates elevated protein translation (Gross et al., 2010;
Osterweil et al., 2010), enhanced AMPAR internalization (Nakamoto et al., 2007),
abnormal spine morphology, PPI (de Vrij et al., 2008) (but also see (Thomas et al.,
2012)), AGS, hyperactivity (Yan et al., 2005), and repetitive behavior (Thomas et al.,
2012) in Fmr1 KO mice. The use of fenobam, a potent negative allosteric modulator of
mGluR5, promisingly attenuates spine abnormality (de Vrij et al., 2008) and impairments
in procedural memory and avoidance discrimination (Vinueza Veloz et al., 2012).
Genetic approaches have further demonstrated the therapeutic role of mGluR1/5 in
FXS. Double mutant mice (heterozygous for mGluR5 and hemizygous for Fmr1) show
normal basal translation, spine density, no significant AGS, and normal fear memory
extinction. However, macroorchidism is not rescued (Dölen et al., 2007). Intriguingly, a
more recent study found that the mGluR5/ Fmr1 double mutants still show AGS,
repetitive behavior, and abnormalities in anxiety and memory (Thomas et al., 2011).
Encouraged by the effects of mGluR5 inhibition, an open label single dose fenobam trial

	
  

16	
  

has been performed with 12 adult patients. While there is mild improvement in PPI, no
significant effect is observed for CPT (continuous performance test) (Berry-Kravis et al.,
2009). New compounds that negatively regulate mGluR5 have been developed and
tested in clinical trials. AFQ056 (Novartis) was found to rescue abnormal spine
morphology and PPI, phenotypes in Fmr1 KO mice (Levenga et al., 2011; Gantois et al.,
2013). In a phase II double blind placebo-controlled crossover trial with AFQ056 on 30
adult males for 28 days, administration of AFQ056 resulted in improvement of
maladaptive behaviors only in a selective subpopulation of FXS patients with full
promoter methylation (Jacquemont et al., 2011). Clinical trials with STX107 (Seaside
therapeutics) also had mostly negative results, a result of which both companies
decided to discontinue further drug development.

1.4.3 mGluR-Independent mechanisms
Inhibition of mGluR1/5 rescues some but not all FXS- related symptoms (Thomas
et al., 2011) and shows therapeutic effects on a sub-population of FXS patients
(Jacquemont et al., 2011). This suggests the existence of mGluR-independent
mechanisms. Similar to mGluR1/5, activation of another group of Gq-coupled receptors,
such as Gq-coupled muscarinic acetylcholine receptors (Gq-mAchR), also triggers

	
  

17	
  

AMPAR internalization and translation-dependent LTD. Gq-mAchR-LTD is significantly
exaggerated in Fmr1 KO mice (Volk et al., 2007). Treating Fmr1 KO mice with an
inhibitor of M1 (one subtype of Gq-mAchR) dampens AGS (Veeraragavan et al., 2011).
It remains to be determined whether simultaneous blocking of mGluR1/5 and GqmAchR offers more robust correction of FXS traits. Several lines of evidence have
demonstrated that alteration of other G-protein coupled receptors are connected to FXS.
For example, genetic or pharmacological inhibition of the Gi-coupled muscarinic M4
receptor rescues limited abnormal behaviors in Fmr1 KO mice (Veeraragavan, S, Bui N,
Perkins J, Yuva-Paylor L, 2011; Veeraragavan et al., 2012). Dopaminergic D1 receptormediated AMPAR surface expression and signaling in prefrontal cortex requires FMRP
and D1 receptor agonist SKF81297 attenuates hyperactivity in Fmr1 KO mice (Wang et
al., 2008).

1.5 Effects of manipulating FMRP targets on FXS
As the basal level of translation is elevated in Fmr1 KO neurons, it is generally
accepted that FMRP suppresses the translation of its target mRNAs. Thus, it is
postulated that the elevated expression of certain ‘‘key’’ FMRP targets may be causal
for FXS, and dampening such ‘‘key’’ targets may be therapeutic. However, this

	
  

18	
  

therapeutic approach is still in its infancy. Only a handful of studies show that
suppressing FMRP targets attenuates cellular abnormalities (Park et al., 2008; Lee et
al., 2011) and certain (but not all) behavioral phenotypes of FXS (Bilousova et al., 2009;
Westmark et al., 2011; Goebel-Goody et al., 2012). Most of the verified FMRP targets
are involved in synaptic plasticity, neurotransmission, and neuronal signalling. Some
targets have a connection to neurological and psychiatric disorders. The therapeutic
value of FMRP targets is investigated using a combination of genetic and
pharmacological approaches. Arc (activity-regulated cytoskeleton-associated protein),
an FMRP target, is associated with the synaptic cytoskeleton network and regulates
AMPA receptor (e.g. GluR1) trafficking (Figure 1) (Park et al., 2008; Waung et al.,
2008). The basal level of Arc expression is elevated in Fmr1 KO neurons (Niere et al.,
2012) and its translation is rapidly stimulated by the activation of mGluR1/5 and is
required for mGluR1/5-LTD (Park et al., 2008; Waung et al., 2008). Genetic deletion of
Arc in wild type and Fmr1 KO mice results loss of mGluR1/5-LTD (Park et al., 2008).
Another FMRP target, APP (amyloid beta precursor protein) (Westmark and Malter,
2007) is also a structural protein that regulates synaptic function as well as
neurodegeneration (Korte et al., 2012). While overexpression of APP in Fmr1 KO mice
increases seizure susceptibility (Westmark et al., 2008), reduction of APP in Fmr1 KO

	
  

19	
  

mice rescues multiple FXS symptoms including AGS, higher density of immature
spines, and the enhanced mGluR1/5-LTD (Westmark et al., 2011). These studies
identify a link between FXS and Alzheimer’s disease (AD), and suggest that therapies
developed for AD to reduce APP level may be used to treat FXS. The expression level
of another FMRP target, STEP (striatal-enriched tyrosine phosphatase) (Figure 1), is
elevated in Fmr1 KO neurons and genetic reduction of STEP rescues some FXS
phenotypes (Goebel-Goody et al., 2012). A number of FMRP targets are functionally
involved in the PI3K (phosphoinositide 3-kinase) and MAPK (mitogen-activated protein
kinase) pathways (see Table 2 and Figure 1), which positively regulate ribosomal
function and translation in an activity-dependent manner. The activity of both PI3K and
MAPK is required for LTD. PI3K activity and the expression of its catalytic subunit p110β
are elevated (Gross et al., 2010; Sharma et al., 2010) in Fmr1 KO neurons. Inhibition of
PI3K by LY294002 suppresses the cellular phenotypes of FXS including exaggerated
basal translation, GluR1 internalization, and spine density (Gross et al., 2010).
Increased expression of an up-stream activator of PI3K (i.e. PIKE or PI3 K enhancer)
and increased activity of a down- stream effector of PI3K (i.e. mTOR or mammalian
target of rapamycin) are observed in the hippocampus of Fmr1 KO mice. Furthermore,
activity of an up-stream suppressor of PI3K (i.e. PTEN or phosphatase and tensin

	
  

20	
  

homolog) is decreased in the hippocampus of Fmr1 KO mice (Sharma et al., 2010).
However, inhibition of mTOR with rapamycin only suppresses mGluR1/5-LTD in wild
type (Hou and Klann, 2004) but not Fmr1 KO mice (Sharma et al., 2010). Treating Fmr1
KO mice with rapamycin does not suppress the elevated basal translation, but does
dampen AGS (Osterweil et al., 2010). ERK1/2 (extracellular signal-regulated kinase
1/2), a component of the MAPK pathway, is an FMRP target. The activity rather than the
expression level of ERK1/2 is elevated in Fmr1 KO mice. Pharmacological inhibition of
ERK1/2 reduces AGS, elevated protein translation, and prolonged epileptiform
discharges in Fmr1 KO mice (Chuang et al., 2005; Osterweil et al., 2010). Osterweil et
al. (Osterweil et al., 2013) reported similar therapeutic effects in Fmr1 KO mouse by
using lovastatin, (a clinically approved cholesterol-lowering drug) to suppress ERK1/2
activity. Although studies on the therapeutic function of ERK1/2 and PI3K have shown
some degree of controversy (Gross et al., 2010; Osterweil et al., 2010), the two
signaling pathways may converge and co-regulate the activity of some FMRP targets.
S6K1 (ribosomal protein S6 kinase 1) is involved in ribosome biogenesis and regulates
protein translation; its activity can be up regulated by ERK1/2- and PI3K-mediated
phosphorylation at different residues (Figure 1) (Zhou et al., 2010). Although the
expression level of S6K1 is normal in Fmr1 KO neurons, phospho-S6K1 is elevated

	
  

21	
  

(Sharma et al., 2010). Genetic deletion of S6K1 in Fmr1 KO mice rescues the enhanced
mGluR1/5-LTD, abnormal spine morphology, and deficits in recognition memory and
social interaction. However, some FXS-related phenotypes such as hyperactivity and
repetitive behavior are not corrected (Bhattacharya et al., 2012). GSK3B is another
FMRP target whose activity can be down regulated by PI3K (Rayasam et al., 2009) and
ERK1/2-mediated phosphorylation (Markou et al., 2008). Since PI3K and ERK1/2
activity are elevated in Fmr1 KO mice, the phosphorylation of both GSK3A and GSK3B
is decreased in FXS (Min et al., 2009a). Thus GSK3 activity is abnormally higher in
Fmr1 KO neurons. Treating Fmr1 KO mice with GSK3 inhibitors lithium and SB- 216763
attenuates AGS, hyperactivity, defective cognitive function, deficits in social interaction,
defective neurogenesis, and abnormal spine morphology (Min et al., 2009a; Yuskaitis et
al., 2010; Liu et al., 2011; Guo et al., 2012). An open label study with 15 FXS males
treated with lithium showed that lithium improves social and maladaptive behavior as
well as auditory memory. However, 7 individuals had side effects of polydipsia and
polyuria (Berry-Kravis et al., 2008). Different subunits of GABA-A receptors are down
regulated in Fmr1 KO mice (El Idrissi et al., 2005; D’Hulst et al., 2006; Curia et al.,
2009). It is not surprising that GABA-A receptor agonists and positive allosteric
modulators (such as diazepam and ganaxolone), which are clinically used as

	
  

22	
  

anticonvulsants, attenuate AGS (Heulens et al., 2012). Another GABA system
modulator, gaboxadol, reduces hyperactivity and PPI in Fmr1 KO mice (Olmos-Serrano
et al., 2011). Although GABA-B receptor mRNA is identified as an FMRP target by highthroughput screening (Darnell et al., 2011), its expression level in FXS is not known.
However, its functional relevance is demonstrated by the fact that GABA-B agonists
baclofen and arbaclofen (STX209) rescue AGS in Fmr1 KO mice. Further, arbaclofen
treatment reduces the enhanced basal translation, AMPA receptor internalization, and
spine density in Fmr1 KO mice (Henderson et al., 2012). The expression of NMDAR
subunits (i.e. NR1 and NR2B) is increased in the neocortex and hippocampus of Fmr1
KO mice (Schutt et al., 2009), but also see (Krueger et al., 2011). FMRP also
suppresses the translation of NR2A (Edbauer et al., 2010). Thus, there is possibly a
combination of excessive NMDAR function and deficient GABA function. Based on this
hypothesis, acamprosate, which acts as an NMDAR antagonist and GABA-A agonist,
was used in an open-label clinical study. Young FXS patients receiving acamprosate
show significant improvement in social behavior and reduction in hyperactivity (Erickson
et al., 2013). Minocycline, an FDA-approved broad-spectrum tetracycline antibiotic,
shows some promising therapeutic effects in human FXS patients. In a pilot open label
study on 50 FXS individuals, minocycline treatment improved cognition, language and

	
  

23	
  

behavior (Utari et al., 2010). In another open label study, 20 FXS individuals treated with
minocycline showed improvement in irritability and other global behavior tests (Paribello
et al., 2010). A double blind, placebo-controlled, crossover trial with minocycline
treatment in FXS children resulted in improvements in anxiety and mood-related
behavior (Leigh et al., 2013). Treating Fmr1 KO neurons and mice with minocycline
corrects

abnormal

spine morphology,

anxiety

phenotype,

defective ultrasonic

vocalization, AGS, and hyperactivity (Bilousova et al., 2009; Rotschafer et al., 2012).
Possible mechanisms include minocycline- mediated inhibition of ribosome function or
MMP9 (matrix metallopeptidase 9), an FMRP target whose expression level is elevated
in FXS (Bilousova et al., 2009; Dziembowska et al., 2013).

1.6 Connecting the mGluR theory and FMRP targets
The mGluR theory and FMRP-mediated translation have been fundamental to the
development of therapeutic approaches for FXS. It is not clear how the dysregulated
translation of FMRP targets contributes to elevated mGluR1/5 signalling in FXS. A
recent study reported that the expression of mGluR5 itself, whose mRNA is identified as
an FMRP target through high through-put screening (Darnell et al., 2011), is elevated in
the prefrontal cortex of FXS patients (Lohith et al., 2013). However, another study has

	
  

24	
  

shown that the expression level of mGluR5 is not changed in the forebrain and
cerebellum of Fmr1 KO mice (Giuffrida et al., 2005). As a major downstream effector of
mGluR1/5 signalling, ERK1/2 shows higher activity in Fmr1 KO neurons but its total
expression level is not changed (Osterweil et al., 2013). Thus, the changes in ERK1/2
may represent an outcome of the elevated mGluR1/5 signaling rather than having a
causal role on the enhanced mGluR5 function. Investigation on the molecules in the
PI3K cascade has found that the expression levels of both PIKE and the p110β subunit
of PI3K are elevated in the absence of FMRP (Gross et al., 2010; Sharma et al., 2010).
Considering that PI3K activation can be triggered by mGluR1/5 stimulation, the
enhanced expression of these FMRP-targets may contribute to the elevated basal
mGluR1/5 activity in FXS.

1.7 Concluding remarks
Since the positional cloning of the FMR1 gene, there have been tremendous
advances in understanding the function of FMRP, which have led to rational designs of
therapeutic approaches. While new agents are being examined in animal models and
clinical trials, successful repurposing of the available drugs such as memantine,
acamprosate, minocycline, fenobam, baclofen, lithium, and lovastatin could benefit FXS

	
  

25	
  

patients without involving the lengthy drug development process. As FMRP regulates
many aspects of neuronal function, simultaneous manipulation of multiple FMRP targets
and/or signaling pathways should also deserve significant consideration. Although the
prevailing theory posits that FMRP suppresses translation and mGluR1/5 signalling,
significant involvement of FMRP in up-regulating protein synthesis and mGluR1/5independent synaptic functions has also been identified. How these newly discovered
functions of FMRP are relevant to FXS etiology remains to be determined.

	
  

26	
  

APPENDIX

	
  

27	
  

Table 1: Human FXS traits that are recapitulated in Fmr1 knockout mice.
FXS symptoms in
human
Intellectual disability

FXS-related behavior in Fmr1 KO mice
Defective spatial learning and memory (Dobkin et al., 2000;
Baker et al., 2010)

Defective passive avoidance memory (Qin et al., 2002,
2005)

Defective contextual memory (Paradee et al., 1999)
Defective recognition memory (Bhattacharya et al., 2012)
Susceptibility to
seizures

Audiogenic seizure (Musumeci et al., 2000; Chen and Toth,
2001)

Enhanced limbic epileptogenesis (Qiu et al., 2009)
Prolonged epileptiform discharges (Chuang et al., 2005)
Hyperactivity

Increased locomotor movement
In open field test (Qin et al., 2002; Liu et al., 2011)

Hyperarousal

Higher acoustic startle responses
to low intensity stimuli (Nielsen et al., 2002)

Abnormal sensorimotor
gating
(reduced PPI)

Variable phenotypes in PPI (Chen and Toth, 2001; Nielsen et

Social anxiety
Defective social
interaction

Deficits in social dominance (Spencer et al., 2005; Goebel-

al., 2002; Frankland et al., 2004; de Vrij et al., 2008; Levenga et al.,
2011; Pietropaolo et al., 2011; Thomas et al., 2011)

Goody et al., 2012)

Decreased interests in social
novelty and social interaction (Yuskaitis et al., 2010; Liu et al.,
2011; Pietropaolo et al., 2011; Bhattacharya et al., 2012)

Defective communication
(tested by ultrasonic vocalization) (Roy et al., 2012)
Perseveration/repetitive Increased marble burying (Zang et al., 2009; Rotschafer et al.,
2012)
behavior
Macroorchidism

Enlarged testes (Dutch-belgian et al., 1994; Zang et al., 2009)

Higher density of
immature spine

Higher density of immature spine (Grossman et al., 2006;
Dölen et al., 2007; Hayashi et al., 2007; de Vrij et al., 2008;
Bhattacharya et al., 2012)

	
  

28	
  

Table 2: FMRP targets that may be functionally involved in FMRP-regulated translation
and FXS-related phenotypes
Gene

Protein

Validation

Changes in

Response to

symbol

name

method

FXS

mGluR1/5

Therapeutic effects

Inhibitors

stimulation
Agap2

App

HT

éprotein

(Darnell et al.,

(Sharma et al.,

2011)

2010)

HT (Darnell

éprotein

étranslation

AGS, hyperactivity, spines,

et al., 2011;

(Westmark et al.,

(Westmark and

mGluR-LTD (Westmark et al.,

Ascano et al.,

2011)

Malter, 2007)

2011)

HT (Darnell

éprotein

étranslation

mGluR-LTD (Park et al.,

et al., 2011;

(Zalfa et al.,

(Park et al., 2008;

2008)

Ascano et al.,

2003)

Waung et al.,

2012),

ê protein

2008)

CoIP (Zalfa

(Krueger et al.,

et al., 2003)

2011)

CaMKII

HT(Darnell

éprotein

étranslation

KN62,

α

et al., 2011),

(Zalfa et al.,

(Hou et al., 2006;

KN93

CoIP (Zalfa

2003; Hou et al.,

Kao et al., 2010;

et al., 2003;

2006)

Osterweil et al.,

PIKE

APP

2012)

, CoIP
(Westmark
and Malter,
2007)

Arc

Camk2a

Arc

Muddashetty

2010)

et al., 2007)

Cyfip2

CYFIP2

HT (Darnell

éprotein

et al., 2011;

(Hoeffer et al.,

Ascano et al.,

2012)

2012)

	
  

29	
  

Table 2 (cont’d)
Dlg4

PSD-95

HT(Darnell

éprotein

étranslation

et al., 2011),

(Todd et al.,

(Todd et al.,

miR-125a

2003)

2003;

(Muddashetty

êprotein

Muddashetty et

et al., 2011),

degradation

al., 2011)

G-

(Tsai et al.,

quadruplex

2012)

(Todd et al.,

ê protein

2003)

(Krueger et al.,
2011)

EF1a

Fmr1

EF1α

FMRP

CoIP (Sung

éprotein

étranslation

et al., 2003)

(Sung et al.,

(Huang et al.,

2003)

2005)

In vitro

ê or lack of

étranslation

(Schaeffer et

expression

followed by
degradation

al., 2001)

(Berwine and
Reenough, 1997;
Hou et al., 2006;
Nalavadi et al.,
2012; Niere et
al., 2012)

Gabbr1

GABA-

HT (Darnell

Protein synthesis, AMPAR

STX209,

B1

et al., 2011)

internalization, abnormal

baclofen

spines, AGS (Pacey et al.,
2009; Henderson et al., 2012)

Gabrd

GABA-

In vitro

êprotein

AGS(Heulens et al., 2012),

Acampros-

Aδ

(Miyashiro et

(Curia et al.,

hyperactivity and

ate,

al., 2003)

2009)

PPI(Olmos-Serrano et al., 2011)

alphaxalone
, gaboxadol,
diazepam

	
  

30	
  

Table 2 (cont’d)
Gria1

AMPA-1

HT, Co-IP

éprotein

étranslation

(Muddashetty

(Muddashetty et

(Muddashetty et

et al., 2007)

al., 2007; Schutt

al., 2007)

et al., 2009)

no change
(Giuffrida et al.,
2005)

Grin1

NR1

HT(Darnell

éprotein

Some clinical efficacy in

Memantine,

et al., 2011),

(Schutt et al.,

human patients (Erickson et

Acampros-

CoIP (Schutt

2009)

al., 2009, 2013)

ate

et al., 2009)

no change
(Giuffrida et al.,
2005)

ê protein
(Krueger et al.,
2011)

Grin2a

NR2A

miR-125b

no change

Some clinical efficacy in

Memantine,

(Edbauer et

(Giuffrida et al.,

human patients (Erickson et

Acampros-

al., 2010)

2005)

al., 2009, 2013)

ate

ê protein
(Krueger et al.,
2011)

Grin2b

NR2B

HT(Darnell

éprotein

Some clinical efficacy in

Memantine,

et al., 2011),

(Schutt et al.,

human patients (Erickson et

Acampros-

CoIP (Schutt

2009)

al., 2009, 2013)

ate

et al., 2009)

no change
(Giuffrida et al.,
2005)

ê protein
(Krueger et al.,
2011)

	
  

31	
  

Table 2 (cont’d)
Gsk3b

GSK3β

HT (Darnell

éactivity

AGS, hyperactivity,

Lithium,

et al., 2011;

(Min et al.,

passive avoidance memory,

SB216763

Ascano et al.,

2009a)

contextual, and cued fear
memory, social preference

2012)

and social anxiety,
defective neurogenesis,
spine abnormality (Min et al.,
2009b; Mines et al., 2010;
Yuskaitis et al., 2010; Liu et al.,
2011; Guo et al., 2012)

Hcn1

éprotein

HCN1

(Brager D.
Akhavan A,
2012)

Homer

Homer1

1

HT (Ascano

Unchanged

Restored mGluR5

et al., 2012)

(Ronesi et al.,

signaling, AGS, anxiety

2012)

phenotype in open field
(Ronesi et al., 2012)

Kcnc1

Kcnd2

Kv3.1;

In vitro

éprotein

Kv3.2

(Darnell et al.,

(Strumbos et al.,

2001)

2010)

HT(Darnell

éprotein(Lee

et al., 2011),

et al., 2011),

toxin

in vitro (Lee

ê protein

HpTx2

et al., 2011)

(Gross et al.,

Kv4.2

LTP (Lee et al., 2011)

2011)

Map1b

MAP1B

HT (Darnell

éprotein

étranslation

et al., 2011;

(Zalfa et al.,

(Hou et al., 2006;

Ascano et al.,

2003; Lu et al.,

Chen and Shen,

2012)

2004; Hou et al.,

2013)

In vitro (Lu

2006)

et al., 2004;
Menon et al.,
2008)

	
  

32	
  

Heteropoda-

Table 2 (cont’d)
Mapk1

MAPK1

HT (Darnell

éphosphoryl

éactivation

Protein synthesis, AGS

U0126, SL

or ERK2

et al., 2011;

ation (Hou et

(Hou et al., 2006;

(Osterweil et al., 2010, 2013)

327,

Ascano et al.,

al., 2006;

Osterweil et al.,

2012)

Osterweil et al.,

2010)

lovastatin

2013)

Mmp9

Mtor

MMP9

mTOR

éprotein

Spine abnormality, anxiety

(Bilousova et al.,

phenotype in elevate plus

2009;

maze, AGS, hyperactivity,

Dziembowska et

communication (Bilousova et

al., 2013)

al., 2009; Rotschafer et al., 2012)

HT (Darnell

éphosphory-

éactivation

et al., 2011;

lation

(Sharma et al.,

Ascano et al.,

éactivity

2010)

2012)

(Sharma et al.,

Minocycline

AGS (Osterweil et al., 2010)

Rapamycin

HT (Darnell

Spine abnormality, LTP,

FRAX486

et al., 2011;

hyperactivity, repetitive

Ascano et al.,

behavior, anxiety, trace fear

2012)

conditioning, AGS (Hayashi

2010)

Pak1

PAK1

et al., 2007; Dolan et al., 2013)

Pik3cb

PI3K

HT (Ascano

éprotein

étranslation

Protein synthesis, spine

LY294002

P110β

et al., 2012)

éactivity

éactivation

abnormality, AMPA

Wortmannin

catalytic

(Gross et al.,

(Gross et al.,

receptor internalization

subunit

2010; Sharma et

2010)

(Gross et al., 2010)

al., 2010)

Pten

PTEN

HT (Darnell

êphosphory-

et al., 2011;

lation (Sharma

Ascano et al.,

et al., 2010)

2012)

Ptpn5

	
  

STEP

HT (Darnell

éprotein

étranslation

AGS, social interaction,

et al., 2011)

(Goebel-Goody

(Zhang et al.,

social anxiety (Goebel-Goody

et al., 2012)

2008)

et al., 2012)

33	
  

Table 2 (cont’d)
Rgs5

RGS5

In vitro
(Miyashiro et
al., 2003)

Rps6kb

S6K1

1

HT (Ascano

éphosphory-

éactivation

Protein synthesis, mGluR-

et al., 2012)

lation

(Sharma et al.,

LTD, dendritic spines,

(Bhattacharya et

2010)

novel object recognition,
social interaction

al., 2012)

(Bhattacharya et al., 2012)

Sapap3/

SAPAP3

éprotein

4

/4

(Schutt et al.,
2009)

êprotein
(Krueger et al.,
2011)

Shank1

SHANK

HT(Darnell

éprotein

étranslation

1

et al., 2011;

(Schutt et al.,

(Schutt et al.,

Ascano et al.,

2009)

2009)

2012),

3’UTR
(Schutt et al.,
2009)

Shank3

SHANK

HT (Darnell

éprotein

3

et al., 2011)

(Schutt et al.,
2009)

Sod1

Tsc2

SOD1

TSC2

In vitro

êprotein

(Bechara et

(Bechara et al.,

al., 2009)

2009)

HT (Darnell

mGluR-LTD, memory

et al., 2011;

deficit (Auerbach et al., 2011)

Ascano et al.,
2012)

	
  

34	
  

Table 2 (cont’d)
Key: HT: high throughput screening; CoIP: coimmunoprecipitation; AGS: audiogenic
seizure; PPI: prepulse inhibition; é: increase; ê: decrease. Therapeutic effects are
observed in Fmr1 KO mice when the corresponding FMRP target is genetically reduced
or pharmacologically inhibited.

	
  

35	
  

Molecular)mechanisms)of)mgluR2LTD)at)the)
synapse)
Glutamate(

mGluR5(

PSD95(

Homer(
PIKE(

Internalization

PI3K(

STEP(
mTOR(

ERK1/2(

NMDAR((NR1,(
NR2A,(NR2B)(

AMPAR(
(AMPA1)(

MAP1B(

Arc(

FMRP(

T421(

p70(S6(kinase(

Regula9on)
of)protein)
transla9on)

T389(

Figure 1: FMRP mRNA targets whose proteins are activated/translated upon
mGluR5 stimulation.

	
  

36	
  

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CHAPTER 2: TYPE I ADENYLYL CYCLASE AS A POTENTIAL THERAPEUTIC
TARGET FOR FRAGILE X SYNDROME

2.1 ABSTRACT
Fragile X syndrome (FXS) is the most common cause of inherited mental
retardation and autism. It is caused by silencing of the FMR1 gene, which encodes the
fragile X mental retardation protein (FMRP). The loss of FMRP, a translational
repressor, leads to excessive translation of its mRNA targets, which results in altered
synaptic signaling and the various phenotypes of FXS. Here we identify type 1 adenylyl
cyclase (AC1), which is neurospecifically expressed, as an mRNA target whose
translation is controlled by FMRP. Our results show that AC1 mRNA translation is
upregulated in the absence of FMRP and is responsive to mGluR5 stimulation only in
the presence of FMRP. Further using a genetic double knockout and acute inhibition of
AC1 by NB001, our study demonstrates the rescue of several cellular and behavioral
phenotypes in the Fmr1 knockout mouse model. Our study thus identifies AC1, a novel
mRNA target whose translation is controlled by FMRP as a potential therapeutic target
for FXS.

	
  

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2.2 Introduction
FXS is the most common inherited form of mental retardation and a leading
cause of autism (Santoro et al., 2012). It is caused by a trinucleotide repeat expansion
in the FMR1 (Fragile X mental retardation 1) gene, which causes transcriptional
silencing of the gene resulting in reduced expression of its protein FMRP (Pixxuti et al.,
1991). FMRP is an mRNA binding protein that selectively binds to mRNA and
suppresses their translation (Darnell et al., 2001, 2011a). In the absence of FMRP the
translation of these mRNAs is enhanced, leading to exaggerated protein synthesis
(Zalfa et al., 2003; Westmark and Malter, 2007; Gross et al., 2010). A seminal study by
Huber et al showed that the protein-synthesis dependent mGluR5 induced LTD is
enhanced in Fmr1 knockout (KO) mice (Huber et al., 2002). This led to the development
of the “mGluR theory of FXS” which posits that many of the cellular, synaptic and
behavioral phenotypes of FXS are due to exaggerated mGluR signaling (Bear et al.,
2004). Subsequent studies have shown that genetic as well as pharmacological
reduction of mGluR5 activity can rescue several abnormal phenotypes in the Fmr1 KO
mouse model (Yan et al., 2005; Dölen et al., 2007; de Vrij et al., 2008). Another valuable
approach to identify new therapeutic targets for FXS has been to study the mRNA that
is regulated by FMRP and mGluR signaling (Krueger and Bear, 2011). Indeed the

	
  

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dysregulated translation of FMRP mRNA targets is vital to the phenotypes of FXS
(Darnell and Klann, 2013; Sethna et al., 2013). Here we observed that the
neurospecific, Ca2+/Calmodulin stimulated type 1 adenylyl cyclase (AC1) mRNA is a top
FMRP binding target in three high-throughput studies (Brown et al., 2001; Darnell et al.,
2011b; Ascano et al., 2012). Since AC1 is stimulated by Ca2+ and calmodulin which are
downstream to the Gq-coupled signaling associated with mGluR5, we hypothesized that
AC1 mRNA translation would be regulated by FMRP and be responsive to mGluR5
signaling. We further hypothesized that if AC1 expression was elevated in Fmr1 KO
mice, several phenotypic abnormalities in these mice would be corrected by genetic or
pharmacological inhibition of AC1.

2.3 Materials and Methods
2.3.1 Animals
WT C57BL/6 mice were obtained from Jackson Labs. Fmr1 KO C57BL/6 mice were
obtained from Dr. C. Westmark and were crossed into the lab WT C57BL/6 strain for at
least 5 generations. AC1 KO C57BL/6 mice were previously reported (Wu et al., 1995).
DKO mice were generated by crossing Fmr1 KO males with AC1 KO females, followed
by crossing of the female progeny with AC1 KO males. This cross-yielded some DKO

	
  

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males and AC1-/-, Fmr1+/- females. Brother-sister crossing of the above progeny
generated DKO females.

Animals were housed in the university laboratory animal

research facility and all the manipulations were in compliance with the guidelines of
Institutional Animal Care and Use Committee at Michigan State University. The mice
had ad libitum access to water and food and were housed under 12 h dark/light cycles.
2.3.2 Biochemical analyses
WT and Fmr1 KO primary hippocampal neurons were established as described in (Zhou
et al., 2010) and grown to DIV14 for western blotting and RNA extraction. Neurons were
treated with 100mM DHPG (RS)-3,5-Dihydroxyphenylglycine, Tocris Cat #0342) for
various time points before they were lysed in Buffer H (50 mM b-glycerophosphate, 1.5
mM EGTA, 0.1 mM Na3VO4, 1 mM DTT). The concentration of protein in the extracts
was determined by Bradford assay and 20ug protein was loaded on the 4-20% SDSPAGE gel (Invitrogen) and transferred to nitrocellulose membranes. The membranes
were probed with anti-Adcy1 antibody (Sigma Cat #SAB4500146) at diluted in 5% BSA
at 1:1000 and anti-b-actin antibody (Sigma Cat #A1978). Western blot signal intensities
were quantitated using ImageJ (NIH, MD, USA). Protein samples from brain tissues
were processed similarly. DIV8 Fmr1 KO hippocampal neurons were used for luciferase
assays. Neurons were transfected using Lipofectamine 2000 (Life Technologies Cat

	
  

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#11668027) 48 hours before determining luciferase activity using the dual glow
luciferase assay protocol (Promega Cat #E2920). WT FMRP PC-Flag 2.17 and pLuci3
plasmids were kindly provided by Dr. Yue Feng (Emory University). Adcy1 3’ UTR
constructs were cloned into the pLuci3 vector between the KpnI and EcoRI sites.
miRIDIAN microRNA mouse mmu-miR-100-5p-hairpin inhibitor was purchased from
Thermo Scientific (Cat # IH-310567-07-0005) and transfected using Lipofectamine 2000
along with the other plasmids. Protein synthesis was determined by SuNSET method
(Schmidt et al., 2009; Bhattacharya et al., 2012). DIV 8 WT and Fmr1 KO hippocampal
neurons were pre-treated with various doses of NB001 (5-{[2-(6-Amino-9H-purin-9yl)ethyl]amino}-1-pentanol) from Toronto Research Chemicals Inc. (Cat # A628913) for
30-min before treating with 5 ug/ml puromycin (Sigma, Cat #P8833) for 30 min. Cells
were lysed processed for western blotting as described above. The membranes were
probed with anti-puromycin antibody (KeraFAST, Cat # EQ0001, 1:1000). b-actin was
used as the loading control. ImageJ was used to measure the combined signal intensity
of proteins with molecular weights ranging from 15 to 250 kDa.
2.3.3 Drugs and treatments
NB001 was dissolved in water to give a 25mM stock solution. The stock was diluted in
saline and i.p. injected into mice at 1mg/kg body weight. In all cases except for open

	
  

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field, the drug was administered 1-hour before testing or before training for passive
avoidance. The drug was administered 30-min before introducing the mouse to the open
field chamber. Control mice were treated similarly but injected with saline.
2.3.4 Behavioral testing
Open field test. 2 month old male mice were placed in the center of a chamber
measuring 28cm X 28 cm with 34 cm high walls and were allowed to move freely for 2hours. Ambulatory movement time, ambulatory movement distance, number of center
entries and the time spent in different areas of the chamber were determined at every
10-min interval during the 2-hour testing period by the TruScan Photo Beam Activity
System (Coulbourn Instruments, Whitehall, PA).
Light-dark test. 3-4 month old male mice were placed in the dark half of the chamber
(Coulbourn Instruments) and the trap door was opened after 1-min. The mice were
allowed to move freely between the two chambers for 5-min. The time spent in each
chamber and the number of crossings into the lit side was recorded.
Passive avoidance. 3-4 month old male mice were introduced into the lit half of the
chamber (Coulbourn Instruments) and allowed to explore it for 1-min before the trap
door was opened. The trap door was closed as soon as the mouse entered the dark
chamber. A foot shock (0.7mA for 2s) was immediately delivered. The mouse was

	
  

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removed from the dark chamber and returned to its home cage 30-seconds after the
foot shock was administered. The mice were tested 24- hours after training. During
testing, the mouse was put in the lit chamber and crossover latency to the dark chamber
was measured. The cutoff for crossover latency was set at 600-seconds.
Audiogenic seizure testing. 21-24 day old male and female mice were placed in a box of
dimensions 30 cm X 17 cm and 12 cm high walls with a flat plastic lid. A personal alarm
(from Streetwise, item # SWPDAL) was taped to the lid of the box and wired to a DC
power supply to keep the sound amplitude constant. The mouse was allowed to
acclimatize to the box for 5-min following which a 120 dB sound was emitted from the
alarm for 2-min. The number of mice undergoing seizure within the 2-min period was
counted. Audiogenic seizure was classified into different stages: wild-running,
clonic/tonic seizure and death (as classified in Dolen et al., 2007)
Marble burying test. 2-3 month old male mice were placed in a 27 cm X 15 cm box with
12cm high walls and 7.6 cm depth of bedding for 1 hour prior to the test. The mouse
was then briefly removed from the testing box and 15 marbles were evenly arranged in
a 5 X 3 pattern on the surface of the bedding. The mouse was reintroduced into its
testing box and was allowed to bury marbles for 10-min. At the end of the testing period,

	
  

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the mouse was removed from the box and the number of marbles that were fully buried,
partially buried and left on the surface was counted.
Social interaction test. 2-3 month old male mice were placed in a 3-chambered social
interaction box (cmX cmX cm) and allowed to explore freely for 5-min. If a mouse
displayed a strong preference for one side during this time, it was omitted from the
study. The test mouse was placed in the center chamber and the entryways to the side
chambers were blocked. A novel male WT mouse was introduced in a confined space in
either of the side chambers. The entryways were then opened and the activity of the test
mouse was recorded. Time spent in each of the chambers, number of entries between
the chambers and time spent sniffing the novel mouse versus the empty enclosure in
the opposite chamber was recorded.
2.3.5 Dendritic spine analysis
2 month old mouse brains were processed according to manufacture instructions using
Fd Rapid GolgiStain Kit (FD NeuroTechnologies. Inc. Cat # PK401). 150um thick
sections were cut using vibratome and images were collected using the 100X objective
on an Olympus FluoView 1000 microscope. Spines starting from 50um from the cell
body up to 100um were analyzed along the apical dendrites of CA1 and primary visual

	
  

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cortex pyramidal neurons. Spines were classified as stubby, thin and mushroom shaped
using NeuronStudio Version 0.9.92 software.
2.3.6 NB001 toxicology studies
2-3 month old male mice were dosed by oral gavage or intraperitoneally twice daily with
either vehicle or 100 mg/kg NB001 over 2 weeks. Clinical observations were performed
twice daily and body weights will be collected at various days of the dosing. Mice were
euthanized on the day following the dosing period and blood was collected for clinical
pathology (Diagnostic Center for Population and Animal Health, Michigan State
University). The liver and kidney were collected at necropsy and tissues were fixed in
10% buffered formalin (INVIVO facility at Michigan State University). H&E staining was
performed on the tissues and tissue morphology was observed.
2.3.7 NB001 pharmacokinetic studies
20 mg/kg NB001 was given by IP injection. Mice were euthanized at various time points
and 0.5-0.8 ml of blood was drawn from a cardiac puncture into a 1 ml syringe, which
was pretreated with NaHeparin. The blood was collected into 1.5 mL microfuge tubes
coated with NaHeparin and put on ice immediately. Samples were centrifuged at 15,000
rpm for 15 min. Blood plasma was collected from the upper layer, leaving the blood cells
behind in the microfuge tube. The plasma was frozen at -80ºC for later analysis. Brains

	
  

63	
  

were taken out and weighed out immediately. Brains were frozen at -80ºC for later
preparation and analysis. The samples were subjected to LC/MS/MS with TurboIonsprayTM Interface used in the positive ion-mode. LC/MS/MS experiment
specifications- column：5 cm x 4.6 mm I.D., packed with 5.0 µm C18 (XBridge, Waters),
mobile phase A- 0.1% formic acid in purified water, mobile phase B- 0.1% formic acid in
acetonitrile, flow rate 1ml/min, injection volume-5ul, run time 6.5 min (Pharmacokinetics
Core at University of Michigan). Mouse liver microsome stability assay was performed at
the Pharmacokinetics Core at University of Michigan.
2.3.8 Statistical analysis
One-way ANOVA followed by post hoc LSD (least significant difference) test or
Student’s t-test was used to compare multiple groups. Student t-test was used to
compare two groups. Two-way ANOVA followed by Student's t test or LSD test was
used to compare different groups in the luciferase assay as well as behavior studies
using NB001 and the SUnSET assay. Chi-square test was used to analyze AGS data.
One-way and Two-way repeated measures ANOVA was used for open field data with
DKO and NB001 respectively. Data were expressed as the mean ± SEM. Differences
with p values < 0.05 were considered significant. SPSS 11.5 for Windows (IBM) was
used for all data analysis.

	
  

64	
  

2.4 Results
2.4.1 AC1 mRNA translation is elevated in the Fmr1 KO hippocampus
The translation of several mRNA targets of FMRP is dysregulated in the Fmr1
KO mouse brain (Table 2). Since AC1 mRNA was identified as a top FMRP binding
target in three highthroughput studies, I investigated whether AC1 mRNA translation
was altered in the brains of Fmr1 KO mice. Hippocampal lysates from Fmr1 KO mice
showed higher expression of AC1 protein (p < 0.05), whereas protein levels were similar
in cortical lysates (Figure 2A). Since the AC1 mRNA levels in the Fmr1 KO lysates were
similar to WT (Figure 2B), we tested whether AC1 mRNA was undergoing increased
active translation in the absence of FMRP. Using linear sucrose gradient fractionation,
we observed that AC1 mRNA was more abundant in the actively translating
polyribosome fractions in the Fmr1 KO hippocampal lysate than in WT lysate (Figure
2C and D, p < 0.05) (Dr. Wei Feng and Dr. Yue Feng). These results show that the
basal level of AC1 protein is overabundant in the Fmr1 KO hippocampus due to
excessive translation of AC1 mRNA in the absence of FMRP.
To test whether mGluR5 stimulation led to translation of AC1, I treated DIV14
hippocampal neurons with 100μM DHPG. Rapid increase in AC1 protein levels, was
observed in WT (Figure 3A p < 0.05) but not Fmr1 KO neurons (Figure 3C). As a control

	
  

65	
  

for the health of the neurons and the efficacy of DHPG, I measured the phosphorylation
of ERK1/2, which is known to be phosphorylated upon DHPG stimulation. DHPG
caused a significant upregulation in ERK1/2 phosphorylation in both WT and Fmr1 KO
neurons (Figure 3B and D, p < 0.05). Interestingly, different studies show that ERK1/2
may or may not be phosphorylated in Fmr1 KO hippocampal slices upon DHPG
stimulation (Prince, T et al., 2007, Ronesi J and Huber K, 2008). My study with DIV14
hippocampal neurons shows that ERK1/2 is phosphorylated upon DHPG treatment in
Fmr1 KO neurons but not as significantly as in WT neurons (Figure 3B and D).
Together, these data show that mGluR5 stimulation and FMRP regulate AC1 protein
expression.
2.4.2 FMRP regulates AC1 translation by microRNA mediated mechanisms
To investigate how FMRP could regulate AC1 mRNA translation, I looked at
FMRP-mediated microRNA (miRNA) mechanisms. FMRP is known to interact with
proteins of the miRNA pathway and also interacts with specific miRNA (Jin et al., 2004).
Using TargetScanMouse (Release 6.2: June 2012), I searched whether known FMRP
binding miRNA had target sites on the mouse AC1 3’ UTR. I narrowed down five
prospective miRNA sites in the 3’ UTR (Figure 4A). For further investigation, a fireflyluciferase reporter construct with either the proximal part (UTR1) or the distal part

	
  

66	
  

(UTR2) of the 3’ UTR was expressed in cultured Fmr1 KO hippocampal neurons (Figure
4A). I observed FMRP dependent reduction in luciferase expression specifically in the
UTR2 containing neurons (Figure 4B, p < 0.05). Since UTR2 contained three
prospective miRNA sites, I transfected Fmr1 KO neurons with luciferase-UTR2
constructs with 21bp deletions in each of the miRNA sites (refer to Figure 4A). While
deletion of the other two miRNA binding sites had no FMRP dependent effect, deletion
of the miR100 site abolished the ability of FMRP to reduce luciferase expression (Figure
4C). To further confirm the involvement of miR100 in FMRP dependent translation of the
luciferase-UTR2 reporter, I co-transfected Fmr1 KO neurons with anti-miR100 to inhibit
miR100 activity. Inhibiting miR100 led to a dramatic increase in the reporter expression
only in the presence of FMRP (Figure 4D, p < 0.01). Further, anti-miR100 could not
increase reporter expression when co-transfected with FMRP and a luciferase-UTR2
construct with a deletion in the miR100 binding site (Figure 4D). Together these data
show that FMRP suppresses translation of AC1 via its 3’ UTR, further, an intact miR100
binding site and miR100 activity is required for this inhibitory effect.
2.4.3 Rescue of several FXS phenotypes in DKO mice
Since AC1 expression was enhanced in Fmr1 KO mice, we wanted to test
whether dampening AC1 activity would be of therapeutic value for fragile X syndrome.

	
  

67	
  

As the major mouse model for studying FXS, the Fmr1 KO mouse recapitulates many of
the symptoms seen in human fragile X patients (Sethna et al., 2013). I dampened AC1
activity in the Fmr1 KO mouse by generating AC1, Fmr1 double-knockout mice (DKO). I
first tested audiogenic seizure (AGS) in Fmr1 KO mice which when exposed to a loud
sound experience seizure (Chen and Toth, 2001). When exposed to a 120dB sound,
Fmr1 KO mice showed the various stages of AGS whereas WT and AC1 KO mice
showed no seizure (Table 3). DKO mice showed greatly reduced seizure occurrence
and there were no seizure- associated deaths in these mice (Table 3, p < 0.05). I
observed repetitive/hyperactive behavior in Fmr1 KO mice in the marble- burying test,
where Fmr1 KO buried more marbles than WT mice (Figure 5A, p < 0.001). This
behavior was restored to WT levels in the DKO mice, while AC1 KO showed normal
marble burying behavior (Figure 5A). I further tested for repetitive/hyperactive behavior
using the light-dark test. Fmr1 KO mice made more transitions between the light and
dark chambers (Figure 5B, p < 0.001) while time spent in the light chamber was similar
to WT (Figure 5C). This behavior was rescued in the DKO mice while the AC1 mutation
had no effect on this behavior (Figure 5B and C). When tested for hyperactivity with the
open-field test, both Fmr1 KO and AC1 KO mice displayed higher ambulatory distance
(Figure 6A, p < 0.001) and more entries (Figure 6B, p < 0.001) into the center area than

	
  

68	
  

WT mice and this seemed to have an additive effect on the DKO mice whose
ambulatory distance and center entries was higher than AC1 and Fmr1 KO mice (Figure
6A and B, p < 0.001). I then examined whether the social interaction deficits in Fmr1 KO
mice would be corrected in the DKO mice. As expected (Mines et al., 2010), Fmr1 KO
mice spent less time sniffing the novel mouse in the social interaction test when
compared to the WT (Figure 6C, p < 0.001) and this behavior was completely rescued in
the DKO mice (Figure 6C) while AC1 KO showed a normal phenotype. All the
genotypes spent considerably more time sniffing the novel mouse rather than the empty
enclosure (Figure 6D, p < 0.05). To test for cognitive deficits, I trained the mice with
passive avoidance training. While all the groups showed similar crossover latency
during training, Fmr1 KO mice had a significantly lower crossover latency compared to
WT when tested 24 hours later (Figure 7, p < 0.005). AC1 KO mice showed a slight
decrease in crossover latency but this was not statistically significant compared to WT
mice while the DKO mice were similar to Fmr1 KO (Figure 7), that is, there was no
rescue in this behavior. Dendritic spine abnormalities are a robust phenotype seen in
Fmr1 KO mice. Here I observed significantly higher total spine density on the apical
dendrites of Fmr1 KO pyramidal neurons in the CA1 area of the hippocampus (Figure
8A and B, p < 0.05). This phenotype was corrected in the DKO neurons, while there

	
  

69	
  

was no difference between AC1 KO and WT spine densities (Figure 8A and B).
Together, these results demonstrate that genetic knockout of AC1 in the Fmr1 KO
mouse rescues several well-characterized behavioral deficits and the dendritic spine
abnormality in FXS model mice.
2.4.4 Pharmacological rescue of FXS phenotypes by an AC1 inhibitor
Encouraged by the above results we tested whether acute suppression of AC1
activity by the newly discovered antagonist NB001 (Wang et al., 2011) could rectify
behavioral and cellular abnormalities in the Fmr1 KO mouse. Fmr1 KO mice receiving
1mg/kg NB001 intraperitoneally, 60-minutes before exposure to a 120dB sound,
experienced significantly fewer occurrences of AGS (Table 4, p < 0.05) when compared
to vehicle (saline) injected Fmr1 KO mice. WT mice injected with vehicle or NB001 did
not show AGS (Table 4). Acute injection 1mg/kg NB001 also restored marble burying
behavior in Fmr1 KO mice to WT levels, although it seems to have a mild but not
statistically significant effect on decreasing the number of marbles buried in WT injected
mice (Figure 9A). Fmr1 KO mice injected with NB001 (1mg/kg) showed reduced
hyperactive/repetitive transitions between the light-dark chambers, which was
equivalent to the number seen in vehicle injected WT mice (Figure 9B). NB001 did not
have an effect on WT mice in this test. Surprisingly, NB001 (1mg/kg) injected WT mice

	
  

70	
  

did not show increased activity in the open field test in contrast to that seen in AC1 KO
mice (Figure 10A and B). Concurrently, NB001 (1mg/kg) administration prior to testing
reduced the higher Fmr1 KO ambulatory activity to WT levels (Figure 10A and B, p <
0.001). Collectively, these results show that acute intraperitoneal administration of
1mg/kg NB001 rescues the repetitive/hyperactive phenotype observed in Fmr1 KO
mice. In the social interaction test, Fmr1 KO mice injected with 1mg/kg NB001, 60minutes before testing, spent significantly more time sniffing the novel mouse, almost
comparable to WT vehicle injected levels (Figure 10C, p < 0.005). When administered
before passive avoidance training, 1mg/kg NB001 surprisingly rescued the memory
deficit observed in Fmr1 KO mice (Figure 11). Interestingly, WT mice injected with
NB001 showed very

mild but not statistically significant memory impairment in the

passive avoidance test (Figure 11). Since enhanced basal protein synthesis is thought
to underlie much of the pathophysiology of FXS and its correction to normal levels is a
potential strategy to treat FXS (Darnell and Klann, 2013), I studied whether NB001
treatment could normalize this abnormality in Fmr1 KO hippocampal neurons. Upon
labeling newly synthesized proteins with puromycin using the SUnSET method (Schmidt
et al., 2009; Bhattacharya et al., 2012), I observed increased basal protein synthesis in
DIV 8 Fmr1 KO neurons as compared to WT neurons (Figure 12A and B, p < 0.05).

	
  

71	
  

Fmr1 KO neurons treated with various doses of NB001 30-minutes prior to the start of
the assay showed protein levels similar to WT neurons, whereas, NB001 had no effect
on WT basal protein synthesis (Figure 12B). These results together demonstrate that
acute administration of NB001 can correct several behavioral deficits as well as
excessive protein synthesis in the Fmr1 KO mouse.
2.4.5 NB001 toxicity and pharmacokinetics
In our study, NB001 showed promising therapeutic value in ameliorating FXS
phenotypes, however, since it is a newly discovered drug, its toxicity has not been
studied. Here we performed some basic toxicology tests in WT male mice by chronically
administering 100mg/kg- a dose ten-times higher than needed for therapeutic efficacy in
mice; twice daily through oral gavage or intraperitoneally for 14 days. Neither oral nor
intraperitoneal NB001 administration affected body weight (Figure 13A), heart/lung,
kidney and liver weight (Figure 13B) (Diagnostic center for Population and Animal
Health) as well as kidney and liver histopathology (Figure 13C). The drug also had no
effect on blood levels of various electrolytes and other proteins (Table 5) (data provided
by Diagnostic center for Population and Animal Health). Together these studies show
that NB001 may be a relatively safe drug when tested in mice. Further we show that
NB001 is very stable in the mouse liver microsome assay (Figure 14A) and can cross

	
  

72	
  

the blood-brain barrier to enter the brain (Figure 14B) (Pharmacokinetics core,
University of Michigan).

2.5 Discussion
Here I validate that Ca2+/Calmodulin dependent type 1 adenylyl cyclase
translation is controlled by FMRP via miR100 binding to its 3’ UTR. Further we observe
that AC1 protein is over abundant in the hippocampus of FXS mice due to increased
translation of its mRNA and is expressed rapidly upon mGluR5 stimulation only in the
presence of FMRP. This is consistent with numerous studies, which show that proteins
regulated by FMRP have increased basal levels in the FXS mouse and are therefore not
responsive to mGluR stimulation (Westmark and Malter, 2007; Park et al., 2008;
Goebel-Goody et al., 2012). It is interesting to note that cyclic AMP (cAMP) levels are
lower in patients with FXS (Kelley et al., 2007), this may be a compensatory mechanism
to counteract the higher mGluR signaling in the absence of FMRP.

Since AC1 is

expressed only in the central nervous system (CNS), it provides a unique
pharmacological target to treat CNS disorders without its effects on peripheral organs
(Wang and Storm, 2003). AC1 KO mice have normal growth, longevity, motor
coordination and have no detectable anatomical or morphological differences in their

	
  

73	
  

brains except for lacking barrel patterning in the sensory-motor cortex (Abdel-Majid et
al., 1998). AC1 KO mice also show normal physiological functions of acute pain, have
normal long-lasting LTP, normal long-term memory for several forms of fear memory,
and perform normally in the Morris water maze (Wang and Storm, 2003). In the current
study, AC1 KO mice exhibit behavior comparable to WT mice except in the open field
test where they show hyperactivity. Using a battery of behavioral tests that are key
phenotypes of FXS mice, I show that knocking out AC1 in the Fmr1 KO mice rescue
several phenotypes. DKO mice have markedly fewer occurrences of AGS and no AGS
induced deaths. Autism-like symptoms such as repetitive/hyperactive behavior and
social deficit that are seen in Fmr1 KO mice are corrected in the DKO mice. Further,
pharmacological inhibition of AC1 using NB001 also corrected these phenotypes, in
addition to correcting the learning deficit in Fmr1 KO mice. Interestingly, differences in
behavior between the DKO mice and NB001 injected Fmr1 KO mice were seen in the
open field test and the passive avoidance test. These differences may be attributed to
one or more of several reasons why a genetic knockdown and pharmacological
inhibition of the same target may produce varying phenotypes (Knight and Shokat,
2007). It is unlikely that NB001 inhibited other ACs in our study because we used a low
dose and the specificity of NB001 for AC1 is at least ten times higher than for other ACs

	
  

74	
  

(Wang et al., 2011). My study also shows that NB001 can reduce the enhanced basal
protein levels in Fmr1 KO neurons, a cellular phenotype that is thought to be critical to
the pathophysiology of FXS (Darnell and Klann, 2013). Since the abnormal dendritic
spine phenotype was corrected in DKO mice, it would be interesting to measure the
spine phenotype upon prolonged exposure to NB001. Finally, our study demonstrates
that prolonged administration of a 100 fold higher dose of NB001 did not have
measurable toxic effects on mice. Very importantly, NB001 could pass through the
blood brain barrier and be detected in the brain following i.p. injection. This together with
the observation that NB001 did not produce any measurable behavior deficits and had
no toxic effects makes it an attractive drug target to pursue for future studies. While
there has been much success in identifying drug targets for FXS in a preclinical setting,
there is limited success in clinical trials, resulting in the recent withdrawal of two
promising drugs (STX209 and AFQ056). This highlights the importance of continuing to
investigate deeper into the molecular mechanisms underlying FXS and identifying new
therapeutic targets. In conclusion, our study identifies AC1 as a novel and promising
therapeutic target for FXS.

	
  

75	
  

APPENDIX

	
  

76	
  

A!
WT ! Fmr1 WT ! Fmr1
KO!
KO!
cortex ! hippocampus!
AC1!

B!
WT !Fmr1 WT! Fmr1
KO!
KO!

β-actin!
cortex ! hippocampus!
1.2!
*!

1!

0.8!
0.6!

Fold change!

Fold change!

1.2!
0.8!

0.4!

0.4!
0.2!

0!

0!

C!

D!
Ratio (1-2)/(3-10)
*

0.5

Ratio

0.4
0.3
0.2
0.1

Fraction!

O
K

W

T

0.0

Figure 2: FMRP represses AC1 translation. (A) Western blot of AC1 protein from
cortex and hippocampus of 21 day old WT and Fmr1 KO mice (n=6 per group),
representative image top panel, quantification bottom. AC1 protein levels are
normalized to β-actin and expressed as relative level to the WT cortex group. * indicates

	
  

77	
  

Figure 2 (cont'd)
p < 0.05 between WT and indicated group determined by Student’s t-test. (B) AC1
mRNA quantitated by qPCR from cortical and hippocampal RNA preparations from 21
day old WT and Fmr1 KO mice. AC1 values normalized to GAPDH and expressed as
levels relative to the WT cortex group, (n=6 per group) (C) Percent distribution of AC1
mRNA determined by qPCR using ribosome fractions obtained from linear sucrose
gradient centrifugation of hippocampal lysates from WT (n=3) and Fmr1 KO (n=3) mice.
(D) Ratio of AC1 mRNA in fractions 1-2 and fractions 3-10 in WT and Fmr1 KO
hippocampal lysates. * indicates p < 0.05 between WT and indicated group determined
by Student’s t-test.

	
  

78	
  

A!
DHPG !

B!
DHPG !
pERK1/2!

β-actin!

tERK1/2!

1.6!
1.2!
0.8!
0.4!
0!

*!

*!

WT!

2!
1.6!
1.2!
0.8!
0.4!
0!

Fold change!

Fold change!

AC1!

β-actin!

tERK1/2!

0.4!
0!

Fmr1
KO!

Fold change!

pERK1/2!

Fold change!

AC1!

0.8!

*!

*!
*!

*!
*!

WT!

D!
DHPG !

C!
DHPG !

1.2!

*!

1.2!

Fmr1
KO!

0.8!
0.4!
0!

Figure 3: Rapid translation of AC1 upon mGluR5 activation in the presence of
FMRP. (A- D) Western blot of AC1 (A,C) and phospho-ERK1/2 (B,D) from DIV14 WT
and Fmr1 KO hippocampal neurons (n=6 per group) treated with 100µM DHPG for
various time points, representative image top panel, quantification bottom. AC1 protein
levels are normalized to β-actin and pERK1/2 levels are normalized to total-ERK1/2. All

	
  

79	
  

Figure 3 (cont'd)
values are represented relative to the 0 min time point. * indicates p < 0.05 between
control and indicated group determined by one-way ANOVA and LSD test.

	
  

80	
  

A!
5’ UTR! Coding region!
5’!
112!

3’ UTR!
3’!
12259!

3469!

5096-8015bp- UTR1 ! 10259-12217bp- UTR2 !
3469!

12259!

miR132!
6440-6462! miR124!
6628-6650!

C!
2!

1.5!

1.5!
1!
0.5!
0!

Relative luciferase activity!

Relative luciferase activity!

B!

miR138!
miR100!
11213-11235!
12014-12036!
miR125!
11263-11285!

#
*!

*!

1!
*!

0.5!
0!

Figure 4: FMRP controls AC1 translation by microRNA-mediated mechanisms. (A)
Schematic representation of AC1 (Adcy1) mRNA 3’UTR. Top. Mouse Adcy1 mRNA,
numbers below represent base-pairs (bp) from the 5’ end. Bottom. 3’ UTR of Adcy1
starting at 3469 to 12259 bp. The UTR1 fragment from 5096 to 8015 bp has an miR132
binding site from 6440 to 6462 bp and an miR124 binding site from 6628 to 6650 bp.

	
  

81	
  

Figure 4 (cont'd)

Relative luciferase activity!

D!
2!

*!

1.5!
1!
0.5!

*!

0!

The UTR2 fragment from 10259 to 12217 bp has one miR138 binding site from 11213
to 11235 bp, one miR125 site from 11263 to 11285 bp and an miR100 site from 12014
to 12036 bp. (B) Relative firefly/renilla luciferase activity from DIV8 Fmr1 KO
hippocampal neurons transfected with UTR1 or UTR2 constructs with or without FMRP
(n=6 per group). Luciferase activity represented relative to the Vector group. * indicates
p < 0.05 between control and indicated group, # indicates p < 0.05 between UTR2 and
indicated group determined by two-way ANOVA followed by LSD test. (C) Relative
luciferase activity in DIV8 Fmr1 KO hippocampal neurons transfected with UTR2 or

	
  

82	
  

Figure 4 (cont'd)
UTR2del100 constructs with or without FMRP (n=6 per group). Values expressed
relative to UTR2. * indicates p < 0.05 between UTR2 and indicated group determined by
two-way ANOVA and Student’s t-test. (D) Relative luciferase activity in DIV8 Fmr1 KO
hippocampal neurons transfected with UTR2 or UTR2del100 constructs along with
antimiR100 with or without FMRP (n=6 per group). Values normalized to the UTR2
group. * indicates p < 0.05 between UTR2 and indicated group, # indicates p < 0.05
between UTR2+antimiR100 and indicated group determined by two-way ANOVA and
LSD test.

	
  

83	
  

Table 3: Rescue of audiogenic seizure in DKO mice

n!

% Wild
running!

% Clonic/
Tonic
seizure!

%
Death!

WT!

10!

0!

0!

0!

Fmr1
KO!

27!

68.5!

62.6!

13.7!

AC1
KO!

10!

0!

0!

0!

DKO!

27!

3.7!

0!

0!

A!
% marbles buried!

80!
Table 3: Audiogenic seizure (AGS) measured in 21-24WT!
day WT, Fmr1 KO, AC1 KO and
#!
Fmr1 KO!
60!
*!
AC1 KO!
DKO mice. Percent of mice experiencing different stages of AGS- wild running,
DKO!
40!
clonic/tonic seizure and death was #!recorded. Chi-square test confirmed p < 0.05
20!
between Fmr1 KO and DKO groups.
0!

Fully
buried!

B!

Partially
buried!

180!

*!

12!

Time in light chamber (s)!

number of entries!

Surface!
C!

16!

120!

8!

#!

4!
0!

	
  

*!

60!

0!

WT! Fmr1 AC1 DKO!
KO! KO!
84	
  

WT! Fmr1 AC1 DKO!
KO! KO!

KO!
AC1
KO!

10!

0!

0!

0!

DKO!

27!

3.7!

0!

0!

A!
% marbles buried!

80!
60!
40!

#!

*!
#!

*!

20!
0!

Fully
buried!

B!

Surface!

Partially
buried!

C!

16!

180!

*!

12!

Time in light chamber (s)!

number of entries!

WT!
Fmr1 KO!
AC1 KO!
DKO!

120!

8!

#!

4!
0!

WT! Fmr1 AC1 DKO!
KO! KO!

60!

0!

WT! Fmr1 AC1 DKO!
KO! KO!

Figure 5: Rescue of hyperactive/repetitive phenotypes in DKO mice. (A) Marble
burying test was performed in 2-3 month old WT (n=10), Fmr1 KO (n=15), AC1 KO
(n=10) and DKO (n=17) mice. % marbles buried fully or partially was measured. (B, C)
Light-dark activity test was performed in in 2-3 month old WT (n=12), Fmr1 KO (n=12),
AC1 KO (n=12) and DKO (n=9) mice. Number of entries into the light-chamber was

	
  

85	
  

Figure 5 (cont'd)
measured (B). Time spent in the light chamber was measured (C). One-way ANOVA
followed by LSD test was used to compute p-values. * indicates p < 0.001 between WT
and indicated group, # indicates p < 0.001 between Fmr1 KO and indicated group.

	
  

86	
  

B!
WT!
180!
Fmr1 KO!
AC1 KO!
DKO!
120!
Number of Center entries!

3000!
2500!
2000!
1500!
1000!
500!

0!

0!

C!
Mouse!
Empty! #!

Sniffing time (s)!

WT!
Fmr1 KO!
AC1 KO!
DKO!

60!

90!
80!
70!
60!
50!
*!
40!
30!
20!
10!
0! WT! Fmr1 AC1
KO! KO!

D!

Mouse!
Empty!

400!

Time in chamber (s)!

Ambulatory distance (cm)!

A!

300!
200!

*!

*!
*!

*!

100!

DKO!

0!

WT! Fmr1 AC1 DKO!
KO! KO!

Figure 6: Rescue of social behavior but not open-field hyperactivity in DKO mice.
(A,B) Ambulatory distance (A) and number of entries into the central area (C) in an open
field was measured in 2-3 month old WT (n=9), Fmr1 KO (n=11), AC1 KO (n=12) and
DKO (n=12) mice. Repeated measures ANOVA and LSD test was used to determine
p<0.05 between WT and Fmr1 KO, WT and AC1 KO and WT and DKO

	
  

87	
  

Figure 6 (cont'd)
groups. (C,D) Sniffing time (C) and time spent in different chambers (D) during the
social interaction test was measured in 2-3 month old WT (n=11), Fmr1 KO (n=11), AC1
KO (n=10) and DKO (n=10) mice. * p < 0.001 between WT and indicated group, # p <
0.001 between Fmr1 KO and indicated group (C) measured by One-way ANOVA and
LSD test. * indicates p < 0.05 between chamber with mouse and empty chamber (D)
measured by Student’s t-test.

	
  

88	
  

Time to enter dark chamber (s)!

700!

WT!
Fmr1 KO!
AC1 KO!
DKO!

600!
500!
400!
300!

*!

200!

*!

100!
0!

Training!

Testing!

Number of spines/um dendrite!

A!
B!
WT! cognitive
Fmr1function
KO!
Figure 7: Impaired
in DKO mice. Passive avoidance testing was
1.5!
WT!
performed in 2-3 month old WT (n=10), Fmr1 KO (n=10), AC1 KO
Fmr1(n=11)
KO! and DKO
AC1 KO! *!
1!
#!
(n=12) mice. Latency to enter the dark chamber was measured 24DKO!
hours after shocking
DKO!
AC1 KO!
the mice. * p < 0.005 between WT and indicated group.
0.5! One-way ANOVA and LSD test
was used to measure the p-value.

	
  

89	
  

0!

Stubby! Thin! Mushroom! Total!

WT!

0!

Training!

Fmr1 KO!

Testing!

B!
Number of spines/um dendrite!

Tim

A!

100!

1.5!

AC1 KO!

DKO!

1!

WT!
Fmr1 KO!
AC1 KO!
DKO!

*!

#!

0.5!

0!

Stubby! Thin! Mushroom! Total!

Figure 8: Rescue of dendritic spine phenotype in DKO mice. (A) Representative
image of Golgi staining of dendritic spines in the apical dendrites from the CA1 area.
Scale bar: 8µm (B) Classification and quantification of dendritic spines n ~ 450 spines
per genotype. * p < 0.05 between WT and indicated group, # p < 0.05 between Fmr1
KO and indicated group measured by One-way ANOVA and LSD test.

	
  

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Table 4: NB001 rescues audiogenic seizure in Fmr1 KO mice

n!

% Wild
running!

% Clonic/
Tonic
seizure!

% Death!

WT
vehicle!

8!

0!

0!

0!

Fmr1 KO
vehicle!

17!

71.4!

64.2!

21.4!

WT
NB001!

6!

0!

0!

0!

Fmr1 KO
NB001!

17!

35.2!

5.8!

5.8!

A!

% marbles buried!

80!
WT vehicle!
Table 4: Audiogenic seizure was measured in 21-24
and Fmr1 KO mice
Fmr1day
KO WT
vehicle!
#!
WT NB001!
60!
*!
Fmr1 KO wild-running,
NB001!
injected with vehicle (saline). Percent mice experiencing
clonic/tonic
#!
40!
seizure and death were reported. p < 0.05 between Fmr1 KO vehicle and Fmr1 KO

*!

20!
NB001 groups computed by Chi-square test.
0!

#!

4!
0!

	
  

WT Fmr1 WT Fmr1
vehicle! KO NB001! KO
vehicle!
NB001!

Time in light chamber (s)!

180!

*!

12!
8!

Partially buried!

C!

16!

number of entries!

B!

Fully buried!Surface!

120!

60!

91	
  

0!

WT Fmr1 WT Fmr1
vehicle! KO NB001! KO
vehicle!
NB001!

WT
NB001!

6!

0!

0!

0!

Fmr1 KO
NB001!

17!

35.2!

5.8!

5.8!

% marbles buried!

A!

80!
60!
40!

#!

*!

20!
0!

Fully buried!Surface!

#!

4!
0!

WT Fmr1 WT Fmr1
vehicle! KO NB001! KO
vehicle!
NB001!

Time in light chamber (s)!

180!

*!

12!
8!

Partially buried!

C!

16!

number of entries!

B!

#!

*!

WT vehicle!
Fmr1 KO vehicle!
WT NB001!
Fmr1 KO NB001!

120!

60!

0!

WT Fmr1 WT Fmr1
vehicle! KO NB001! KO
vehicle!
NB001!

Figure 9: NB001 rescues hyperactive/repetitive phenotypes in Fmr1 KO mice. (A)
Marble burying test was performed in 2-3 month old WT and Fmr1 KO mice injected
with vehicle (WT=9, Fmr1 KO=12) or 1mg/kg NB001 (WT=9, Fmr1 KO=9). % marbles
buried fully or partially was measured. (B,C) Light-dark activity test was performed in 2-3
month old WT and Fmr1 KO mice injected with vehicle (WT=11, Fmr1 KO=13) or

	
  

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Figure 9 (cont'd)
1mg/kg NB001 (WT=10, Fmr1 KO=15). Number of entries into the light-chamber was
measured (B). Time spent in the light chamber was recorded (C). * p < 0.001 between
WT and indicated group, # p < 0.001 between Fmr1 KO and indicated group. p -values
measured by Two-way ANOVA and Student’s t-test.

	
  

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2500!

B!
WT vehicle!
160!
Fmr1 KO vehicle!

2000!

WT NB001!
Fmr1 KO NB001!

1500!
1000!
500!

Number of Center entries!

Ambulatory distance (cm)!

A!

0!

Mouse!
Empty!

80!

*#!

60!
40!

120!
80!
40!
0!

*!

20!

D!
Time in chamber (s)!

Sniffing time (s)!

C!

WT vehicle!
Fmr1 KO vehicle!
WT NB001!
Fmr1 KO NB001!

0! WT
Fmr1 WT Fmr1
vehicle! KO NB001! KO
vehicle!
NB001!

350!

Mouse!
Empty!

300!
250!
200!

*!

*!

*!

*!

150!
100!
50!
0!

WT Fmr1 WT Fmr1
vehicle! KO NB001! KO
vehicle!
NB001!

Figure 10: NB001 rescues open-field hyperactivity and social deficit in Fmr1 KO
mice. (A, B) Ambulatory distance (A) and number of entries into the center area (B) in
an open field was measured in 2-3 month old WT and Fmr1 KO mice injected with
vehicle (WT=10, Fmr1 KO=10) or 1mg/kg NB001 (WT=10, Fmr1 KO=10). Repeated

	
  

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Figure 10 (cont'd)
measures ANOVA was used to determine significance between WT: Fmr1 KO and
Fmr1 KO: DKO groups. (C,D) Sniffing time during the social interaction test was
measured in 2-3 month old WT and Fmr1 mice injected with vehicle (WT=12, Fmr1
KO=12) or 1mg/kg NB001 (WT=10, Fmr1 KO=12). Two-way ANOVA and Student’s ttest was used to compute p values. * p < 0.005 between WT and indicated group, # p <
0.005 between Fmr1 KO and indicated group (C). * p < 0.05 between chamber with
mouse and empty chamber (D).

	
  

95	
  

Time to enter dark chamber (s)!

700!
600!
500!

WT vehicle!
Fmr1 KO vehicle!
WT NB001!
Fmr1 KO NB001!

400!
300!

*!

200!
100!
0!

Training!

Testing!

Puromycin-labeled proteins!

A!
B!
Figure NB001!
11: NB001
a cognitive deficit in Fmr1 KO mice. Passive avoidance
WT!rescues
Fmr1!WT!Fmr1!
-! -! +! +!
1.8!
WT!
testing was performed in 2-3 month old WT and Fmr1
with vehicle
Fmr1 KO!
* KO mice injected
Fold change!

#
#
1.2!
(WT=12, Fmr1 KO=12) or 1mg/kg NB001 (WT=10,
Fmr1 KO=18), latency
# to enter the
dark chamber was measured 24 hours after shocking
the mice. * p < 0.005 between WT
0.6!
and indicated group measured by Two-way ANOVA and Student’s t-test.
0!
Control! NB
NB
NB
5uM! 20uM! 100uM!
β-actin!

	
  

96	
  

Tim

100!
0!

Training!

A!

Testing!

B!

β-actin!

1.8!

Fold change!

Puromycin-labeled proteins!

NB001! WT! Fmr1!WT!Fmr1!
-! -! +! +!

1.2!

WT!
Fmr1 KO!

*
#

#
#

0.6!
0!

Control! NB
5uM!

NB
NB
20uM! 100uM!

Figure 12: NB001 normalizes exaggerated protein synthesis in Fmr1 KO neurons.
(A) Representative image of western blot. Basal protein synthesis measured in DIV8
WT and Fmr1 KO neurons (n=6 per group) with different doses of NB001. (B)
Quantification of protein bands between 150 kb and 20 kb, normalized to actin levels
and expressed relative to the WT control group. Student’s t-test was used to determine
p-values. * shows p < 0.05 between indicated group and WT, # shows p<0.05 between
indicated group and Fmr1 KO.

	
  

97	
  

A!

Control!
B!
NB001 Oral!
4!
NB001 IP!

Weight Relative to Brain!

Body Weight (g)!

35!
30!
25!
20!
15!
10!
5!
0!

2!
1!
0!

C!

Control!
NB001 Oral!
NB001 IP!

3!

Heart
&
Lung!

Control!

NB001 (oral)!

Control!

NB001 (oral)!

Liver!

Kidney!

Liver!

Kidney!

Figure 13: NB001 toxicity studies. 100mg/kg NB001 or saline was repeatedly
administered orally (Saline=3, NB001=5) or intraperitoneally (Saline=3, NB001=5) to 2
month old mice for 15 days (A) Body weight at different days. (B) Heart/lung, liver and

	
  

98	
  

Figure 13 (cont'd)
kidney weight normalized to brain weight after 15 days of administration. (C) H&E
staining and histology of liver and kidney tissue after 15 days of oral administration.

	
  

99	
  

B!

160!

NB001 brain
concentration (ng/g)!

% NB001 Remaining!

A!

120!
80!
40!
0!

40!
30!
20!
10!
0!

0! 3! 5! 10! 15! 30! 60!
Time (min)!

0.5! 1!
2!
4!
Time (hour)!

7!

Figure 14: Pharmacokinetics of NB001. (A) Mouse liver microsome assay of NB001.
(B) Pharmacokinetic stability assay of NB001 in the brain.

	
  

100	
  

Table 5: Chronic high dose NB001 administration does not alter blood chemistry in mice

Urea Nitrogen (mg/dL)!
Sodium (mmol/L)!
Potassium (mmol/L)!
Chloride (mmol/L)!
TCO2 (mmol/L)!
Anion Gap (mmol/L)!
Na/K Ratio!
Osmolarity (mOs/L)!
Glucose (mg/dL)!
Calcium (mg/dL)!
Phosphorus (mg/dL)!
Magnesium (mg/dL)!
Iron (ug/dL)!
Albumin (g/dL)!
ALT (U/L)!
AST (U/L)!
ALP (U/L)!
Amylase (U/L)!
Chol (mg/dL)!
Hemolysis Chem!
Lipemia Chem!
Icterus Chem!

Control!
21.67 ±1.33!
151.33 ±0.33!
4.77 ±0.03!
108.33 ±0.67!
13.00 ±0.58!
35.00 ±0.58!
31.67 ±0.33!
321.00 ±1.00!
183.50 ±15.50!
8.60 ±0.00!
7.37 ±0.18!
3.33 ±0.12!
107.00 ±44.00!
2.80 ±0.00!
22.67 ±1.45!
40.33 ±1.86!
72.00 ±2.89!
576.67 ±12.25!
95.67 ±4.81!
Normal!
Normal!
Normal!

NB001 oral!
25.00 ±0.71!
152.50 ±1.04!
4.55 ±0.13!
110.25 ±1.03!
13.50 ±0.29!
33.25 ±0.75!
33.75 ±0.75!
322.25 ±2.06!
154.25 ±10.86!
8.83 ±0.09!
6.98 ±0.22!
3.13 ±0.10!
135.00 ±16.41!
2.98 ±0.05!
24.00 ±1.47!
39.75 ±1.11!
66.00 ±2.27!
613.50 ±12.01!
104.75 ±2.56!
Normal!
Normal!
Normal!

NB001 i.p.!
24.50 ±0.87!
150.50 ±1.50!
4.73 ±0.22!
112.25 ±0.85!
13.50 ±1.04!
29.50 ±2.36!
32.00 ±1.47!
321.00 ±4.00!
216.50 ±8.50!
8.83 ±0.20!
6.50 ±0.24!
2.60 ±0.11!
115.50 ±4.50!
2.65 ±0.13!
18.50 ±0.96!
32.50 ±1.55!
62.75 ±4.48!
586.50 ±29.02!
100.00 ±5.96!
Normal!
Normal!
Normal!

Table 5: Measurement of ions, metabolites and enzymes in blood plasma of mice
injected with saline (control n=3), orally fed NB001 (NB001 oral n=5) and injected with
NB001 (NB001 i.p. n=5) after repeated administration of 100mg/kg NB001 twice daily
for 15 days.

	
  

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REFERENCES

	
  

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CHAPTER 3: TRIFLUOPERAZINE INHIBITS Gq-COUPLED RECEPTOR SIGNALING
AND RESCUES CELLULAR AND BEHAVIORAL ABNORMALITIES IN A MOUSE
MODEL OF FRAGILE X SYNDROME

3.1 ABSTRACT
Aberrantly elevated basal protein synthesis and over-activation of the Gq-coupled
glutamate and acetylcholine receptors have been considered as potential mechanisms
underlying the pathophysiology in Fragile X syndrome (FXS). Identification of functional
components in Gq signaling that affect protein synthesis may aid in development of
novel therapeutic targets for FXS. Through pharmacological inhibition, we tested the
function of calmodulin (CaM) in intracellular signaling stimulated by the activation of the
Gq-coupled metabotropic glutamate receptor 1/5 (mGluR1/5) and muscarinic cholinergic
receptors (Gq-mAchR). CaM inhibitors suppressed mGluR1/5- and Gq-mAchRmediated Arc upregulation and the activation of ERK1/2, whose activity is required for
protein synthesis and synaptic long-term depression (LTD). Further, we used acute
injection of a known CaM inhibitor- trifluoperazine (an FDA approved drug for treating
psychosis) and examined FXS-related behaviors and elevated protein synthesis in a
mouse model of FXS. Low dose administration of trifluoperazine at 0.05 mg/kg

	
  

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attenuated audiogenic seizures and corrected multiple FXS-related symptoms including
hyperactivity, repetitive behavior, and defective passive avoidance memory in Fmr1
knockout mice. Trifluoperazine also normalized the elevated protein synthesis in FXS
neurons to the wild type level. Our data suggest CaM as a new therapeutic target. Our
study also suggests a potential new application of trifluoperazine in FXS.

	
  

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3.2 Introduction
Fragile X syndrome (FXS), caused by mutations in the FMR1 (fragile X mental
retardation 1) gene, is the most common form of inherited mental retardation and a
leading cause of autism (Santoro et al., 2012). Most cases of FXS occur due to a
significant expansion of the CGG repeats in the 5’ untranslated region of the FMR1
gene. This inhibits its transcription, thus preventing the expression of its gene product
FMRP (fragile X mental retardation protein). FXS patients exhibit numerous neurological
abnormalities including learning disability, higher susceptibility to seizures, hyperactivity,
and perseveration (Bagni et al., 2012).
Biochemical studies have demonstrated that FMRP binds to roughly 4% of
mRNAs (Brown et al., 2001; Darnell et al., 2011; Ascano et al., 2012). Neurons lacking
FMRP show enhanced basal protein synthesis, supporting the idea that FMRP is a
translational suppressor of its mRNA targets (Darnell and Klann, 2013). The lack of
FMRP-mediated suppression of translation in FXS patients may cause alterations in
synaptic functions that require activity-dependent protein synthesis. In a seminal study,
Huber et al. reported that long-term depression (LTD) triggered by the activation of the
Gq-coupled mGluR1/5 is enhanced in Fmr1 knockout (KO) mice (Huber et al., 2002).
Strikingly, protein synthesis inhibitors only suppress the mGluR1/5-mediated LTD in wild

	
  

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type (WT) but not Fmr1 KO mice (Hou et al., 2006; Nosyreva and Huber, 2006). As
mGluR1/5 activation leads to translational upregulation, it is hypothesized that overactivation of mGluR1/5 signaling may be causal for both enhanced translation and LTD
in FXS (Bear et al., 2004). In support of this idea, inhibition of mGluR5 has shown
significant therapeutic effects to rescue several FXS-related symptoms in mouse
models (Yan et al., 2005; de Vrij et al., 2008; Thomas et al., 2012). However, the
efficacy of mGluR5 inhibitors in a human clinical trial was limited to a sub-population of
FXS patients (Jacquemont et al., 2011).
mGluR5-based therapies may be enhanced when used in conjunction with
strategies targeting other etiological factors. Similar to the effects of mGluR1/5
activation, activation of the Gq-mAchR (such as the M1 receptor) also triggers protein
synthesis and LTD. Interestingly, the M1-mediated LTD in Fmr1 KO mice is also
enhanced and resistant to protein synthesis inhibitors (Volk et al., 2007). Inhibition of
the Gq-mAchR rescues certain but not all FXS symptoms in Fmr1 KO mice
(Veeraragavan et al., 2011). Studies on the pathophysiology associated with the
exaggerated mGluR1/5 and Gq-mAchR signaling in FXS raise an important question:
can we achieve more robust therapeutic efficacy by simultaneously inhibiting both
receptors through dampening a common downstream signaling target?

	
  

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Our strategy was to block intracellular signaling following the activation of both
mGluR1/5 and muscarinic receptors. We found that inhibition of calmodulin (CaM)
activity suppressed signal transduction triggered by the activation of both mGluR1/5 and
muscarinic receptors. Further, systemic administration of a clinically used CaM inhibitor,
trifluoperazine, attenuated audiogenic seizures in Fmr1 KO mice. Trifluoperazine also
corrected hyperactivity, repetitive behavior, and defective passive avoidance memory in
Fmr1 KO mice. Moreover trifluoperazine suppressed the enhanced mRNA translation in
Fmr1 KO neurons. Our results suggest that inhibiting signal propagation of multiple Gqcoupled receptors may be considered as a new strategy to treat FXS.

	
  

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3.3 Materials and Methods
3.3.1 Animals
Fmr1 KO mice on C57BL/6 background were obtained from Dr. James Malter and Dr.
Cara Westmark. Animals were housed in the University Laboratory Animal Research
facility. All manipulations were approved by the Institutional Animal Care and Use
Committee at Michigan State University. The mice had ad libitum access to water and
food and were housed under 12 h dark/light cycle.
3.3.2 Neuronal cell culture and Western blotting
Primary hippocampal cultures were established from neonatal rats (Sprague Dawley) or
wild type (WT) and Fmr1 KO mice as described (Zhou et al., 2010). Control and treated
neurons were lysed and proteins were extracted in Laemmli buffer. Equal amounts of
cell extract were separated by SDS-PAGE followed by transferring to nitrocellulose
membranes. The following antibodies were used to detect the corresponding targets.
Arc antibody was obtained from Synaptic Systems (Cat # 156011, 1:1000 dilution). βactin antibody was from Sigma (Cat #A5441, 1:10,000 dilution). Anti-phospho-ERK1/2
(Cat #9101, 1:1000 dilution), anti-ERK1/2 (Cat #9102, 1:1000 dilution), anti-S6K1 (Cat
#9202, 1:1000 dilution), anti-phospho-S6K1 (at Thr421/Ser424) (Cat #9204, 1:1000

	
  

112	
  

dilution), and anti-phospho-S6K1 (at Thr389) (Cat # 9234, 1:1000 dilution) were from
Cell Signaling.
For quantification, the level of Arc was normalized to the level of β-actin. The level of
phosphorylated ERK1/2 and S6K1 was normalized to the level of total ERK1/2 and
S6K1, respectively. The relative intensity of the Western blot signal in the no treatment
control group, was defined as 1. The signal in the treatment samples was normalized to
the control group. All bands were quantified using ImageJ (NIH, MD, USA).
3.3.3 Electrophysiology
Field excitatory postsynaptic potentials (fEPSP) in the CA1 region of the hippocampus
were measured in anaesthetized mice (2- to 3-month old) (100 mg/kg Nembutal sodium
solution for the initial dose, another dose of 10 mg/kg was given 30 min later) or acute
brain slices (obtained from 1-month old mice) as described (Zhang et al., 2011;
Bhattacharya et al., 2012). To induce LTD in vivo, 0.5 ul of 2.5 mM mGluR1/5 agonist
DHPG ([RS]-3,5-Dihydroxyphenylglycine, Tocris Cat #0342) (with the infusion rate of
0.05 ul/min) was infused to the CA1 region of the anaesthetized mice. To determine the
effects of calmodulin inhibition on mGluR1/5-LTD, 0.5 ul 1.75 mM W13 (N-[4Aminobutyl]-5-chloro-2-naphthalenesulfonamide hydrochloride, Tocris Cat #0361) or 0.5
ul 1.75 mM W13 plus 2.5 mM DHPG (with the infusion rate of 0.05 ul/min) was delivered

	
  

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to the CA1 region. To determine the mGluR1/5-LTD in vitro, DHPG (100 uM) or DHPG
(100 uM) plus W13 (70 uM) was perfused to the recording chamber. The ratio of fEPSP
after drug administration to baseline fEPSP was used to determine the degree of
synaptic depression.
3.3.4 Behavioral tests
To measure audiogenic seizures (AGS), twenty one- to twenty four-day old male and
female mice were placed in a box (30 cm L by 17 cm W by 12 cm H) with a flat plastic
lid. A personal alarm (from Streetwise, item # SWPDAL) was taped to the lid of the box
and wired to a DC power supply to keep the sound amplitude constant. The mouse was
allowed to acclimatize to the box for 5 min, following which a 120 dB sound was emitted
from the alarm for 2 min. The number of mice undergoing seizure within the 2-min
period was counted. Audiogenic seizures were classified into different stages: wild
running, clonic/tonic seizure and death.
To determine animal activity in the open field, two-month old male mice were placed
in the center of an open field chamber measuring 28 cm by 28 cm with 34-cm high walls
and were allowed to move freely for 2 hours. Total ambulatory movement distance, and
ambulatory distance in the center area of the open field were determined every 10-min

	
  

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during the 2-hour testing period by the TruScan Photo Beam Activity System (Coulbourn
Instruments, Whitehall, PA).
To perform the marble burying test, two- to three-month old male mice were placed
in a box (27 cm by 15 cm box with 12-cm high walls) with 7.6 cm depth of bedding for 1
hour prior to the test. The mouse was then briefly removed from the testing box and 15
marbles were evenly arranged in a 5 by 3 pattern on the surface of the bedding. The
mouse was reintroduced into the testing box and was allowed to bury marbles for 10
min. At the end of the testing period, the mouse was removed from the box and the
number of marbles that were fully buried, partially buried, and left on the surface was
counted.
To examine animal activity in the light-dark test, three- to four-month old male mice
were placed in the dark half of the light-dark chamber and the trap door was opened 1
min later. The mice were allowed to move freely between the dark and the lit chambers
for 5 min. The time spent in the lit chamber and the number of crossings into the lit side
were recorded.
To examine passive avoidance learning, three- to four-month old male mice were
introduced into the lit half of the passive avoidance chamber (Coulbourn Instruments,
Whitehall, PA) and allowed to explore for 1 min before the trap door was opened. The

	
  

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trap door was closed as soon as the mouse entered the dark chamber. A mild foot
shock (0.7 mA for 2 sec) was immediately delivered. The mouse was removed from the
dark chamber and returned to its home cage 30 sec after the foot shock was delivered.
The mice were tested 24 hours after training. During testing, the mouse was put in the lit
chamber and crossover latency to the dark chamber was recorded. If mice stayed in the
lit chamber for more than 600 sec, they were manually removed from the chamber, and
600 sec was used as their crossover latency.
3.3.5 Administration of Trifluoperazine
For drug administration, Trifluoperazine dihydrochloride from Sigma (Cat #T8516) was
dissolved in water to give a 5 mg/ml stock solution. The stock was diluted in saline and
i.p. injected into mice at 0.05 mg/kg. In all cases except for open field, the drug was
administered 1 hour before testing or before training for passive avoidance. For open
field analysis, the drug was administered 30 min before testing. Control mice were
treated similarly but injected with vehicle.
3.3.6 Measurements of protein synthesis in cultured neurons
Protein synthesis was determined by the SUnSET method (Schmidt et al., 2009;
Bhattacharya et al., 2012). DIV (days in vitro) 14 hippocampal neurons were pre-treated
with trifluoperazine for 30 min followed by 5 ug/ml puromycin (Sigma, Cat #P8833)

	
  

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treatment for 30 min. Cells were lysed in Buffer H (50 mM b-glycerophosphate, 1.5 mM
EGTA, 0.1 mM Na3VO4, 1 mM DTT). The samples were sonicated and centrifuged. An
aliquot of the supernatant was used to determine protein concentration, and the rest
was denatured in Laemmli buffer. 20 ug protein was separated by 4-20% SDS-PAGE
(Invitrogen) and transferred onto nitrocellulose membranes. The membranes were
probed with anti-puromycin antibody (KeraFAST, Cat # EQ0001, 1:1000). The relative
amount of loading was determined by the level of β-actin. ImageJ was used to measure
the combined signal intensity of proteins with molecular weights ranging from 15 to 250
kDa.
3.3.7 Statistical analysis
For data analysis, one-way ANOVA followed by LSD (least significant difference) posthoc test was used to compare multiple groups. Two-way repeated measures ANOVA or
two-way repeated measures ANOVA followed by Student’s t-test were used to analyze
the electrophysiology data or the open field data. Chi-squared test was used to analyze
the AGS data. Two-way ANOVA followed by pairwise comparison or Student’s t-test
was used to compare different groups in the light-dark, passive avoidance, marble
burying tests as well as the SUnSET assay. Data were expressed as the mean ± SEM.

	
  

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Differences with p values less than 0.05 were considered significant. SPSS 11.5 for
Windows (IBM) was used for all data analysis.

3.4 Results
3.4.1 Calmodulin activity is required for mGluR1/5-mediated intracellular
signaling
Overactivation of mGluR1/5 has been considered as a cellular mechanism
underlying the pathophysiology of FXS. Thus, it is hypothesized that dampening
mGluR1/5 signaling may be of therapeutic value for FXS (Bear et al., 2004). To
effectively

manipulate

mGluR1/5-mediated

signal

transduction,

I

investigated

components in the signaling cascade. By using cultured rat hippocampal neurons, I first
confirmed that stimulation with the mGluR1/5 agonist DHPG caused significant
phosphorylation of ERK1/2 (Figure 15A, p < 0.05), whose phosphorylation level is
elevated in Fmr1 KO neurons (Michalon et al., 2012). The activity of S6K1 (p70-S6
kinase 1) is positively regulated by phosphorylation and involved in regulating ribosomal
biogenesis. In vitro studies have shown that ERK1/2 can directly phosphorylate S6K1 at
Thr421/Ser424. Consistently, activation of mGluR1/5 stimulated phosphorylation of
S6K1 at Thr421/Ser424 (p < 0.05) as well as Thr389 (p < 0.05) (Figure 15B), a target

	
  

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site of PI3K (Zhou et al., 2010). DHPG also caused significant upregulation of Arc
(Figure 15C, p < 0.05), whose function is implicated in mGluR1/5-mediated LTD (Park et
al., 2008). DHPG-induced Arc translation is also required for mGluR1/5-mediated LTD
(Waung et al., 2008).
Because increased Ca2+ release from the internal storage is one of the major
downstream cellular events following Gq activation, we expected that inhibiting the Ca2+
effector molecule CaM might block the propagation of mGluR1/5 signaling. I pre-treated
neurons with a CaM inhibitor W13 followed by DHPG stimulation. W13 blocked DHPGstimulated phosphorylation of ERK1/2 (Figure 16A, p < 0.05) and S6K1 (Figure 16B, p <
0.05) as well as the upregulation of Arc (Figure 16C, p < 0.05). Treatment with W13
alone decreased the level of ERK1/2 phosphorylation (Figure 16A) whereas the level of
Arc expression and S6K1 phosphorylation remained unchanged (Figure 16B and 16C).

3.4.2 CaM activity is required for mGluR1/5-mediated LTD
We next determined the effects of CaM inhibition on synaptic functions following
the activation of mGluR1/5. After infusion of DHPG to the CA1 region of the
hippocampus in anesthetized mice, we observed significant LTD (68.4 ± 4.6%); infusion
of the vehicle did not change synaptic responses (97.9 ± 3.4%) (Figure 17A, p < 0.005).

	
  

119	
  

Infusion of W13 alone (99.4 ± 7.1%) or W13 plus DHPG (111.1 ± 7.6%) caused
transient synaptic depression but not LTD (Figure 17B). One-way ANOVA followed by
post hoc Bonferroni test indicated that W13 suppressed mGluR1/5-mediated LTD (p <
0.01) (Figure 17C) (Dr. Ming Zhang).
In acute hippocampal slices, application of DHPG induced significant LTD, which
was significantly suppressed by W13 (p < 0.001) (Figure 17D) (Dr. Hanoch Kaphzan
and Dr. Eric Klann). These results implicate CaM as a functional component in the
mGluR1/5 signaling cascade.

3.4.3 ERK1/2 activity is required for mGluR1/5-mediated upregulation of Arc
expression
I found that CaM activity was required for both ERK1/2 activation and Arc
upregulation following mGluR1/5 stimulation. To determine whether W13 inhibits Arc
translation through suppressing ERK1/2 activity, I pre-treated neurons with the MEK
inhibitor U0126. Because MEK directly regulates ERK1/2 phosphorylation, U0126
suppressed the level of phospho-ERK1/2 in both un-stimulated and DHPG stimulated
neurons (Figure 18A, p < 0.05). I next found that U0126 also significantly blocked
DHPG-stimulated Arc upregulation (Figure 18B, p < 0.05). A previous study using

	
  

120	
  

hippocampal slices showed that U0126 suppresses mGluR1/5-LTD (Gallagher et al.,
2004). Here, we observed that U0126 inhibited hippocampal mGluR1/5-LTD in
anesthetized mice (Figure 18C, p < 0.005) (Dr. Ming Zhang). These data imply that the
attenuation of ERK1/2 activity by CaM inhibition is relevant to the suppression of Arc
upregulation and mGluR1/5-LTD.

3.4.4 Trifluoperazine attenuates audiogenic seizures and corrects hyperactivity,
repetitive behavior, and defective passive avoidance memory in Fmr1 KO mice
We reasoned that suppression of mGluR1/5 signaling through CaM inhibition
might have therapeutic value for FXS treatment. As W13 is not suitable for systemic
administration, we searched for available reagents that could be administered at
peripheral sites and cross the blood-brain barrier. We chose trifluoperazine, because it
is a well-documented CaM inhibitor and approved for clinical use by FDA to treat
psychotic conditions and anxiety in humans.
FXS and autism patients show higher susceptibility to seizures. As the major FXS
animal model, Fmr1 KO mice analogously exhibit significant audiogenic seizure (AGS)
(Musumeci et al., 2000; Chen and Toth, 2001). On exposing mice to a sound at 120 dB,
I observed wild running, clonic/tonic seizures, and death in juvenile Fmr1 KO but not

	
  

121	
  

wild type (WT) animals (Table 6). Fmr1 KO mice receiving intraperitoneal injection of
trifluoperazine at 0.05 mg/kg had lower occurrence of AGS (Table 6, p < 0.05).
Hyperactivity is observed in both human FXS patients and Fmr1 KO mice (Qin et
al., 2002). During the 2-hour open field test, Fmr1 KO mice displayed higher total travel
distance (Figure 19A1, p < 0.01; Figure 19A2, p < 0.01), and higher locomotor activity in
the center area of the arena (Figure 19B1, p < 0.01; Figure 19B2, p < 0.01) than the WT
animals. Administration of trifluoperazine (at 0.05 mg/kg) to Fmr1 KO mice corrected the
overall hyperactivity (Figure 19 4A1, drug effect: p < 0.01, genotype-drug interaction: p <
0.05; Figure 19A2, drug effect: p < 0.01, genotype-drug interaction: p < 0.01) and the
higher activity in the center area (Figure 19B1, drug effect: p < 0.01, genotype-drug
interaction: p < 0.01; Figure 19B2, drug effect: p < 0.01, genotype-drug interaction: p <
0.01).
Fmr1 KO mice show significant repetitive behavior, recapitulating perseverative
symptoms in FXS and autism patients. I examined animal behavior in the marbleburying test, which has been considered to measure repetitive behavior and barely
involves novelty-induced anxiety (Thomas et al., 2009). I first confirmed that Fmr1 KO
mice buried more marbles than WT animals (Figure 20A, p < 0.05) leaving less number
of marbles on the surface (Figure 20A, p < 0.05) (Zang et al., 2009). We further

	
  

122	
  

examined Fmr1 KO mice with the light-dark test. The mutant mice showed more
transition between the light and dark chambers than WT animals, indicating both
hyperactivity and repetitive behavior (p < 0.001) (Figure 20B1). Fmr1 KO and WT mice
spent comparable time in the light and dark chamber (Figure 20B2) (Dr. Qi Ding).
Administration of trifluoperazine (at 0.05 mg/kg) corrected both marble-burying (Figure
20A, drug effect: p < 0.05, genotype-drug interaction: p < 0.05 for the fully buried
marbles; drug effect: p < 0.05, genotype-drug interaction: p < 0.05 for the surface
marbles) and light-dark test (Figure 20B1, drug effect: p = 0.26, genotype-drug
interaction: p < 0.005) phenotypes in Fmr1 KO mice, without affecting the behavior of
WT mice.
We next trained Fmr1 KO and WT mice with passive avoidance test following
intraperitoneal injection of trifluoperazine (0.05 mg/kg) or vehicle. All groups of animals
showed similar crossover latency during training. When tested 24 hours later, the
vehicle-treated WT mice showed significantly longer crossover latency than the vehicletreated Fmr1 KO mice (Figure 21, p < 0.005). This confirms the impaired passive
avoidance memory in the FXS mice (Qin et al., 2002). Although trifluoperazine did not
have an effect on WT animals, the crossover latency in the trifluoperazine-treated Fmr1
KO mice increased to WT levels (Figure 21, drug effect: p < 0.01, genotype-drug

	
  

123	
  

interaction: p < 0.05) (Dr. Qi Ding). Thus, trifluoperazine corrected this cognitive deficit
in FXS mice.

3.4.5 Trifluoperazine blocks intracellular signaling triggered by the activation of
Gq-coupled receptors
Although previous studies have demonstrated that trifluoperazine inhibits CaM
activity, it is important to examine its effects on mGluR1/5 signaling. I found that
trifluoperazine blocked DHPG-induced phosphorylation of ERK1/2 (Figure 22A, p < 0.05
) and Arc upregulation (Figure 22B, p < 0.05). Similar to the effects of W13 (Figure 16A
and C), trifluoperazine also suppressed the basal level of ERK1/2 activity (Figure 22A)
in non-stimulated neurons.
A previous study reported that activation of Gq-coupled muscarinic cholinergic
receptors (i.e., M1, M3, and M5) by carbachol (CCh) induces translation-dependent
LTD, which is also enhanced in Fmr1 KO mice (Volk et al., 2007). Since the activation of
either mGluR1/5 or M1/3/5 leads to the activation of Gq signaling, it is not surprising that
mGluR1/5- and M1/3/5-mediated LTD share similar mechanisms. Here, I confirmed that
carbachol stimulates ERK1/2 phosphorylation in hippocampal neurons (Figure 22C and
D). Neurons co-treated with W13 and carbachol did not show upregulation of ERK1/2

	
  

124	
  

phosphorylation (Figure 22C, p < 0.05). Trifluoperazine also blocked ERK1/2
phosphorylation in carbachol-treated neurons (Figure 22D, p < 0.05). These results
demonstrate that CaM is a common downstream target of both mGluR1/5 and Gqcoupled muscarinic cholinergic receptors, and pharmacological inhibition of CaM is
effective in dampening the signaling triggered by activation of multiple Gq-coupled
receptors.

3.4.6 Trifluoperazine corrects the enhanced protein synthesis in Fmr1 KO
neurons
Enhanced basal protein synthesis in neurons is thought to be the core molecular
abnormality associated with FXS and manipulations that correct the enhanced protein
synthesis are potential strategies to treat FXS (Darnell and Klann, 2013). Here, I labeled
newly synthesized proteins in neurons with puromycin using the SUnSET method
(Schmidt et al., 2009; Bhattacharya et al., 2012). I observed enhanced protein synthesis
in Fmr1 KO neurons compared to WT neurons (Figure 23A and B, genotype effect: p <
0.05). Treatment with different concentrations of trifluoperazine specifically reduced the
level of puromycin-labeled proteins in Fmr1 KO neurons to the WT level (Figure 23B)
while it did not affect protein synthesis in WT neurons (Figure 23B).

	
  

125	
  

In summary, this study suggests CaM as a new potential therapeutic target.
Administration of a known CaM inhibitor, trifluoperazine, blocked Gq-coupled receptor
signaling and attenuated both cellular and behavioral symptoms associated with FXS.

3.5 Discussion
Due to lack of effective medical treatments for FXS, there is a significant need for
novel therapeutics. Following the discovery that showed enhanced mGluR1/5-mediated
LTD in Fmr1 KO mice, mGluR1/5 has been considered the most viable target for FXS
therapy. By using the FXS mouse model, several studies have demonstrated
therapeutic effects of mGluR5 antagonists. For example, the mGluR5 antagonist MPEP
reduces AGS and hyperactivity in the center area of the open field arena (Yan et al.,
2005). A negative allosteric mGluR5 modulator fenobam improves associative motor
learning in Fmr1 KO mice, but has significant negative effects on WT controls (Veloz et
al., 2012). In a recent study, Thomas et al. reported that MPEP and JNJ16259685 (an
mGluR1 antagonist) both reduced AGS in Fmr1 KO (Thomas et al., 2012). Considering
that FXS is a complex neurological disorder, it is not surprising that inhibition of mGluR5
alone is not effective to rescue many of the FXS-related behavioral and synaptic
abnormalities in mice (Suvrathan et al., 2010; Thomas et al., 2012; Franklin et al., 2014)

	
  

126	
  

as well as in human patients. In a small open-label single dose trial that involved 12
FXS patients, fenobam improved pre-pulse inhibition but showed no obvious effects on
the continuous performance test (Berry-Kravis et al., 2009). Disclosure of a recent
placebo-controlled phase II clinical trial using the mGluR5 antagonist AFQ056 revealed
therapeutic effects only on patients with full promoter methylation (Jacquemont et al.,
2011). It is important to note that these human studies only involved a single dosing
regime for a fairly short period of time. Further trials using multiple doses and longer
treatment durations should be seriously considered. Nevertheless, these results indicate
the possibility that inhibition of mGluR5 alone may not be robust enough to correct
multiple symptoms associated with FXS. Other drug targets should be considered in
conjunction with mGluR1/5.
One interesting mGluR1/5-independent pathophysiology in FXS is mediated by
the overactivation of the Gq-coupled muscarinic acetylcholine receptors (Gq-mAchRs
consisting of M1, M3, and M5). Volk et al. showed that the mAchR agonist carbachol
induced translation-dependent LTD that is mainly mediated through the activation of the
Gq-mAchR M1 (Volk et al., 2007). Because both mGluR1/5 and M1 are coupled to Gq, it
is not surprising that enhanced LTD in Fmr1 KO mice is observed following the
stimulation with either DHPG or carbachol. Further, Veeraragavan et al. demonstrated

	
  

127	
  

that administration of the M1 antagonist dicyclomine rescued AGS in Fmr1 KO mice
(Veeraragavan et al., 2011). However, it is evident that suppression of Gq-coupled M1
receptor alone is not robust enough to correct the multiple FXS symptoms. For instance,
dicyclomine non-specifically reduces marble-burying behavior in both WT and Fmr1 KO
animals, and does not correct some core behavioral abnormalities such as hyperactivity
(Veeraragavan et al., 2011).
In this study, we demonstrated that blocking CaM activity with two different
known inhibitors dampened signal transduction triggered by both mGluR1/5 and
muscarinic cholinergic receptors. We hypothesized that inhibiting CaM activity, would
offer therapeutic effects that are more robust than blocking a single type of Gq-coupled
receptor. In support of this hypothesis, acute administration of trifluoperazine attenuated
multiple FXS-related phenotypes ranging from audiogenic seizures to hyperactivity,
repetitive behavior, and certain aspect of cognitive defects. Interestingly, trifluoperazine
suppressed the basal level of ERK1/2 phosphorylation, which is enhanced in the brains
of FXS patients (Wang et al., 2012) and Fmr1 KO mice (Michalon et al., 2012). Such a
pharmacological effect may represent a possible mechanism underlying its therapeutic
efficacy, as it has been reported that lovastatin indirectly dampens ERK1/2
phosphorylation and corrects several cellular and behavioral FXS phenotypes

	
  

128	
  

(Osterweil et al., 2013). It has also been reported that either direct or indirect inhibition
of ERK1/2 suppresses the enhanced mRNA translation in FXS (Osterweil et al., 2010;
Osterweil et al., 2013). Although the causal role of enhanced protein synthesis in FXS is
not clear, we found that trifluoperazine specifically dampened protein synthesis and
affected behavioral phenotypes in Fmr1 KO mice without showing major effects on WT
mice, except for the locomotor activity in the open field test (Figure 19A2 and 19B2).
Trifluoperazine belongs to the phenothiazine group, and has been primarily used
to treat schizophrenia and severe anxiety. Due to its extrapyramidal effects such as
akathisia, dystonia, and Parkinsonism (Marques et al., 2004), adjustment in the dosing
regime should be seriously considered for any treatment of FXS patients. Several preclinical studies suggested new applications of trifluoperazine and other phenothiazines
in treating allodynia (Luo et al., 2008; Shirahama et al., 2012), addiction (Ye et al., 2004;
Tang et al., 2006), and cancer (Gutierrez et al., 2014). Luo et al. showed that, possibly
through inhibition of CaM and its down-stream target CaM-dependent protein kinase II,
trifluoperazine at 0.25 to 0.5 mg/kg attenuated inflammatory pain in mice (Shirahama et
al., 2012). Another study found that trifluoperazine at 0.1 mg/kg and 0.3 mg/kg reduced
oxaliplatin-induced mechanical allodynia (Shirahama et al., 2012). It was reported that
the anti-allodynia doses (ranging from 0.1 mg/kg to 0.5 mg/kg) did not have significant

	
  

129	
  

effects on rotarod performance (Chen et al., 2009) and spontaneous locomotion (Ye et
al., 2004) in rodents. Encouraged by these animal studies, an open-label human phase I
study was performed with trifluoperazine (Molokie et al., 2013). Compared to the
recommended dose used for Schizophrenia (i.e., 15-20 mg/day), lower trifluoperazine
(ranging from 0.5 to 10 mg/day) was given to adults with sickle cell disease. Patients
reported no adverse effects, as judged by the Extrapyramidal Symptom Rating Scale,
after receiving trifluoperazine at < 5 mg/day. Promisingly, dose at as low as 1 mg/day
had analgesic effects in 2 out of 3 patients. Considering that the pre-clinical dose to
attenuate FXS (0.05 mg/kg in this study) is lower than the pre-clinical doses used for
allodynia (i.e. > 0.1 mg/kg), daily administration of less than 1 mg/day of trifluoperazine
in human FXS may show therapeutic efficacy without extrapyramidal effects.
Because all available small molecule drugs have multiple pharmacological
effects, it is difficult to conclude that the therapeutic effects of trifluoperazine are
mediated exclusively through CaM inhibition. The primary application of trifluoperazine
in Schizophrenia shows strong effects on correcting the positive symptoms possibly
through its inhibition on dopamine receptors. It is known that the anti-dopamine effect of
trifluoperazine decreases locomotor activity but does not improve cognitive function. I
found that while trifluoperazine at 0.05 mg/kg dampened locomotor activity in WT mice

	
  

130	
  

in the open field test, it reduced the hyperactivity in Fmr1 KO mice to the WT level.
Notably, 0.05 mg/kg trifluoperazine rescued the cognitive impairment in Fmr1 KO mice,
and did not affect locomotion activity of the WT mice in the light/dark test. Furthermore,
a previous study showed hypo-dopaminergic function in FXS and that a dopamine
receptor agonist rescued hyperactivity in Fmr1 KO mice (Wang et al., 2008). These
lines of evidence suggest that the therapeutic effects of trifluoperazine may not be
mainly mediated through its anti-dopamine functions. However, molecular and genetic
manipulations of CaM are needed for future studies to further examine the significance
of CaM inhibition in FXS therapy. Considering that CaM activity may be essential to
regulate many cellular targets and neuronal functions, the degree of pharmacological
inhibition and the dose of CaM inhibitors should be carefully considered.
In summary, our study identifies new pharmacological effects of trifluoperazine
on mGluR1/5- and muscarinic cholinergic receptor-mediated intracellular signaling, and
suggests targeting CaM as a novel strategy to treat FXS. Repurposing existing FDAapproved drugs may offer benefits to FXS patients without involving the lengthy and
expensive process of de novo drug development. Our study suggests a potential new
application of trifluoperazine in FXS, and encourages future human studies using low
dose trifluoperazine.

	
  

131	
  

APPENDIX

	
  

132	
  

C!
DHPG !

A!
DHPG !

Arc!

pERK1/2!

β-actin!
ERK1/2!

Relative
level!

Relative
level!

2

3
2
1

1

B!
DHPG !
pS6K421!
pS6K389!
S6K !

Relative
level!

pS6K421!

pS6K389!

2!
1!

Figure 15: mGluR5 stimulation leads to phosphorylation of ERK1/2 and S6 kinase
and Arc translation. (A-C), DIV (days in vitro) 8 rat hippocampal neurons were treated
with 100 µM DHPG for 10, 30 and 60 min. The levels of pERK1/2 (A), pS6K (at
Thr421/Ser424 and Thr389) (B), and Arc (C) were determined by Western blot.
Representative images are shown in the upper panels and quantifications are shown in

	
  

133	
  

Figure 15 (cont'd)
the lower panels. The level of pERK1/2 and pS6K was normalized to the level of total
ERK1/2 and total S6K, respectively. The level of Arc was normalized to the level of βactin. The relative protein level in the no treatment control group was defined as 1, and
all samples were normalized to the control group. All data were collected from 6
independent samples. *: p < 0.05 between control and the DHPG-treated samples
determined by One-way ANOVA and LSD test.

	
  

134	
  

A!
DHPG!
W13!

-!
-!

+!
-!

+!
+!

-!
+!

+!
+!

-!
+!

#!

#!

β-actin!

ERK1/2!

2!

Relative
level!

Relative
level!

+!
-!

Arc!

pERK1/2!

3!
2!
1!

B!
DHPG! -!
W13! -!
pS6K421!

C!
DHPG! -!
-!
W13!

1!

$!

+!
-!

+!
+!

-!
+!

pS6K389!
S6K !
Relative
level!

2!
1!

#!

#! #! #!

Figure 16: Calmodulin activity is required for mGluR1/5 signaling. (A-C) DIV8 rat
hippocampal neurons were pre-treated with 75 µM W13 or vehicle control for 30 min,
after which the cells were treated with 100 µM DHPG or vehicle for 30 min. The levels of
pERK1/2 (A), pS6K (at Thr421/Ser424 and Thr389) (B), and Arc (C) were determined

	
  

135	
  

Figure 16 (cont'd)
by Western blot. Representative images are shown in the upper panels and
quantifications are shown in the lower panels. The level of pERK1/2 and pS6K was
normalized to the level of total ERK1/2 and total S6K, respectively. The level of Arc was
normalized to the level of β-actin. The relative protein level in the no treatment control
group was defined as 1, and all samples were normalized to the control group. All data
were collected from 6 independent samples. *: p < 0.05 between control and the DHPGtreated samples. $: p < 0.05 between the indicated group and the control group as well
as the DHPG-treated group. #: p < 0.05 between the indicated group and the DHPGtreated group. The p value was determined by one-way ANOVA and post hoc LSD test.

	
  

136	
  

B!

A!

fEPSP slope
(% baseline)!

fEPSP slope (%
baseline)!

W13 n=5!
W13+DHPG n=5!

150!

WT PBS n=6!
WT DHPG n=14!

150!

100!

100!
50!

50!

DHPG!
0!

-20!

0!

20!
40!
Time (min)!

C!

0!
-20!

60!

0!

20!
40!
Time (min)!

60!

fEPSP slope
(% baseline)!

150!

DHPG n=12!
W13+DHPG n=14!

100!

100!

*!

50!
0!

fEPSP slope
(% baseline)!

D!

50!
DHPG!
W13!

0!
PBS!

DHPG! W13! W13+DHPG!

-20! 0! 20! 40! 60! 80!
Time (min)!

Figure 17: Calmodulin activity is required for mGluR1/5-mediated synaptic LTD.
(A-C), DHPG, vehicle, W13, or W13 plus DHPG was infused to the CA1 region of the
anesthetized mice. The changes of fEPSP during the whole recording period (A and B),
and the averages during the last 5 min of recording (C) are presented as mean +/- SEM.
(D) DHPG or W13 plus DHPG was applied to acute hippocampal slices, and changes of
fEPSP were recorded and presented as mean +/- SEM. *: significantly different (p <
0.05) from other three groups.

	
  

137	
  

A!

B!
DHPG! -!
U0126! -!

+!
-!

+!
+!

-!
+!

DHPG!
U0126!

pERK1/2!

Arc!

ERK1/2!

β-actin!

-!

+!

+!

-!

-!

-!

+!

+!

Relative
level!

2!

Relative
level!

2!

3!

1!

1!

$!

$!

#!

#!

C!

fEPSP slope
(% baseline)!

150!

DHPG n=9!
DHPG+ U0126 n=6!

100!
50!
DHPG!
0!
-20!

0!

20!
40!
Time (min)!

60!

Figure 18: Inhibition of ERK1/2 suppresses mGluR1/5-mediated Arc upregulation
and LTD. DIV 8 rat hippocampal neurons were treated with 10 µM MEK inhibitor U0126
for 30 min, following which the cells were stimulated with 100 µM DHPG for 30min.
Protein samples were analyzed by Western blot and are presented as normalized
protein levels relative to the no treatment control. (A) U0126 suppressed ERK1/2
phosphorylation in both control and DHPG-stimulated neurons. (B). U0126 inhibited

	
  

138	
  

Figure 18 (cont'd)
DHPG-stimulated Arc upregulation. Representative results are shown in the upper
panels, and quantification (n = 6 for each group) is shown in the lower panels. *: p <
0.05 between control and DHPG-treated samples. $: p < 0.05 between the indicated
group and the control group as well as the DHPG-treated group. #: p < 0.05 between
the indicated group and the DHPG-treated group. The p value was determined by oneway ANOVA and post hoc LSD test. (C) U0126 suppressed DHPG-induced LTD in
anesthetized mice. 0.5 ul DHPG (2.5 mM) or 0.5 ul DHPG (2.5 mM) plus U0126 (2 mM)
was infused to the CA1 region. The relative fEPSP value was normalized to the baseline
level.

	
  

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Table 6: TFP rescues audiogenic seizure in Fmr1 KO mice

n!

%Wild % seizures! %Death!
running!

WT!

5!

0!

0!

0!

Fmr1!

27!

55.5!

29.6!

3.7!

WT+TFP!

5!

0!

0!

0!

Fmr1+TFP!

29!

34.5!

17.2!

3.4!

Table 6: Audiogenic seizures were tested with 21- to 24-day old mice. The percentage
of animals showing wild running, seizures, and death is presented for WT and Fmr1 KO
mice receiving vehicle or trifluoperazine. Chi-square test was used to determine
significance between Fmr1 KO vehicle and TFP groups.

	
  

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Center distance (cm)!

B1!
40
0!
30
0!
20
0!
10
0!
0

2000!
1000!
0!

50! 70! 90! 110!
min!
WT!

Fmr1+TFP!
Fmr1! WT+TFP!

B2!

3500!
3000!
2500!
2000!
1500!
1000!
500!

10! 30!

50! 70! 90! 110!
min!

#

Fmr1+TFP!

10! 30!

3000!

WT+TFP!

0

4000!

Fmr1+TFP!

200!

#!

WT+TFP!

400!

5000!

Fmr1!

600!

6000!

Fmr1!

WT+TFP n=9!

WT!

Fmr1 n=12!

A2! 7000!

WT!

Fmr1+TFP n=9!

Total distance (cm)!

Distance (cm)!

800!

WT n=10 !

Total center
distance (cm)!

A1!

0!

Figure 19: Trifluoperazine attenuates the hyperactivity phenotype in Fmr1 KO
mice. Two- three-month old WT and Fmr1 KO mice receiving vehicle or trifluoperazine
were subjected to a 2-hour open filed test. The total travel distance (A1 and A2) and
center distance (B1 and B2) are presented. The activity for each 10-min bin is presented
in A1 and B1. Cumulative activity during the whole 2-hour testing is presented in A2 and
B2. Two-way ANOVA followed by Student’s t-test were used to

	
  

141	
  

Figure 19 (cont'd)
determine the p values. *: p < 0.05 between the indicated group and vehicle-treated WT
mice. #: p < 0.05 between vehicle- and trifluoperazine-treated Fmr1 KO mice.

	
  

142	
  

#!
60!

20!
Fully buried! Surface! Partially buried!

0

Time in light
chamber (s)!

80!
40!
0!

Fmr1! WT+TFP! Fmr1+TFP!

WT!

N=17!

120!

N=20!

N=19!

N=17!

N=20!
WT!

N=17!

# of light chamber
entries!

#!

10!
5

N.S.!

B2!

15!

N=17!

0!

B1!

#!

40!

WT n=12!
Fmr1 n=17!
WT+TFP n=13!
Fmr1+TFP n=15!

N=19!

% marbles!

A!

Fmr1! WT+TFP!Fmr1+TFP!

Figure 20: Trifluoperazine rescues repetitive behavior in Fmr1 KO mice. 2-3 month
old WT and Fmr1 KO mice were injected with vehicle or trifluoperazine 60 min before
testing. (A) The percentage of marbles fully buried, on the surface and partially buried
was determined. (B) WT and Fmr1 KO mice (2 to 3 months of age) receiving vehicle or
trifluoperazine were subjected to the light/dark test. The total number of entries to the lit
chamber (B1) and time spent in the lit chamber (B2) were scored. Two-way ANOVA
followed by Student’s t-test or pairwise comparison was used to determine the p value.

	
  

143	
  

Figure 20 (cont'd)
*: p < 0.05 between the indicated group and vehicle treated WT mice. #: p < 0.05
between indicated group and vehicle-treated Fmr1 KO mice. N.S.: not significant.

	
  

144	
  

Crossover latency (s)!

600!

WT n=14!
Fmr1 n=17!
WT+TFP n=11!
Fmr1+TFP n=9!

#!

400!

*!

200!
0!

Training!

Testing!

Figure 21: Trifluoperazine rescues the impaired passive avoidance memory in
Fmr1 KO mice. Two- to three-month old WT and Fmr1 knockout mice were injected
with vehicle or trifluoperazine 60 min before passive avoidance training. Mice were
tested 24 hours after training, and crossover latency was scored. Two-way ANOVA
followed by pairwise comparison was used to determine the p value. *: p < 0.05
between the indicated group and the vehicle-treated WT mice, #: p < 0.05 between
indicated group and vehicle-treated Fmr1 KO mice.

	
  

145	
  

-!
-!

+!
-!

+!
+!

-!
+!

B!
DHPG!
TFP!

*!

Relative
level!

200!

-!
+!

#!

#!

*!

200!

#!

*!

100!

100!
0!

0!
+!
-!

+!
+!

D!
CCh!
TFP!
pERK1/2!

-!
-!

+!
-!

+!
+!

ERK1/2!

*!

#!

Relative
level!

-!
-!

ERK1/2!

Relative
level!

+!
+!

β-actin!

300!

1.2!
0.8!
0.4!
0!

+!
-!

Arc!

ERK1/2!

C!
CCh!
W13!
pERK1/2!

-!
-!

Relative
level!

A!
DHPG!
TFP!
pERK1/2!

1.2!
0.8!

*!
#!

0.4!
0!

Figure 22: Trifluoperazine suppresses mGluR1/5- and muscarinic receptormediated intracellular signaling. DIV 8 rat hippocampal neurons were treated with 20
µM trifluoperazine (A, B and D) or 75 uM W13 (C) for 30 min before the 30-min
treatment with 100 µM DHPG (A and B) or 100 µM carbachol (C and D). Samples
harvested immediately after the stimulation were analyzed by Western blot. The
representative results showing the levels of p-ERK1/2, total ERK1/2, Arc, and b-actin

	
  

146	
  

Figure 22 (cont'd)
are presented in the upper panels. Quantification (n = 6 from 2 independent
experiments for each treatment) is presented in the lower panels, all samples are
normalized to the control. One-way ANOVA and post hoc LSD test were used to
determine the p value. *: p < 0.05 between the indicated group and the non-treated
control group. #: p < 0.05 between the indicated group and the DHPG- or carbacholtreated group.

	
  

147	
  

WT! Fmr1!WT!Fmr1!

-!

-! +! +!

Puromycin-labeled
proteins!

A!
TFP!

β-actin!

B!

Relative level!

2.5!
2!

1.5!

*!

#!
#!

#!

WT!
#!

Fmr1!
#!

#!

1!

0.5!
0!

Control! TFP! TFP! TFP!
100nM! 500nM! 1uM!

TFP! TFP! TFP!
2uM! 10uM! 20uM!

Figure 23: Trifluoperazine suppresses enhanced basal protein translation in Fmr1
KO neurons. Basal protein synthesis was determined in DIV 14 WT and Fmr1 KO
mouse neurons by the SUnSET method. Cells were pre-incubated with vehicle or
trifluoperazine for 60 min before the addition of puromycin into the culture media. Equal
amounts of protein were loaded on the gel for Western blot analysis. (A) Representative

	
  

148	
  

Figure 23 (cont'd)
image shows the level of puromycin-labeled proteins is higher in Fmr1 KO than WT
neurons, and 100 nM trifluoperazine dampens protein synthesis in Fmr1 KO but not WT
neurons. (B) Quantification (n = 6 from 2 independent experiments for each treatment)
shows that the enhanced protein synthesis in Fmr1 KO but not WT neurons was
suppressed by different concentrations of trifluoperazine. All the samples are relative to
control WT levels. Student’s t-test were used to determine the p value. *: p < 0.05
between Fmr1 KO and WT neurons. #: p < 0.05 between the treated and non-treated
Fmr1 KO neurons.

	
  

149	
  

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150	
  

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CHAPTER 4: CONCLUDING REMARKS AND FUTURE DIRECTIONS
This research identifies two novel therapeutic targets for fragile X syndrome,
using the Fmr1 KO mouse model.
The results in Chapter 2 show that type-1 adenylyl cycalse (AC1) mRNA
translation is repressed by FMRP. This results in AC1 protein overexpression in the
absence of FMRP. Further AC1 protein is rapidly translated in response to mGluR5
stimulation and this response is lost in the absence of FMRP. Using a genetic knockout
and pharmacological inhibition, this study shows that abolishing or reducing AC1 activity
in Fmr1 KO mice rescues cellular, synaptic and behavioral phenotypes related to mental
retardation and autism. We currently do not understand the cellular mechanisms that
result in AC1 translation upon mGluR5 stimulation and whether mGluR5 stimulated
translation of AC1 is regulated by FMRP. One possible mechanism worth investigating
would be to check whether the status of FMRP phosphorylation can control AC1 mRNA
translation via microRNA mediated mechanisms. Muddhashetty et al., demonstrated
that FMRP phosphorylation promotes the formation of an AGO2-miR125a inhibitory
complex on PSD-95 mRNA, further mGluR5 stimulation leads to dephosphorylation of
FMRP which results in release of the complex from the mRNA and rapid translation of
PSD-95. These studies could be performed using phosphomimic and phosphodeficient

	
  

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FMRP and the luciferase assays described in Chapter 2. FMRP may also regulate AC1
via other mechanisms that we did not test. Co-immunoprecipitation of AC1 mRNA with
FMRP would aid in identifying its binding sites on the mRNA. Since mouse AC1 mRNA
is 12.26 kb long, this could be challenging. Bioinformatically identifying potential binding
sites such as G-quartet forming structures or the proposed RNA recognition elements of
FMRP: ACUK and WGGA (where K is G or U and W is A or U), may aid in this search.
Another aspect that needs to be studied is the mechanistic contribution of AC1 in
mGluR or other Gq signaling pathways that are altered in the absence of FMRP.
Interestingly, previous work from our lab shows that mGluR5 induced LTD is absent in
AC1 KO mice (Dr. Ming Zhang). Given our current findings, and the knowledge that
Fmr1 KO mice show enhanced mGluR5 LTD, it would be interesting to study how
DHPG would affect LTD in the DKO mice. We tested ERK1/2 (phosphorylation)
activation by AC1 as one plausible mechanism that could link AC1 to mGluR5 LTD.
Since AC1 is a Ca2+/Calmodulin stimulated protein, and mGluR5 is a Gq coupled protein
which can activate Ca2+/Calmodulin signaling, we hypothesized that AC1 may indirecly
phosphorylate ERK1/2 by activating Rap1 upon mGluR5 stimulation. However, I
observed increased ERK1/2 phosphorylation upon mGluR5 stimulation in AC1 KO
neurons, making this conclusion difficult. It is possible that AC1 could indeed modulate

	
  

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ERK1/2 singaling in this pathway, but our preliminary studies could not reveal this
mechanism. Further studies into these mechanisms are warranted.
Finally, our experiments show that an acute single dose of NB001 can rescue
various phenotypes in the Fmr1 KO mouse. This is indeed encouraging, and the next
critical step would be to check how a repeated dose and preferably orally administered
regimen will affect these phenotypes. Since FXS is also a developmental disorder, it
would be interesting to study whether prenatal exposure to NB001 could ameliorate FXS
phenotypes.
Chapter 3 demonstrates that calmodulin is a signaling component in the mGluR5
pathway and is crucial for mGluR5 induced LTD. It is important to note that these
studies were performed using a pharmacological inhibitor of calmodulin and studies
using genetic inhibition are important for confirming these results. Since there are no
available inhibitors of calmodulin that can be systemically administered and cross the
blood-brain barrier, we used trifluoperazine which is a known calmodulin inhibitor.
Trifluoperazine is a 'typical' antipsychotic used for treatment of schizophrenia. Its use is
often associated with extrapyrimidal side effects in patients and with the development of
better antipsychotics, it is no longer extensively used in the clinic. However we used a
low dose of this drug in our studies and demonstrated that this FDA approved drug can

	
  

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rectify cellular and behavioral phenotypes associated with FXS in the Fmr1 KO mouse.
It is important to note that trifluoperazine is not a specific calmodulin inhibitor and like
many other drugs used clinically; the mechanistic basis of this phenotypic rescue is not
known. To aid in understanding whether calmodulin is a good target for FXS, we need to
investigate whether a highly specific calmodulin inhibitor and/or acute genetic inhibition
of calmodulin can cause phenotypic rescue in Fmr1 KO mice. Towards this, we can
inject W13 (a high specificity calmodulin inhibitor), express dominant- negative
calmodulin or deliver siRNA to the mouse brain and observe the phenotypes in Fmr1
KO mice. If calmodulin is indeed a good therapeutic target for FXS, these studies would
further the development of better and safer calmodulin inhibitors and aid in drug
development. Our study using this FDA approved drug is nonetheless important
because there is currently no cure for FXS and often a cocktail of various drugs are
administered to manage the symptoms, antipsychotics being one of them. Thus low
doses of trifluoperazine may benefit current FXS patients without them having to wait for
the process of the lengthy clinical trials which are associated with the development of
new drugs.

	
  

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