Autism Spectrum Disorder Causes, Mechanisms, and Treatments: Focus on Neuronal Synapses

REVIEW ARTICLE published: 05 August 2013 MOLECULAR NEUROSCIENCE doi: 10.3389/fnmol.2013.00019 Autism spectrum disorder causes, mechanisms, and treatments: focus on neuronal synapses

Hyejung Won 1,WonMah1,2 and Eunjoon Kim 1,2*

1 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, South Korea 2 Center for Synaptic Brain Dysfunctions, Institute for Basic Science, Daejeon, South Korea

Edited by: Autism spectrum disorder (ASD) is a group of developmental disabilities characterized Nicola Maggio, The Chaim Sheba by impairments in social interaction and communication and restricted and repetitive Medical Center, Israel interests/behaviors. Advances in human genomics have identified a large number of Reviewed by: genetic variations associated with ASD. These associations are being rapidly verified by a Carlo Sala, CNR Institute of Neuroscience, Italy growing number of studies using a variety of approaches, including mouse genetics. These Lior Greenbaum, Hadassah Medical studies have also identified key mechanisms underlying the pathogenesis of ASD, many Center, Israel of which involve synaptic dysfunctions, and have investigated novel, mechanism-based *Correspondence: therapeutic strategies. This review will try to integrate these three key aspects of ASD Eunjoon Kim, Center for Synaptic research: human genetics, animal models, and potential treatments. Continued efforts in Brain Dysfunctions, Institute for Basic Science, and Department of this direction should ultimately reveal core mechanisms that account for a larger fraction Biological Sciences, Korea Advanced of ASD cases and identify neural mechanisms associated with specific ASD symptoms, Institute of Science and Technology, providing important clues to efficient ASD treatment. Kuseong-dong, Yuseong-ku, Daejeon 305-701, South Korea Keywords: autism spectrum disorder, therapeutics, genetics, animal model, synapse, synaptopathy e-mail: [email protected]

INTRODUCTION TO AUTISM SPECTRUM DISORDER disorder (ADHD)-like hyperactivity, intellectual disability, and Autism spectrum disorder (ASD) is a group of developmen- mood disorders such as anxiety and aggression (Goldstein and tal disabilities characterized by abnormal social interaction and Schwebach, 2004; Simonoff et al., 2008; Geschwind, 2009). Some communication, and stereotyped behaviors with restricted inter- monogenic syndromes including fragile X syndrome and Rett est. Autism was ﬁrst reported by Kanner (1943) with a clinical syndrome also have autistic features, while we should be cau- description of 11 children showing “extreme aloneness from the tious to directly interpret the disorders as autism since the major very beginning of life, not responding to anything that comes symptoms for these syndromes are intellectual disabilities. to them from the outside world.” He proposed the behavioral combination of autism, obsessiveness, stereotypy, and echolalia PREVALENCE as childhood schizophrenia. However, until the 1980s, ASD was An early study conducted in the UK in 1966 reported a preva- not accepted as an individual developmental disorder with a bio- lence rate of autism of 4.5 in 10,000 children (Lotter, 1966). The logical origin. In the early 1980s, studies demonstrated the high estimated prevalence increased to 19 in 10,000 American chil- heritability of ASD and its association with other genetic syn- dren in 1992 and rose steeply to 1 in 150 in 2002 (Autism et al., dromes (Gillberg and Wahlstrom, 1985; Wahlstrom et al., 1986), 2007) and 1 in 110 in 2006 (Autism et al., 2009)(seealsodata providing compelling evidence for a genetic etiology of ASD from the US Centers for Disease Control and Prevention [CDC]). and fueling the conceptualization of autism as a distinct neu- The currently accepted prevalence of ASD, based on consistent rodevelopmental disorder. From the deﬁnition of “childhood or reports of ASD prevalence by multiple sources in different pop- early-onset schizophrenia” put forward by Kanner, autism was ulations, is ∼1% worldwide, placing this disorder as one of the renamed “infantile autism” in 1980, “autism disorder” in 1987 most common pervasive developmental disorders and elevating and, more recently, “autism” or the umbrella term “ASD”. public concerns.

DIAGNOSIS GENETICS Currently, ASD is included in the diagnostic category of a On the basis of numerous studies that have been undertaken neurodevelopmental disorders in the Diagnostic and Statistical to elucidate the pathogenic mechanisms underlying ASD, it is Manual of Mental Disorders V (Grzadzinski et al., 2013). The widely accepted that ASD is a disorder with strong genetic com- diagnosis of autism is mainly based on the presence of two major ponents. In support of this notion, the concordance rates for aforementioned symptoms: social-communication deﬁcits, and autism reach up to 90% in monozygotic twins and 10% in dizy- restricted and repetitive interests/behaviors (Grzadzinski et al., gotic twins (Rutter, 2000; Folstein and Rosen-Sheidley, 2001; 2013). These symptoms must be shown from early childhood Veenstra-Vanderweele et al., 2003). of individuals with ASD. But autism is also associated with However, autism is an etiologically heterogeneous disorder various comorbidities, including sensory and motor abnormal- in that no single genetic mutation accounts for more than ities, sleep disturbance, epilepsy, attention deﬁcit/hyperactivity 1–2% of ASD cases (Abrahams and Geschwind, 2008). Thus

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far, linkage and candidate-gene analyses, genome-wide associa- ANIMAL MODELS FOR ASD tion studies (GWAS), and assessments of chromosomal variations Animal models of human diseases need to satisfy three major have uncovered a wide range of genes with predisposing muta- criteria; face validity, construct validity, and predictive validity. tions and polymorphisms associated with ASD (International Animal models for ASD should display behavioral abnormalities, Molecular Genetic Study of Autism, 1998, 2001; Abrahams and including impaired sociability, impaired social communication, Geschwind, 2008; Glessner et al., 2009; Ma et al., 2009; Wang and repetitive and restricted behaviors (face validity). These mod- et al., 2009; Weiss et al., 2009; Anney et al., 2010; Pinto et al., els should share analogous genetic or anatomical impairments 2010; Devlin and Scherer, 2012; Moreno-De-Luca et al., 2013) with humans (construct validity), and show similar responses (see Tables 1, 2 for examples). Moreover, recent advancements in to the medications used to treat ASD in humans (predictive exome sequencing and next-generation sequencing have enabled validity). the discovery of an overwhelming number of de novo muta- Dedicated efforts of many behavioral neuroscientists including tions that confer a risk for ASD (Iossifov et al., 2012; Neale Jacqueline Crawley led to the establishment of several well-known et al., 2012; O’Roak et al., 2012a,b; Sanders et al., 2012). These assays for rat/mouse models of ASD (Silverman et al., 2010b). mutations include rare mutations or copy number variations Examples include 3-chambered test to assess sociability and social in synaptic proteins such as Shanks/ProSAPs (Durand et al., novelty recognition of rodents, ultrasonic vocalization (USV) test 2007; Berkel et al., 2010; Sato et al., 2012) and neuroligins to measure the communication patterns of rodents, T-maze test (Jamain et al., 2003). for restricted interests, and home cage behavior or marble bury- However, how these mutations lead to ASD phenotypes is ing assay for repetitive behaviors. Through these assays, many poorly understood. In addition, many ASD-related genes are also genetic and non-genetic animal models of ASD have been char- associated with other neuropsychiatric disorders. For example, acterized and used to identify the etiology of ASD and develop IL1RAPL1 and OPHN1 are associated with X chromosome-linked novel treatments (see Tables 3–6 for four different groups of ASD intellectual disability (Billuart et al., 1998a; Carrie et al., 1999). models). Additional examples include schizophrenia for RELN, GluR6, Although animal models are useful for exploring ASD mech- GRIN2A, GRIN2B,andCNTNAP2 (Bah et al., 2004; Friedman anisms and testing novel interventions, we should be cautious et al., 2008; Shifman et al., 2008; Demontis et al., 2011), child- in interpreting the results from animal models of ASD because hood absence epilepsy for GABRB3 (Feucht et al., 1999), ADHD what we are observing in animals are behavioral features that look and depression for 5-HTT (Manor et al., 2001; Caspi et al., 2003), similar to some of the ASD symptoms in humans. This notion and major depression for TPH2 (Zill et al., 2004). Dissecting partly stems from the fact that the brains of humans and rodents the neural mechanisms underlying diverse symptoms/disorders are fundamentally different. For instance, there are small but sig- caused by single genetic defects is one of the key directions for niﬁcant differences in gene expression patterns in the cerebral neuropsychiatric research. cortex in different species (Zeng et al., 2012), suggesting that the

Table 1 | Examples of ASD-associated chromosomal loci and candidate genes from GWAS.

Chromosomal loci Candidate genes Sample size Design Population References

4p, 7q, 16p GPR37, PTPRZ1, 87 affected sib pairs Family 99 Caucasian families International EPHB6, PTN, CASP2, and 12 non-sib (66 from the UK, 11 from Molecular Genetic GRM8, EAG in 7q region affected relative pairs Germany, 10 from the Study of Autism, Netherlands, 5 from USA, 5 from 1998 France, 2 from Denmark)

2q, 4q, 5p, 6q, 7q, GABRB3 in 15q11-q15 51 families including at Family 51 Caucasian families Philippe et al., 1999 10q, 15q11-q15, region, MACS GRIK6, least two siblings or (18 from Sweden, 15 from France, 16p, 18q, 19p, Xp GPR6 in 6q region half-siblings affected 6 from Norway, 5 from the USA, 3 by autism from Italy, 2 from Austria and 2 from Belgium)

5p14.1 CDH9, CDH10 in 5p14.1 943 families Family Autism Genetic Resource Wang et al., 2009 region Exchange (AGRE)

5p14.1 CDH9 and CDH10 in 487 families Family 487 Caucasian families (80 Ma et al., 2009 5p14.1 region multiplex families, 407 singleton familes)

5p15, 6q27,20p13 TAS2R1 and SEMA5A in 1031 multiplex families Family AGRE and US National Institute Weiss et al., 2009 5p15 region for Mental Health (NIMH)

20p12.1 MACROD2 in 20p12.1 1558 families Family Autism Genome Project (AGP) Anney et al., 2010

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Table 2 | Examples of ASD-associated human genetic variations.

Genes CNV/SNV Sample size Design Population References

MET rs1858830 743 autism families, Case/control, family Italian and American Campbell et al., 702 unrelated autism population 2007 patients/189 unrelated controls

WNT2 linkage disequilibrium in 75 autism-affected sibling Trio Families recruited from three Wassink et al., 2001 Wnt 3UTR, R299W, L5R pair families (ASP) regions of the United States (Midwest, New England, and mid-Atlantic states)

rs3779547,rs4727847, 170 autism patients/214 Case/control Japanese population Marui et al., 2010 rs3729629 normal controls

RELN 5 UTR polymorphic GGC 371 families Family Caucasian Skaar et al., 2005 repeats

172 autism trios, 95 Case/control, trio Italian and American Persico et al., 2001 unrelated autism population patients/186 unrelated controls

EN2/ rs1861972, rs1861973 518 families Family AGRE and National Institutes Benayed et al., ENGRAILED-2 of Mental Health (NIMH) 2005; Gharani et al., 2004

HOXA1 A218G 57 probands, 166 relatives Probands/relatives Not identiﬁed Ingram et al., 2000b

CHD8 de novo frameshift, 209 trios Trio Simons Simplex Collection O’Roak et al., 2012a nonsense mutations (SSC)

GRIK2 (GluR6) M867I 59 ASP, 107 trios Family Families recruited from 7 Jamain et al., 2002 countries (Austria, Belgium, France, Italy, Norway, Sweden, US)

GRM8 R859C, R1085Q, R1100Q, 196 multiplex families Family AGRE Serajee et al., 2003 intrachromosomal segmental duplication

GRIN2A rs1014531 219 sibling pairs, 32 Family International Molecular Barnby et al., 2005 (GluN2A) families with extended Genetics Study of Autism relative pairs Consortium (IMGSAC)

GRIN2B de novo protein truncating 209 trios Trio Simons Simplex Collection O’Roak et al., 2012a (GluN2B) and splicing mutations (SSC)

GABRB3 Linkage disequilibrium 138 families, mainly trio Family 104 Caucasian, 6 African Cook et al., 1998 American, 13 Asian American, 5 Hispanic

Transmission disequilibrium 70 families Trio AGRE, Seaver Autism Buxbaum et al., Research Center (SARC) 2002

5-HTT Transmission disequilibrium 86 trios Trio 68 Caucasian, 5 African Cook et al., 1997 American, 3 Hispanic American, 10 Asian American

TPH2 rs4341581, rs11179000 88 autistic subjects, 95 Case/control people from Utah Coon et al., 2005 unrelated controls

(Continued)

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Table 2 | Continued

Genes CNV/SNV Sample size Design Population References

NRXN1 rs1363036, rs930752, 1491 families Family Autism Genome Project Autism Genome hemizygous CNV deletion (AGP) Consortium Project et al., 2007 of coding exons of NRXN1

L18Q, L748I, rs1045874 57 ASD subjects, 27 OCD Case/control Developmental Genome Kim et al., 2008 subjects, 30 Tourette Anatomy Project (DGAP) syndrome subjects

NLGN3 R451C 36 sibling pairs, 122 trios Trio not identiﬁed Jamain et al., 2003

NLGN4 Frameshift mutation by 1bp insertion (1186InsT)

SHANK1 de novo deletion 1614 ASD subjects, 15000 Case/control 1158 Canadian, 456 European Sato et al., 2012 controls

SHANK2 CNV deletion for premature 396 ASD cases, 184 MR Case/control Canadian for ASD, German Berkel et al., 2010 stop, R26W, P208S, R462X, cases, 659 controls for MR T1127M, A1350T, L1008_P1009dup

R443C, R598L, V717F, 851 ASD cases, 1090 Case/control Paris Autism Research Leblond et al., 2012 A729T, E1162K, G1170R, controls International Sibpair (PARIS) V1376I, D1535N, L1722P

SHANK3 R12C, A198G, R300C, 227 families Family PARIS Durand et al., 2007 G1011V, R1066L, R1231H, de novo frameshift mutation, de novo truncating mutation

CNTNAP2 rs2710102 476 trios Trio AGRE Alarcon et al., 2008

rs779475 72 families Family NIMH Arking et al., 2008

Nonsynonymous variants, 635 patients, 942 controls Case/control 587 white, 24 white-Hispanic, Bakkaloglu et al., I869T 7 unknown, 6 Asian, 6 more 2008 than one race, 3 African-American, 1 Native Hawaiian, 1 more than one race-Hispanic

rs17236239 184 families Family Speciﬁc Language Vernes et al., 2008 Impairment Consortium (SLIC)

ILRAPL1 Frameshift 142 ASD case, 189 Case/control 85 French Canadians, 47 Piton et al., 2008 controls European Caucasians, 10 OPHN1 Frameshift non-Caucasians Piton et al., 2011

SYNGAP1 CNV deletion 996 ASD cases, 1287 Case/control European Pinto et al., 2010 controls

TM4SF2 Nonsynonymous variants, 142 ASD case, 189 Case/control 85 French Canadians, 47 Piton et al., 2011 P172H controls European Caucasians, 10 non-Caucasians same cell types in different species may have different functions. create difficulties in translating the ASD-related mechanisms Moreover, the size, structural complexity, and neural connectivity identified in model organisms into human applications. However, of the human brain are much greater than those in rodent brains. some fundamental aspects of the neural mechanisms identified These functional and anatomical differences between species may in animal models such as alterations in synaptic transmission,

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Table 3 | ASD models with chromosomal abnormality.

Mouse Molecular function Phenotype Suggested References mechanism Social Social Repetitive Other interaction communication behavior phenotypes

15q11-13 Ube3a, Gabr Impaired Reduced calls Behavioral NA Altered serotonergic Nakatani et al., 2009 duplication inﬂexibility signaling

16p11.2 CNV Kif22, Mapk3 NA NA Climbing Altered diurnal Hypothalamic Horev et al., 2011 deﬁcits rhythm Deﬁcits

22q11.2 Dgcr2, Comt, Dgcr8 NA NA NA Hyperactivity, Altered microRNA Stark et al., 2008 microdeletion Sensorygating biogenesis deﬁcits

excitation-inhibition balance, and neuronal excitability might be In support of this notion, selective deletion of Tsc1 (tuberous conserved across species and translatable. In addition, given that sclerosis 1) in cerebellar Purkinje cells is sufficient to cause all stem cell technologies are rapidly improving, it is becoming eas- core autism-like behaviors in mice in association with reduced ier for the changes observed in rodent neurons to be compared excitability in Purkinje cells (see also Table 4 for summary of with those in human neurons derived from individuals with syndromic ASD models) (Tsai et al., 2012). In addition, mice −/− neuropsychiatric disorders (Brennand et al., 2011). lacking the neuroligin-3 gene (Nlgn3 mice), another autism model with an Nlgn3 deletion identified in autistic patients, POTENTIAL MECHANISMS UNDERLYING ASD show occluded metabotropic glutamatergic receptor (mGluR)- Mechanisms underlying autism have been extensively studied dependent long-term depression (LTD) at synapses between par- using various approaches. Neuroanatomical studies have reported allel fibers and Purkinje cells in association with motor coordina- macrocephaly and abnormal neuronal connectivity in autistic tion deficits (see also Table 5 for summary of synaptopathy ASD individuals, while genetics studies using mouse models have models) (Baudouin et al., 2012). Both synaptic and behavioral implicated a variety of neuronal proteins in the development of perturbations are rescued by Purkinje cell-specific re-expression ASD. More recently, defects in a number of synaptic proteins have of Nlgn-3 in juvenile mice, suggesting the interesting possibility been suggested to cause ASD via alterations in synaptic struc- that altered neural circuits can be corrected after completion of ture/function and neural circuits, suggesting that “synaptopathy” development. is an important component of ASD. The cerebral cortex is another brain region frequently affected in ASD. Abnormal enlargement or hyperplasia of the cerebral cor- NEUROANATOMICAL ABNORMALITIES tex has been reported in MRI studies on young children with A change frequently observed in the brains of individuals with ASD (Sparks et al., 2002; Herbert et al., 2003). Because frontal ASD is the overgrowth of the brain termed macrocephaly, which and temporal lobes are important for higher brain functions is observed in ∼20% of autistic children (Bolton et al., 2001; including social functioning and language development, these Courchesne, 2002; Courchesne et al., 2003, 2007; Fombonne anatomical anomalies are likely to underlie the pathophysiology et al., 1999; Hazlett et al., 2005). Aberrations in cytoarchitec- of autism. tural organization in autistic brains are observed during early The amygdala and hippocampus are subcortical brain regions brain development in regions including the frontal lobe, parieto- associated with ASD (Aylward et al., 1999; Schumann et al., 2004; temporal lobe, cerebellum, and subcortical limbic structures Schumann and Amaral, 2006). Some studies have reported that (Fombonne et al., 1999; Bolton et al., 2001; Courchesne, 2002; the autistic amygdala exhibits early enlargement, whereas others Courchesne et al., 2003, 2007; Hazlett et al., 2005). have reported a reduction in neuron numbers and amygdala vol- The cerebellum is a strong candidate for anatomic abnormal- ume. Increases and decreases in the volume of hippocampus are ities in autism (Courchesne, 1997, 2002). Magnetic resonance also associated with ASD. imaging (MRI) studies have found hypoplasia of the cerebellar Aberrant connectivity is an emerging theory to account for vermis and hemispheres, and autopsy studies have reported a anatomical abnormalities in autism. Neuroimaging techniques, reduction in the number of cerebellar Purkinje cells. In line with such as diffusion tensor imaging (DTI) and functional MRI these anatomical changes, cerebellar activation is significantly (fMRI), have suggested that ASD involves abrogation of white reduced during selective attention tasks (Allen and Courchesne, matter tracts in brain regions associated with social cognition, 2003), whereas it is enhanced during a simple motor task (Allen such as the prefrontal cortex, anterior cingulate cortex, and supe- et al., 2004). Although the putative role of the cerebellum in rior temporal regions (Barnea-Goraly et al., 2004; Minshew and ASD has been restricted to sensory and motor dysfunctions, it is Williams, 2007). Alterations in connectivity across diverse brain becoming increasingly clear that the cerebellum is associated with regions associated with language, working memory, and social thecoresymptomsofautism. cognition have also been linked to autism. In general, it appears

Frontiers in Molecular Neuroscience www.frontiersin.org August 2013 | Volume 6 | Article 19 | 5 Won et al. ASD causes, mechanisms, and treatments Crusio, 2006 2011; Tsai et al., 2012 2001, 2002 2002; Moretti et al., 2005 2008 et al., 2011 Kwon et al., 2006 Bernardet and Auerbach et al., Costa et al., Shahbazian et al., Ehninger et al., Penagarikano Han et al., 2012 Syndrome References Duclos syndrome Potential therapeutics MPEP FXS NANA Neurofibromatosis Cowden/Lhermitte- BDNF,IGF-1 Rett syndrome CDPPB Risperidone CDFE Clonazepam Dravet’s syndrome mechanism mGluR hyperfunction hyperactivation hyperactivation, Macrocephaly Decreased GABAergic transmission interneurons, Abnormal neuronal migration GABAergic transmission Anxiety Ataxia Cerebellar deficits Rapamycin Tuberous sclerosis problem, Lethality Learning deficits mGluR hypofunction Rapamycin, inflexibility Repetitive behavior Other phenotypes Hindlimb clasping Respiratory burying Phenotype Suggested Social communication Increased scent marking Social interaction Impaired (enhanced interaction) Impaired NAImpaired Increased calls Grooming, Behavioral Hand flappingImpaired Learning deficits, NAImpaired NA NA NA Learning deficits Ras signaling Learning deficits PI3K pathway Impaired Reduced calls Grooming Seizure Reduced number of + channel Impaired NA Grooming Seizure Decreased + function regulator repressor Tumor suppressor suppressor suppressor interaction, K channel cluster KO Cb Table 4 | Syndromic ASD models. Mouse Molecular MeCP2 KO Transcriptional FMR1 KO Translational TSC1 HT, TSC1 TSC2 HTNF1 HTPTEN KO Tumor Tumor ImpairedCNTNAP2 KO Increased calls Neuron-glia Increased marble Scn1a KO Na

Frontiers in Molecular Neuroscience www.frontiersin.org August 2013 | Volume 6 | Article 19 | 6 Won et al. ASD causes, mechanisms, and treatments ; Guo et al., 2009; Clement et al., 2012 Won et al., 2012 Peca et al., 2011 References Blundell et al., 2010 Hines et al., 2008 Tabuchi et al., 2007 Radyushkin et al., 2009; Baudouin et al., 2012 Jamain et al., 2008 Hung et al., 2008; Silverman et al., 2011 Bozdagi et al., 2010 Schmeisser et al., 2012 Wang et al., 2011 Sadakata et al., 2007 NA therapeutics NA NA NA NA Ampakine, IGF-1 NA BDNF Premature spine maturation and hyperexcitability NMDAR hypofunction CDPPB, DCS transmission transmission Cerebellar deficit Impaired glutamatergic transmission transmission NMDAR hyperfunction NA transmission Decreased density of PV interneuron, Reduced BDNF release Learning deficits, Hyperactivity, Anxiolytics, Abnormal sensory-motor gating Other phenotypes Anxiety Hyperactivity Decreased brain volume, deficits Anxiety Anxiety (repeated single beam break within 1 s) Repetitive behavior behavioral flexibility Grooming Hyperactivity, NA Grooming Learning deficits NMDAR hypofunctionReduced calls D-cycloserine Normal NA Decreased brain volume NA NA Stereotypy communication NA NA Hyperactivity, Pattern change Minimal impairment aggression Not impaired Not impaired Not impaired Anxiety, Motor Normal social interaction, Impaired social recognition, Propensity toward social isolation Social interaction Social Impaired, Maternal neglect -dependent + 2 molecule protein protein for Ras small GTPase secretion activating protein KOKO Impaired Reduced calls Jumping Impaired Hyperactivity, Pattern change Grooming Learning deficits Impaired glutamatergic 7 9 KO Impaired Reduced calls, − − exon7 exon6 exon4 Table 5 | Synaptopathy ASD models. Mouse MolecularNLGN1 function KONLGN2 Tg Synaptic adhesion NLGN3 R451C KINLGN3 KONLGN4 KO Phenotype Impaired ImpairedShank1 KO NA SynapticShank2 scaffolding Increased calls Impaired NAShank2 Impaired, Less Reduced Jumping callsShank3 HT Suggested mechanism Normal Enhanced learning Potential Shank3B KO Seizure (EEG) Increased GABAergic Shank3 Increased GABAergic Cadps2 KO Ca ImpairedSyngap1 HT Impaired GTPase-activating Reduced calls NA NA Grooming NA Anxiety Impaired glutamatergic Striatal dysfunction NA

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that autism subjects display local over-connectivity and long- range or inter-regional under-connectivity (Herbert et al., 2003, 2004; Baron-Cohen and Belmonte, 2005; Herbert, 2005; Just et al., 2007). Potential ASD-related neural circuitries have also been pro- −/− posed based on animal studies. Shank3b mice, which exhibit autistic-like behaviors, have striatal dysfunctions (Table 5)(Peca References Takayanagi et al., 2005; Crawley et al., 2007 Brielmaier et al., 2012 Salinger et al., 2003; Lalonde et al., 2004 Lijam et al., 1997; Long et al., 2004 Santini et al., 2013 Gkogkas et al., 2013 et al., 2011). In addition, a shift in the balance between excitation and inhibition (E-I balance) toward excitation in the mouse medial prefrontal cortex (mPFC) induced by optogenetic stimulation causes sociability impairments (Yizhar et al., 2011). These results suggest that the striatum and mPFC are components of therapeutics Oxytocin estrogen 4EGI-1 4EGI-1 sh-NLGNs ASD-related neural circuits. Although various neuroanatomical defects are observed in autistic brains, a direct linkage between neuroanatomical anomalies and behavioral symptoms of ASD remains to be elucidated. Uncovering the detailed circuitries underlying autistic behaviors wouldhelpusunderstandhighercognitivefunctions,suchas language and sociability. Impaired Wnt signaling NA signaling Lissencephaly Neonatal E/I imbalance in PFC, Increased LTP in striatum and hippocampus translation, Increased excitation EXTRACELLULAR FACTORS It has been found that growth factors and neurotrophic factors are associated with ASD. Genetic and protein expression studies have shown that MET, a transmembrane receptor for hepatocyte growth factor (HGF) with tyrosine kinase activity, is associated with ASD. Genetic variations including rs1858830 deficits Ataxia Impaired reversal learning, Impaired fear extinction NA Increased NLGNs in the promoter region that abrogate MET transcription are associated with ASD in Italian and American families and case/control studies, and the levels of MET mRNA and protein are reduced in the cortex of autistic patients (Campbell et al., 2007, 2006). However, this association between rs1858830 and ASD failed to replicate in another study (Sousa et al., 2009). By binding to MET, HGF acts as a neurotrophic factor for neu- marble burying Repetitive behavior Other phenotypes Grooming, Increased marble burying rons to influence neurite outgrowth and dendritic morphology (Figure 1)(Powell et al., 2001, 2003; Sun et al., 2002; Gutierrez et al., 2004), implicating abnormal neuronal structures in ASD pathology. WNT2 is a secreted growth factor that has been linked to communication Reduced calls Not impairedReduced NA calls NAnumber and duration Impaired oxytocin Learning deficits, ASD. Acting through the canonical Wnt pathway, WNT2 triggers a signal transduction cascade mediated by Dishevelled (Dvl1). WNT2 is a critical regulator of multiple biological functions, including embryonic development, cellular differentiation, and cell-polarity generation. It also regulates neuronal migration, axon guidance, and dendrite branching (Figure 1)(Logan and Nusse, 2004). Multiple lines of evidence have implicated the Social interaction Social Impairedaggression Not impairedImpaired NAImpaired, Increased social dominance Not impairedImpaired Not impaired Sensory gating Increased call Learning deficits Cerebellar deficitsWNT2 NA locus in ASD: the WNT2 gene is located at the autism- susceptibility chromosomal locus 7q31 (Vincent et al., 2000; Warburton et al., 2000), and single nucleotide polymorphisms (SNP; rs3779547, rs4727847, and rs3729629, in a case/control study in a Japanese population) and several WNT2 locus variants (R299W and L5R, in autism-affected sibling pair [ASP] and

pathway transcription factor glycoprotein mTOR downstream signal trio families) are associated with autism (Wassink et al., 2001; Marui et al., 2010), although a subsequent study in Han Chinese trios failed to replicate the SNP association with ASD (Chien et al., 2011). While the majority of Wnt2−/− mice are lethal (Goss et al., 2009), null mutants of Dvl1 show deﬁcits in nest

Table 6 | Non-synaptopathy ASD models. Mouse Molecular functionDvl1 KOOxtr KO Wnt signaling Engrailed-2 KO Oxytocin receptor Homeodomain Impaired, Less Reeler4E-BP2 KO Extracellular matrix Phenotype Translational control, eIF4E O/Ebuilding and home-cage Impaired Suggested mechanism Potential huddling NA (see also Grooming, Increased Table 6 for summary

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FIGURE 1 | Signaling pathways and possible treatments associated with blue arrows, respectively. Possible treatments and their target molecules are ASD. Molecules whose mutations or polymorphisms are associated with indicated by red texts in orange boxes. SynGAP1, which directly interacts ASD are indicated in red. Stimulations and inhibitions are indicated by red and with PSD-95, could not be placed next to PSD-95 for simplicity.

of non-synaptopathy ASD models) (Lijam et al., 1997; Long autism pathogenesis, evidence for its role in ASD is becoming et al., 2004). Moreover, the Wnt signaling pathway is associated clear. with and is regulated by chromodomain-helicase-DNA-binding Reelin is also involved in autism. Reelin is a large secreted protein 8 (CHD8; Figure 1), de novo mutations of which are extracellular matrix glycoprotein that acts as a serine protease for repeatedly detected in autistic patients (Neale et al., 2012; O’Roak the extracellular matrix, a function that is essential for neuronal et al., 2012b; Sanders et al., 2012). migration, cortical patterning, and brain development (Figure 1) Brain-derived neurotrophic factor (BDNF) is associated with (Forster et al., 2006). The RELN gene is located in an autism sus- ASD. BDNF is a member of the neurotrophin family of growth ceptibility locus on chromosome 7q22, and triplet GGC repeats factors that supports neurogenesis, axodendritic growth, neu- in 5 untranslated regions (5UTR) in the RELN gene have been ronal/synaptic differentiation, and brain dysfunctions (Figure 1) associated with autism in a Caucasian population (Persico et al., (Huang and Reichardt, 2001; Martinowich et al., 2007). Elevated 2001; Skaar et al., 2005)(Table 2). Expression levels of Reelin levels of BDNF were reproducibly found in the sera of Japanese are decreased in postmortem autism brains (Fatemi et al., 2005). and American autistic individuals (Connolly et al., 2006; Miyazaki Reelin has also been implicated in pathogenesis of various neu- et al., 2004). Another clue comes from calcium-dependent secre- ropsychiatric disorders, including schizophrenia, bipolar disor- tion activator 2 (CADPS2), a calcium binding protein in the der, lissencephaly, and epilepsy (Fatemi, 2001). Reeler mice, with presynaptic nerve terminal that interacts with and regulates a 150-kb deletion of the Reln gene, exhibit deﬁcits in motor coor- exocytosis of BDNF-containing dense-core vesicles (Figure 1) dination, increased social dominance, and learning and memory (Cisternas et al., 2003). CADPS2, located at the autism- impairments (Table 6)(Salinger et al., 2003; Lalonde et al., 2004). susceptibility locus on chromosome 7q31, is abnormally spliced in autism patients, and Cadps2−/− mice exhibit social interac- TRANSCRIPTION FACTORS tion deﬁcits, including maternal neglect (Table 5)(Sadakata et al., Syndromic forms of ASD frequently involve transcription fac- 2007). Hence, although it is unclear how BDNF contributes to tors. This is likely because defective transcription factors have

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significant influences on many genes and their downstream segmental duplication) (Serajee et al., 2003), and the N-methyl- molecules, affecting diverse neuronal functions. D-aspartic acid receptor (NMDAR) subunit GluN2A (rs1014531) MeCP2 (X-linked gene methyl CpG binding protein 2) is one (Barnby et al., 2005), and GluN2B (de novo protein truncat- of the best examples. It is a member of a large family of methyl- ing and splice mutations) (O’Roak et al., 2012a,b)(Table 2). CpG binding domain (MBD) proteins that selectively binds to Decreased levels of glutamine and abnormal levels of glutamate methylated DNA and represses gene transcription (Figure 1) were observed in the plasma of autistic children (Rolf et al., (Bienvenu and Chelly, 2006). Its downstream targets encom- 1993; Moreno-Fuenmayor et al., 1996). In addition, neuropatho- pass ASD-related genes such as BDNF and CDKL5.Mutations logical studies of postmortem autism brains show perturba- in MeCP2 are the major cause of Rett syndrome, a progres- tions in the glutamate neurotransmitter system (Purcell et al., sive neurodevelopmental disorder with autistic features (Amir 2001). et al., 1999; Bienvenu and Chelly, 2006; Chahrour and Zoghbi, AbnormalGABAergicsystemisalsoproposedasapoten- 2007). Mecp2-null mice, an animal model for Rett syndrome, tial mechanism for ASD. Reduced expression levels in a rate- recapitulate most symptomatic traits of Rett syndrome such as limiting enzyme for GABA synthesis, glutamic acid decarboxylase respiratory dysfunction, forelimb and hindlimb clasping stereo- (GAD), and GABA receptors with altered subunit composition typy, motor dysfunction, tremor, hypoactivity, anxiety, cognitive were observed in autistic brains (Fatemi et al., 2002, 2010). impairments, and altered sociability (Table 4)(Shahbazian et al., Furthermore, linkage disequilibrium and transmission disequi- 2002; Moretti et al., 2005). librium between GABRB3, a gene encoding the β3 subunit of Engrailed-2 is a homeodomain transcription factor associated GABAα receptors, with Angelman syndrome and autism has been with ASD. Engrailed-2 is involved in a diverse range of biological reported (Cook et al., 1998; Bass et al., 2000; Buxbaum et al., processes from embryological development and segmental polar- 2002)(Table 2). ity to brain development and axon guidance (Figure 1)(Brunet The serotonergic system would also play a role in ASD et al., 2005; Joyner, 1996). The Engrailed-2 gene on human pathogenesis by modulating the E-I balance. Serotonin levels in chromosome 7q36 is in the autism susceptibility locus, and an blood or urine are increased in subjects with autism (Cook and association between two intronic SNPs rs1861972 and rs1861973 Leventhal, 1996; Burgess et al., 2006), and various genes in the at Engrailed-2 locus and ASD has been repeatedly identified in serotonin system are linked to autism. Among them are genes 518 ASD families (Gharani et al., 2004; Benayed et al., 2005) encoding the serotonin transporter 5-HTT (transmission dise- (Table 2). However, these SNPs were not found to be associ- quilibrium at the 5-HTT locusin86autismtrios)(Cook et al., ated with ASD in Han Chinese trios (Wang et al., 2008). This 1997), and a rate-limiting enzyme for serotonin synthesis TPH2 association between Engrailed-2 and ASD was further confirmed (two intronic SNPs rs4341581 and rs11179000 at introns 1 and 4, by animal model studies, which showed Engrailed-2 null mice respectively, have been associated with autism) (Coon et al., 2005) display social dysfunction and cognitive impairments (Table 6) (Table 2). (Brielmaier et al., 2012). Because Engrailed-2 is expressed upon Neurexins and neuroligins are synaptic cell adhesion activation of WNT2-Dvl1 signaling, it appears that the WNT2- molecules enriched at pre- and post-synaptic membranes, Dvl1-Engrailed-2 pathway, which regulates neuronal migration respectively (Figure 1)(Craig and Kang, 2007; Sudhof, 2008). and axonal guidance, may significantly contribute to ASD patho- Specific interactions between neurexins and neuroligins regulate genesis via neuroanatomical abnormalities. In addition, a base various aspects of both excitatory and inhibitory synaptic devel- substitution (A218G) mutant of HOXA1,anotherhomeobox opment and function, affecting the E-I balance in postsynaptic gene, was reported in autistic individuals (Ingram et al., 2000b), neurons. Many mutations in genes encoding neurexins (includ- indicating the importance of homeobox genes in normal brain ing hemizygous CNV deletions and missense mutations) and function and ASD. neuroligins (e.g., R451C for NLGN3 and a frameshift insertion mutation for NLGN4) have been associated with ASD, intellectual EXCITATORY AND INHIBITORY IMBALANCE disability, and schizophrenia (Jamain et al., 2003; Laumonnier Mutations identified in important synaptic molecules includ- et al., 2004; Autism Genome Project et al., 2007; Kim et al., ing neuroligins (Jamain et al., 2003), neurexin (Autism Genome 2008; Walsh et al., 2008)(Table 2). Neuroligin3 knockin mice Project et al., 2007; Kim et al., 2008)andShank(Durand et al., with the R451C mutation found in autistic patients recapitulate 2007; Berkel et al., 2010; Sato et al., 2012) in autistic subjects autistic features including moderately impaired sociability have prompted investigations into exploring the roles of synaptic (Table 5)(Tabuchi et al., 2007). Notably, inhibitory transmission dysfunctions in ASD pathogenesis. This “synaptopathy” model of was enhanced in the cortical regions of the mutant brains of autism has provided much insight into the field (Table 5). these mutant mice, suggesting that disrupted E-I balance may Defects in synaptic proteins would lead to defective transmis- contribute to ASD. sions at excitatory and inhibitory synapses, disrupting the E-I SHANK family genes encode scaffolding proteins enriched balance in postsynaptic neurons, a key mechanism implicated in in the postsynaptic density (PSD), a postsynaptic membrane ASD. In line with this, ASD has been genetically associated with specialization composed of multi-synaptic protein complexes diverse glutamate receptors, including the kainite receptor sub- (Figure 1)(Sheng and Kim, 2000). The Shank family contains unit GluR6 (M867I in the intracytoplasmic C-terminal region of three known members, Shank1, Shank2 and Shank3, also known GluR6) (Jamain et al., 2002), the metabotropic glutamate recep- as ProSAP3, ProSAP1, and ProSAP2, respectively. The idea that tor 8 (GRM8) (R859C, R1085Q, R1100Q, and intrachromosomal Shanks are involved in the etiology of ASD firstly emerged

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from Phelan-McDermid syndrome (PMS) or 22q13 deletion syn- mice show schizophrenia-like phenotypes including hyperactiv- drome, a neurodevelopmental disorder caused by a microdeletion ity, impaired sensory-motor gating, impaired social memory on chromosome 22 (Boeckers et al., 2002; Wilson et al., 2003; and fear conditioning, and preference to social isolation (Guo Phelan and McDermid, 2012). The association between SHANK et al., 2009)(Table 5). In a more recent study, Syngap1 het- and ASD became evident by identifying numerous mutations erozygous mice showed premature dendritic spine development including de novo frameshift, truncating, and missense muta- together with enhanced hippocampal excitability and abnormal tions on SHANK3 locus in autistic individuals (Durand et al., behaviors, suggesting that over-paced excitatory synaptic devel- 2007)(Table 2). Mutations in SHANK2 and SHANK1 including opment during a critical time window of postnatal brain devel- de novo CNV deletions and missense mutations in Canadian and opment causes intellectual disability and ASD (Clement et al., European populations were also identified in individuals with 2012). ASD and intellectual disability (Berkel et al., 2010; Leblond et al., Several genes associated with X chromosome-linked intel- 2012; Sato et al., 2012). lectual disability (XLID) and synaptic regulations have been Multiple lines of transgenic mice with Shank mutations found associated with ASD. One of them is interleukin 1 receptor acces- in human patients have been reported. Shank3 heterozygous sory protein-like 1 (IL1RAPL1) that encodes a synaptic trans- mice show sociability deficits and reductions in miniature excita- membrane protein (Carrie et al., 1999). Recently, a systematic tory postsynaptic currents (mEPSC) amplitude and basal synap- sequencing screen of X chromosomes of ASD-affected individ- tic transmission (Bozdagi et al., 2010); mice with deletion of uals has identified a de novo frameshift mutation in IL1RAPL1 exon 4–9 of Shank3 are socially impaired and exhibit alterations (Piton et al., 2008). IL1RAPL1 plays an important role in the in dendritic spine morphology and activity-dependent surface formation and stabilization of excitatory synapses by recruit- expression of AMPARs (Wang et al., 2011); Shank1−/− mice ing the scaffolding protein PSD-95 to excitatory postsynaptic display reduced basal synaptic transmission in the hippocam- sites through the JNK signaling pathway (Figure 1)(Pavlowsky pal CA1 region and reduced motor function and anxiety-like et al., 2010). In addition, IL1RAPL1 induces the presynaptic dif- behavior, although they show normal sociability (Hung et al., ferentiation through its trans-synaptic interaction with protein 2008; Silverman et al., 2011); mice expressing Shank2-R462X in tyrosine phosphatase δ (PTPδ)(Figure 1)(Valnegri et al., 2011b; hippocampal CA1 neurons exhibit cognitive dysfunction accom- Yoshida et al., 2011). This interaction between IL1RAPL1 and panied by reduced mEPSC amplitude and changes in neuronal PTPδ recruits RhoGAP2 to the excitatory synapses and induces morphologies (Table 5)(Berkel et al., 2012). dendritic spine formation (Valnegri et al., 2011b). Interestingly, CNTNAP2, a neuronal transmembrane protein, is a member IL1RAPL1 regulates the development of inhibitory circuits in the of the neurexin family localized at juxtaparanodes of myeli- cerebellum, an ASD-related brain region, and disrupts the exci- nated axons. Here, CNTNAP2 regulates neuron-glia interactions tatory and inhibitory balance, as determined by a study using and potassium channel clustering in myelinated axons (Figure 1) Il1rapl1−/− mice (Gambino et al., 2009). These results suggest (Poliak et al., 1999). Several SNPs (e.g., rs2710102, rs7794745, that IL1RAPL1 is involved in the regulation of excitatory synaptic rs17236239) and nonsynonymous variants (e.g., I867T) in development and the balance between excitatory and inhibitory CNTNAP2 locus were found to be associated with ASD, lan- synaptic inputs. guage impairment, and cortical dysplasia-focal epilepsy syndrome Another XLID gene related with ASD is OLIGOPHRENIN- in humans (Alarcon et al., 2008; Arking et al., 2008; Bakkaloglu 1 (OPHN1), which encodes a GTPase-activating protein that et al., 2008; Vernes et al., 2008)(Table 2).Inacase-controlasso- inhibits Rac, Cdc42, and RhoA small GTPases. Since the initial ciation study in Spanish autistic patients and controls, however, report of the association of a truncation mutation of OPHN1 CNTNAP2 SNPs rs2710102 and rs7794745 did not associate with with XLID (Billuart et al., 1998a,b), additional studies have ASD (Toma et al., 2013). Cntnap2−/− mice recapitulate all three associated nonsynonymous rare missense variants in OPHN1 core symptoms of autism, and displayabnormalneuronalmigra- with ASD (e.g., H705R) and schizophrenia (e.g., M461V) (Piton tion, reduced number of GABAergic interneurons, and abnormal et al., 2011). OPHN1 regulates dendritic spine morphogene- neuronal synchronization (Table 4)(Penagarikano et al., 2011). sis through the RhoA signaling pathway (Govek et al., 2004) Excessive grooming and hyperactivity in these mice were restored and activity-dependent synaptic stabilization of AMPA recep- by the treatment of the antipsychotic risperidone (Table 4), sug- tors (Nadif Kasri et al., 2009). OPHN1 also interacts with the gesting the possibility of therapeutic intervention for certain transcription repressor Rev-erba to regulate expression of circa- symptoms of autism. dian oscillators (Valnegri et al., 2011a). Importantly, Ophn1−/− SynGAP is a GTPase-activating protein for the Ras small mice show immature spine morphology, impaired spatial mem- GTPase. SynGAP directly interacts with PSD-95, and nega- ory and social behavior, and hyperactivity (Khelfaoui et al., 2007). tively regulates the Ras-MAPK signaling pathway, excitatory These results suggest that OPHN1 regulates excitatory synaptic synapse development, and synaptic transmission and plasticity development and function. (Figure 1)(Chen et al., 1998; Kim et al., 1998). In humans, TM4SF2 or tetraspanin 7 (TSPAN7), another X-linked gene de novo mutations of SYNGAP1 have been associated with which encodes a membrane protein which belongs to trans- intellectual disability and autism (Hamdan et al., 2011). In membrane 4 superfamily (TM4SF), plays important roles in the addition, a genetic case/control study in European popula- cell proliferation, activation, growth, adhesion, and migration tions associates a rare de novo copy number variation in (Maecker et al., 1997). TM4SF proteins form a complex with inte- SYNGAP1 with ASD (Pinto et al., 2010). Syngap1 heterozygous grin, which regulates cell motility and migration by modulating

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the actin cytoskeleton (Berditchevski and Odintsova, 1999). A which binds to target mRNAs and regulates their translation balanced translocation and mutations (a nonsense mutation and and transport of mRNA into dendrites and synapses (Figure 1) a P172H missense mutation) of TM4SF2 was firstly discovered (Bassell and Warren, 2008). In the absence of FMRP,target mRNA in the individuals with XLID (Zemni et al., 2000). In subsequent translation becomes excessive and uncontrolled, leading to an studies, the P172H missense mutation was found in individu- aberrant activity-dependent protein synthesis. Fmr1 mutant mice als with XLID (Maranduba et al., 2004)andASD(Piton et al., show enhanced protein synthesis-dependent mGluR-mediated 2011). A microduplication in the locus of TM4SF2 was revealed, LTD and dendritic spine elongation, together with cognitive but this duplication was also present in unaffected controls, sug- deficits, social anxiety and impaired social interaction (Table 4) gesting that it may be a neutral polymorphism (Cai et al., 2008). (Bernardet and Crusio, 2006). Interestingly, target molecules of In neurons, TM4SF2 regulates excitatory synaptic development FMRP include Shank3, GluN2A, mTOR, TSC2, NF1, neuroligin2, and AMPA receptor trafficking by binding to the synaptic PDZ and neurexin1 (Darnell et al., 2011), which are associated with protein PICK1 (Figure 1)(Bassani et al., 2012). ASD pathogenesis. It should be noted that the ASD-related signaling molecules SYNAPTIC SIGNALING mentioned above are also associated with NMDAR and mGluR Disrupted synaptic signaling may be a key determinant of ASD. signaling pathways. NMDARs and mGluRs play critical roles in Components in mGluR- or NMDAR-dependent signaling cas- the regulation of synaptic function and plasticity at excitatory cades have recently been implicated in ASD. synapses. NF1 interacts with the NMDAR complex and regulates Neurofibromin 1 (NF1), tuberous sclerosis complex GluN2A phosphorylation (Figure 1)(Husi et al., 2000). FMRP (TSC1/TSC2), and phosphatase and tensin homolog (PTEN) and TSC have profound effects on mGluR-dependent LTD and protein synthesis, which are upregulated in Fmr1−/y mice, while are genes associated with neurological diseases with common +/− autistic symptoms including neurofibromatosis (Rasmussen and downregulated in Tsc2 mice (Auerbach et al., 2011). FMRP Friedman, 2000), tuberous sclerosis (vanSlegtenhorstetal., is also in the downstream of mGluR signaling (Figure 1)(Bassell 1997), and Cowden/Lhermitte-Duclos syndrome (Pilarski and and Warren, 2008). Eng, 2004). They are tumor suppressors sharing a common Defects in NMDAR function and associated signaling are also function; they negatively regulate the mammalian target of observed in nonsyndromic ASD models with Shank mutations. rapamycin (mTOR) signaling pathway. Although Tsc1 null mice Shank proteins are physically connected to both NMDARs and are embryonically lethal (Wilson et al., 2005), mutant mice mGluRs, suggesting that Shank may regulate signaling pathways downstream of NMDAR or mGluR activation, and the functional with loss of Tsc1 in cerebellar Purkinje cells display autistic-like −/− behaviors (Tsai et al., 2012), and Tsc2 heterozygote mice exhibit interaction between the two receptors (Figure 1). Shank2 abnormal social communication (Young et al., 2010); Nf1 mutant mice with the deletion of exons 6 and 7 display autistic-like mice show aberrant social transmission of food preference and behaviors and reductions in NMDAR function and associated signaling, without affecting mGluR-dependent LTD (Won et al., deficits in hippocampus-dependent learning (Costa et al., 2001, −/− 2002); Pten deficient mice show altered social interaction and 2012), while Shank2 mice with exon 7 deletion show similar macrocephaly with hyperactivation of mTOR pathway (Table 4) behavioral abnormalities with NMDAR hyperfunction (Table 5) (Kwon et al., 2006). (Schmeisser et al., 2012). Although how similar exon deletions in Signaling molecules in the downstream of mTOR in the mTOR Shank2 lead to comparable behavioral abnormalities but differ- pathway play crucial roles in ASD pathogenesis. Upon phospho- ent changes in NMDAR function remains to be determined, these rylation by mTORC1, 4E-BP proteins are detached from eIF4E to results point to that Shank2 is an important regulator of NMDAR promote eIF4E-dependent protein translation (Figure 1)(Richter function, and that NMDAR function and NMDAR-associated and Sonenberg, 2005). A SNP at eIF4E promoter region which signaling are associated with ASD. increases its promotor activity was found in autism patients (Neves-Pereira et al., 2009). Implications of mTOR downstream NEUROIMMUNE RESPONSE signaling in ASD were demonstrated as 4E-BP2 knockout mice The implication of the immune system on autism was initially and eIF4E overexpression mice display autistic-like behaviors. proposed in 1976 based on that some autistic children do not 4E-BP2 knockout mice show enhanced translational control of have detectable Rubella titers in spite of previous vaccination neuroligins and increased excitatory transmission in the hip- (Stubbs, 1976). Levels of serum IgG and autoantibodies to neu- pocampus (Table 6)(Gkogkas et al., 2013), while eIF4E over- ronal and glial molecules were elevated in autistic patients (Singh expressing transgenic mice show impaired excitatory/inhibitory et al., 1997; Croonenberghs et al., 2002), proposing involvement balance in the mPFC and increased LTD in the hippocampus and of autoimmune responses in autism. In addition, plasma or cere- striatum (Table 6)(Santini et al., 2013). Autistic features of these brospinal fluid (CSF) levels of pro-inflammatory cytokines and mutant mice were ameliorated by 4EGI-1 infusion, which inhibits chemokines including MCP-1, IL-6, IL-12, IFN-γ and TGFβ1 the eIF4E–eIF4G interaction. were increased in autistic individuals (Ashwood and Van de Fragile X syndrome is the most common cause of intellec- Water, 2004; Ashwood et al., 2006). tual disability and autism. It is mostly caused by the expansion Astrocytes and microglia are two glial cell types important for of CGG trinucleotide repeats in the promoter region of the immune responses in the brain as well as regulation of neuronal FMR1 gene, which enhances the methylation of the promoter functions and homeostasis (Fields and Stevens-Graham, 2002). and represses generation of FMR1-encoded protein (FMRP), Postmortem analyses demonstrated abnormal glial activation and

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neuroinflammatory responses in autistic brains (Vargas et al., mGLuR POSITIVE ALLOSTERIC MODULATORS 2005). Transcriptome analysis of autistic postmortem brain tis- mGluR1 and mGluR5 are group I mGluRs that are postsynap- sues has also revealed upregulation in the expression of genes tically expressed in broad brain regions, including the cerebral belonging to immune and inflammatory networks (Voineagu cortex, striatum, hippocampus, nucleus accumbens, and inferior −/− et al., 2011). Reactive astrocytes were also detected in Cntnap2 colliculus (Testa et al., 1995). Upon activation, group I mGluRs brains, a well-established autism model (Penagarikano et al., enhance calcium release from intracellular stores resulting in neu- 2011). These results clearly suggest the association between neuronal depolarization, augmentation of neuronal excitability, and roimmune defects with ASD, although further details remain to activation of intracellular signaling cascades such as PKA, PKC, be determined. MAPK, ERK, and CREB (Niswender and Conn, 2010). mGluR5 is physically linked to NMDARs via Homer-Shank/ProSAP- NON-GENETIC MODELS OF ASD GKAP/SAPAP-PSD-95 interactions (Naisbitt et al., 1999; Tu et al., Although we have thus far described genetic factors underlying 1999), and is functionally coupled to NMDARs via aforemen- ASD, environmental factors also have strong influences on ASD. tioned signaling molecules including PKC (Niswender and Conn, Epidemiologic studies suggest that maternal exposure to stress, 2010). Through these structural and biochemical interactions, viral or bacterial infection, thalidomide, and valproic acid can mGluR5 activation is thought to potentiate NMDAR function increase the risk for ASD in offspring (Grabrucker, 2012). (Awad et al., 2000; Attucci et al., 2001; Mannaioni et al., 2001; Maternal immune activation (MIA) induced by poly(I:C), the Pisani et al., 2001; Alagarsamy et al., 2002; Rosenbrock et al., synthetic doublestrand RNA polyriboinosinic-polyribocytidilic 2010). acid, in pregnant mice leads to the development of core ASD- Positive allosteric modulators of mGluR5 receptors were first like phenotypes in the offspring, including impaired sociability, developed to alleviate symptoms of schizophrenia (Gregory decreased USV, and increased repetitive behaviors (Malkova et al., et al., 2011). Although antipsychotics are available for pos- 2012). MIA by lipopolysaccharide (LPS) treatment during preg- itive symptoms of schizophrenia, such as hallucinations, no nancy can also induces ASD-like phenotypes in rodent offspring, medications are currently available for negative symptoms or including impaired social interaction (Hava et al., 2006; Kirsten cognitive impairments. Two main hypotheses have been pro- et al., 2010)andreducedUSV(Baharnoori et al., 2012). IL-6 posed for schizophrenia: dopaminergic hyperactivity and NMDA is thought to play a critical role in this process, as IL-6 knock- hypofunction. Dopaminergic hyperactivity can be treated by out mice do not show poly(I:C) induced social deficits (Smith dopamine receptor-antagonistic antipsychotics such as risperi- et al., 2007), and IL-6 levels are significantly elevated in the cere- done, but NMDA hypofunction is difficult to modulate given the bellum of autistic subjects (Wei et al., 2011). Although further expected side effects of enhancing NMDAR functions. details remain to be determined, the underlying mechanisms may Therefore, the concept of augmenting NMDAR signal- include IL6-dependent regulation of excitatory and inhibitory ing via mGluR potentiation was proposed to improve nega- synaptic transmission and neuroprotection (Sallmann et al., 2000; tive symptoms of schizophrenia (Uslaner et al., 2009; Stefani Biber et al., 2008; Dugan et al., 2009). and Moghaddam, 2010). mGluR positive allosteric modulators Prenatal exposure to teratogens can increase the risk for ASD increase the function of NMDAR only when they are occupied in animals, as in humans. Thalidomide (THAL) and valproic by the endogenous ligand glutamate (Figure 1). mGluR posi- acid (VPA) cause rat offspring to display brain morphological tive allosteric modulators have significant advantages over the abnormalities observed in ASD, including altered cerebellar struc- conventional mGluR agonist, (RS)-3,5-dihydroxyphenylglycine tures and reduced number of cranial motor neurons (Rodier (DHPG). While DHPG has poor specificity toward particular et al., 1997; Ingram et al., 2000a). Behaviorally, VPA-exposed mGluR subtypes, mGluR positive allosteric modulators offer rats show decreases in prepulse inhibition, stereotypy, and social high subtype specificity. Some positive allosteric modulators have behaviors (Schneider and Przewlocki, 2005). VPA-exposed rats high brain blood barrier penetrance, which enables the sys- display elevated serotonin levels and abnormal serotonergic neu- temic administration of the drugs. Furthermore, whereas direct rons (Anderson et al., 1990; Narita et al., 2002; Miyazaki et al., mGluR agonists cause rapid receptor desensitization, mGluR 2005), decreased parvalbumin-positive interneurons in the neo- positive allosteric modulators potentiate mGluR function with cortex (Gogolla et al., 2009), and elevated NMDA receptor levels minimal desensitization, because they bind to an allosteric site and enhanced LTP (Rinaldi et al., 2007), suggesting that these on the receptor distinct from the orthosteric glutamate bind- mechanisms may contribute to the development of ASD-like ing site. These properties of positive allosteric modulators are phenotypes. predicted to minimize their excitotoxicity and enable high-dose administrations. POTENTIAL TREATMENTS FOR ASD A large number of mGluR5 allosteric modulators have been Currently, only two medicines have been approved for ASD by developed (Williams and Lindsley, 2005; Gregory et al., 2011). US FDA; risperidone (Risperdal®) and aripiprazole (Abilify®), Of these, CDPPB, ADX47273, MPPA, and VU0092273 read- which act as dopamine/5-HT receptor antagonists (McPheeters ily cross the blood-brain barrier, and CDPPB, particular, has et al., 2011). These drugs are useful for correcting irritability and been examined in various behavioral assays and model ani- stereotypy, but not sociability defects. Recently, a number of can- mals. In CHO (Chinese hamster ovary) cells expressing human didate ASD medications for treating social abnormalities have mGluR5, CDPPB treatment was shown to enhance mGluR5 been suggested (Figure 1). activity in a concentration-dependent manner (Kinney et al.,

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2005). Behaviorally, CDPPB alleviates prepulse inhibition and effect 1 h after intraperitoneal administration (Peterson, 1992). hyperactivity produced by amphetamine, suggesting that CDPPB D-cycloserine shows dose-dependent elimination (higher elimi- could be a potential antipsychotic agent. nation rates with lower doses) and a half-life of 7–15 h in humans BecauseNMDARsplayanessentialroleinlearningand and 23 min in mice (lwainsky, 1988; Wlaz et al., 1994). memory, indirect potentiation of NMDARs by mGluR5 posi- When glycine levels are low, D-cycloserine amplifies the tive allosteric modulators may facilitate synaptic plasticity and activity of the NMDAR complex and enhances synaptic plastic- learning and memory. Indeed, CDPPB and ADX47273 enhance ity and cognitive function. D-cycloserine alleviates senescence- theperformanceofwild-typemiceintheMorriswatermaze associated behavioral defects (Flood et al., 1992)andfacilitates test, a hippocampus-dependent learning and memory paradigm memory acquisition, consolidation, and retrieval (Quartermain (Ayala et al., 2009). In addition, VU-29 and ADX47273 poten- et al., 1994). While low doses (10–20 mg kg−1)ofD-cycloserine tiate two forms of NMDAR-dependent synaptic plasticity—LTP have cognition-enhancing effects (Monahan et al., 1989; Flood and LTD—in the CA1 region of the hippocampus (Ayala et al., et al., 1992; Schuster and Schmidt, 1992; Sirvio et al., 1992; 2009). DFB-treated rats make fewer errors in the Y-maze spa- Quartermain et al., 1994), higher doses (>100 mg kg−1)exert tial alternation task (Balschun et al., 2006), and CDPPB and anticonvulsant effects in tonic convulsion models (Peterson, ADX47273 enhance performance in novel object recognition 1992; Peterson and Schwade, 1993). and five-choice serial reaction time tasks (Liu et al., 2008; Putative effects of D-cycloserine on ASD have been suggested Uslaner et al., 2009). CDPPB not only improves learning and by previous studies. Mice with a neuroligin1 (Nlgn1) deficiency memory performance of wild-type mice, but also reverses cog- exhibit abnormally increased grooming behavior, and this behav- nitive dysfunction and behavioral inflexibility induced by the ioral anomaly is reversed by D-cycloserine treatment (Table 5) NMDAR antagonist MK-801 (Uslaner et al., 2009; Stefani and (Blundell et al., 2010). Low-dose D-cycloserine alleviates nega- Moghaddam, 2010). These results suggest that mGluR5 positive symptoms of schizophrenia-affected individuals (Goff et al., tive allosteric modulators have the potential to improve cog- 1999), and reduces social withdrawal and increases social respon- nitive impairments associated with brain disorders including siveness in autistic patients (Posey et al., 2004). Moreover, D- schizophrenia and autism. cycloserine partially rescues social deficits of Shank2−/− mice, Indeed, CDPPB has recently shown promise as a potential supporting the role of NMDAR functionality in autism (Table 5) treatment for ASD. In a study using Tsc2+/− mice, a mouse model (Won et al., 2012). of tuberous sclerosis characterized by intellectual disability and autism, Mark Bear and colleagues showed that cognitive impair- BENZODIAZEPINES ments observed in these mice could be alleviated by CDPPB Recently, benzodiazepines were suggested as putative therapeutic administration (Table 4)(Auerbach et al., 2011). In addition, −/− agents for Dravet’s syndrome, which is a developmental dis- social deficits of Shank2 mice are rescued by CDPPB treat- order with myoclonic infantile seizure, ADHD-like inattention ment (Won et al., 2012), implicating hypofunction of mGluRs and hyperactivity, motor impairment, sleep disorder, anxiety-like and NMDARs in social impairment, and suggesting mGluR posi- behaviors, cognitive defects, autism-like social dysfunction, and tive allosteric modulators as novel therapeutics for the treatment restricted interests. Mice heterozygous for a deletion of the α- of social deficits (Table 5). More recently, CDPPB was shown to subunit of the type 1 voltage-gated sodium channel (Scn1α+/− reverse defects in social novelty recognition induced by neonatal mice), an animal model for Dravet’s syndrome, recapitulate most phencyclidine treatment (Clifton et al., 2012). features of the disorder, including epilepsy, ataxia, sleep disorder, anxiety-like behaviors, hippocampus-dependent learning D-CYCLOSERINE impairments, sociability deficits, and excessive repetitive groom- Although it is well established that NMDARs critically regulate ing behaviors (Table 4)(Yu et al., 2006; Kalume et al., 2007; Han normal brain functions, the excitotoxicity and poor bioavail- et al., 2012). In Scn1α+/− mouse brains, expression of the voltage- ability of direct NMDAR agonists have hampered attempts to gated sodium channel type-1 (Nav1.1) is decreased in GABAergic control brain activity by modulating NMDARs (Quartermain interneurons, and GABAergic transmission onto postsynaptic et al., 1994). D-cycloserine is a high-affinity partial ago- neurons was reduced. This would cause a shift in the balance nist of NMDA-coupled, strychnine-insensitive glycine receptors between excitation and inhibition in postsynaptic neurons toward (Figure 1)(Hood et al., 1989). Similar to glycine, D-cycloserine excitation, which may be corrected by stimulating GABA recep- also binds to the glycine site of NMDARs as a partial agonist, tors in these neurons. Indeed, it was shown that both behavioral potentiating NMDARs by increasing the frequency of channel abnormalities and aberrant GABAergic transmission are rescued opening. In addition, because NMDARs are not maximally poten- by low-dose administration of clonazepam (Table 4)(Han et al., tiated by endogenous glycine, there is room for D-cycloserine 2012). to further potentiate NMDARs. These properties enable D- Clonazepam, a type of benzodiazepine, is a positive allosteric cycloserine to act as a positive modulator of NMDARs. modulator of GABAA receptors that exerts sedative, hypnotic, D-cycloserine is a viable drug candidate because it is a par- anxiolytic, anticonvulsant, and muscle relaxing effects (Figure 1) tial agonist, displaying efficacy of 40-50% relative to glycine, and (Rudolph and Knoflach, 2011). Similar to the action of mGluR has low toxicity and decent bioavailability (Hood et al., 1989). positive allosteric modulators, clonazepam potentiates GABA Although the brain penetrance of D-cycloserine is not high, it signaling only when GABAA receptors are bound by their can nonetheless infiltrate the blood-brain barrier, exerting a peak endogenous ligand, GABA. Therefore, these results indicate that

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normalization of disrupted E-I balance may be a novel and and inappropriate speech (King et al., 2001). Memantine is also promising strategy for treating symptoms of ASD. effective in improving language and social behavior and clinical global impressions (CGI) scale in autistic patients (Chez et al., mGLuR NEGATIVE ALLOSTERIC MODULATORS 2007; Erickson et al., 2007; Niederhofer, 2007). The potential of mGluR of negative allosteric modulation as a With regard to mechanisms of memantine and amantadine therapeutic strategy in ASD was first proposed based on studies in underlying the treatment of ASD remains, both medications Fmr1−/y mice, an animal model for fragile X syndrome (Bakker are highly likely to exert their therapeutic effects by suppress- et al., 1994). The enhanced mGluR5-dependent LTD and pro- ing NMDAR function and modulating excitotoxicity in autistic tein synthesis observed in Fmr1−/y mice provided a conceptual subjects. However, care should be taken because other possibili- framework for the mGluR theory of fragile X pathogenesis (Bear ties exist. For instance, memantine treatment promotes excitatory et al., 2004; Bassell and Warren, 2008). Synaptic protein synthesis synapse formation and maturation and cell adhesion proper- is stimulated by local mRNA translation, a process that depends ties of cerebellar granule cells (CGCs) of Fmr1 knockout mice on group I mGluR activation. FMRP, encoded by the Fmr1 (Wei et al., 2012). In addition, memantine exerts neuroprotective gene, is a repressor of mRNA translation; thus, mGluR-mediated activities by promoting glia-derived neurotrophic factor (GDNF) protein synthesis could be enhanced in the absence of FMRP. release and preventing migroglial inflammatory responses (Wu Therefore, attempts have been made to correct fragile X syn- et al., 2009). Memantine can also act as a non-competitive antag- drome by suppressing abnormally enhanced mGluR5-dependent onist for 5-HT receptors (Rammes et al., 2001) and nicotinic synaptic plasticity and protein synthesis. acetylcholine receptors (Aracava et al., 2005), while it functions Two approaches have been used to normalize behavioral as an agonist for D2 dopamine receptors (Seeman et al., 2008). and neuronal deficits of Fmr1−/y mice: genetic crossbreeding +/− with Tsc2 mice, which exhibit suppressed mGluR activity, IGF-1 and acute administration of an mGluR antagonist (Auerbach A new approach for alleviating phenotypic traits of ASD in ani- et al., 2011). Administering the mGluR5 antagonist 2-methyl-6- mal models has come from research on Rett syndrome. Rett −/ (phenylethynyl) pyridine hydrochloride (MPEP) to Fmr1 y mice syndrome is an X-linked neurological disorder caused by muta- normalizes defective phenotypes, including cognitive deficits, tions in the MeCP2 gene. MeCP2 is a transcriptional repres- perturbed mGluR-dependent LTD and protein synthesis, and sor and activator, which binds widely across the genome and excessive filopodia-like long and thin spines (Figure 1, Table 4) influence a large number of genes (Chahrour et al., 2008). (Yan et al., 2005; de Vrij et al., 2008). In line with this, mGluR One of the best characterized targets of MeCP2 is BDNF, a negative allosteric modulators are now in clinical trials for fragile neurotrophic factor that regulates neuronal development and Xsyndromepatients(Krueger and Bear, 2011). synaptic plasticity (Figure 1)(Greenberg et al., 2009). Bdnf The therapeutic potential of mGluR antagonists in ASD has conditional knockout mice show features analogous to Rett also been suggested. Repetitive grooming behaviors in BTBR and syndrome, including smaller brain size and hindlimb-clasping valproic acid (VPA) mouse models of autism are significantly alle- behavior (Chang et al., 2006). Mice with double knockout of viated by MPEP treatment (Silverman et al., 2010a; Mehta et al., Bdnf and MeCP2 show earlier onset of Rett-like symptoms, 2011). Impairments in social interaction of BTBR mice are also whereas overexpression of Bdnf in MeCP2 knockout mice delays ameliorated by MPEP administration (Silverman et al., 2010a). the onset and relieves the electrophysiological defects of MeCP2 GRN-529, a selective negative allosteric modulator of mGluR5 mutants. Moreover, restoring Bdnf expression through ampakine developed by Pfizer, was shown to fully rescue excessive repet- administration alleviates respiratory problems of MeCP2 mutant itive grooming behavior and social dysfunction in BTBR mice mice (Ogier et al., 2007). Although BDNF appears to have and jumping stereotypy in C58/J mice (Silverman et al., 2012). significant effects in Rett syndrome model animals, it poorly These findings suggest that mGluR negative allosteric modulators penetrates the blood-brain barrier, limiting its therapeutic have novel therapeutic potential in autism, in addition to fragile application. Xsyndrome. Another growth factor associated with Rett syndrome is insulin-like growth factor 1 (IGF-1) (Figure 1). IGF-1 is a NMDAR ANTAGONISTS polypeptide hormone with structural similarity to insulin. While NMDAR antagonists including amantadine and its close ana- it has a profound effect on overall cell growth, it also plays logue memantine are now in clinical trials for autistic patients an important role in regulating neuronal functions by promot- (Nightingale, 2012; Spooren et al., 2012). Amantadine and ing axonal outgrowth (Ozdinler and Macklis, 2006), neuro- and memantine are non-competitive antagonists for NMDARs with synaptogenesis (O’Kusky et al., 2000), and activity-dependent multiple clinical uses (Chen et al., 1992; Blanpied et al., 2005). cortical plasticity (Tropea et al., 2006). IGF-1 binds to IGF- Memantine is currently being used for Alzheimer’s disease, while binding proteins (IGFBP1–6), resulting in extension of the half- it is also useful for viral infection and Parkinson’s disease. Because life of IGF-1 (Hwa et al., 1999). Upon binding to its cognate both drugs are weak NMDAR antagonists with moderate affinity, receptor, IGF-1 activates Ras-MAPK and PI3K-Akt pathways prolonged receptor blockade during treatment is unlikely to cause (Fernandez and Torres-Aleman, 2012), signaling cascades that are significant side effects. also activated by BDNF. In a double-blind, placebo controlled study, amantadine- Because IGF-1 crosses the blood-brain barrier, it may be treated group show significant improvements in hyperactivity aviablealternativetoBDNFasatherapeuticagentforRett

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syndrome. Indeed, IGF-1 and IGFBP have been implicated in OXYTOCIN Rett syndrome and autism: IGFBP3 levels are abnormally ele- Oxytocin is a nine amino acid neuropeptide hormone synthesized vated in MeCP2 mutant mice and Rett syndrome patients (Itoh by magnocellular neurons in paraventricular and supraoptic et al., 2007), and the concentration of IGF-1 in CSF is reduced nuclei of the hypothalamus and secreted from the posterior pitu- in autistic individuals (Riikonen et al., 2006). The therapeutic itary gland into the circulation (Figure 1). Oxytocin acts through utility of IGF-1 in Rett syndrome was originally suggested by oxytocin receptors (OXTRs), which are abundantly expressed Mriganka Sur and coworkers, who reported that lethality, hypoac- in the amygdala, hippocampus, and hypothalamus (Gould and tivity, and respiratory problems of MeCP2-null mice are partially Zingg, 2003). Oxytocin is associated with various social behav- rescued by IGF-1 treatment in association with normalization iors including affiliation, maternity, aggression, and pair bonding of impaired spine density, synaptic transmission, and cortical (Lee et al., 2009; Caldwell, 2012; Feldman, 2012). Given the plasticity (Table 4)(Tropea et al., 2009). IGF-1 also reverses the prominence of oxytocin in the regulation of social behavior, reduction in excitatory synapse number and density of neurons the association of oxytocin with autism pathogenesis has been derived from Rett patients (Marchetto et al., 2010). extensively examined. Several SNPs of OXTRs are associated with ASD (Wu et al., RAPAMYCIN 2005; Jacob et al., 2007; Yrigollen et al., 2008; Liu et al., 2010). Rapamycin is an immunosuppressant originally identified as Oxtr knockout mice display autistic-like behaviors; they emit an antifungal agent in isolates from Streptomyces hygroscopicus fewer USVs upon social isolation, show defects in social recog- (Sehgal et al., 1975; Vezina et al., 1975; Baker et al., 1978; Singh nition and discrimination, and are less aggressive (Table 6) et al., 1979). Rapamycin strongly binds to FK506-binding pro- (Takayanagi et al., 2005; Crawley et al., 2007). Supporting the tein (FKBP); this complex then binds and inhibits mTOR, a pharmacotherapeutic potential of oxytocin, nasal administration serine/threonine kinase implicated in transcription, cytoskeleton of oxytocin improves social interactions and communications dynamics, ubiquitin-dependent protein degradation, autophagy, (Andari et al., 2010; Kosaka et al., 2012), reduces repetitive and membrane trafficking (Figure 1)(Dennis et al., 1999). mTOR behaviors (Hollander et al., 2003), and enhances social cognition signaling has profound effects on neuronal cells in addition to (Hollander et al., 2007) in autism-affected individuals. cancer cells (Busaidy et al., 2012), immune cells (Araki et al., 2011), and cells that regulate lifespan (Powers et al., 2006; PERSPECTIVES Harrison et al., 2009). In the nervous system, mTOR regu- HOMEOSTATIC MECHANISMS UNDERLYING ASD lates axon guidance, dendrite arborization, synaptogenesis, and Tuberous sclerosis and fragile X syndrome are disorders with synaptic plasticity (Troca-Marin et al., 2012). common symptoms including intellectual disabilities, seizures, Perturbations in mTOR signaling have significant impacts on and autism. While their genetic determinants are different normal brain functions. Patients with Alzheimer’s disease and (TSC1/TSC2 for tuberous sclerosis and FMR1 for fragile X syn- Drosophila tauopathy models show enhanced mTOR signaling in drome), their gene products both regulate protein synthesis the brain (Li et al., 2005; Khurana et al., 2006). Hyperactivation in neurons (Bassell and Warren, 2008; Ehninger et al., 2009). of the Akt-mTOR pathway is observed in hippocampal neurons Interestingly, animal models of tuberous sclerosis (Tsc2+/− mice) of Ts1Cje mice, which models Down syndrome (Troca-Marin and fragile X syndrome (Fmr1−/y mice) display abnormal protein et al., 2011). Animal models and patients of Parkinson’s disease synthesis in opposite directions (Auerbach et al., 2011). exhibit enhanced levels of REDD1, which inhibits mTOR activity Tsc2+/− mice exhibit diminished mGluR-dependent LTD and (Malagelada et al., 2006). mTOR is observed in inclusion bod- protein synthesis in the hippocampus, whereas Fmr1−/y mice ies from Huntington’s disease patients and corresponding mouse show excessive mGluR-dependent LTD and protein synthesis. models (Ravikumar et al., 2004). Importantly, rapamycin treat- Consistent with this, cognitive impairments of the two animal ment alleviates several pathogenic traits observed in in vivo and models are corrected by drugs that modulate mGluR5 in the oppo- in vitro models of Alzheimer’s disease (Khurana et al., 2006; site manner (CDPPB for Tsc2+/− mice and MPEP for Fmr1−/y Harrison et al., 2009), Parkinson’s disease (Pan et al., 2009; Tain mice). In addition, crossbreeding of these two mouse lines res- et al., 2009), and polyglutamine diseases (Ravikumar et al., 2004; cues behavioral impairments and synaptic dysfunctions. These Berger et al., 2006; Pandey et al., 2007). results strongly suggest that mGluR5-mediated synaptic plasticity The therapeutic utility of rapamycin in ASD was suggested and protein synthesis in the normal range is important and that in 2008 based on studies in Tsc2+/− mice (Ehninger et al., deviationineitherdirectionfromanormalrangecancausebrain 2008). The mTOR pathway is associated with TSC because TSC1 dysfunctions that yield similar behavioral manifestations. and TSC2 are upstream inhibitory regulators of mTOR activity Another such example comes from two mouse models with (Han and Sahin, 2011). In this study, the learning and memory different mutations in the same gene. Shank2−/− mouse lines deficits, lethality, aberrant brain overgrowth, and altered synaptic lacking exons 6 and 7 (Won et al., 2012)orexon7only plasticity of Tsc2+/− mice were ameliorated by acute treatment (Schmeisser et al., 2012), both of which mimic mutations found with rapamycin (Table 4). The social dysfunction and behav- in humans (Berkel et al., 2010), display similar autistic-like ioral inflexibility of Purkinje cell-specific Tsc1 mutant mice were behaviors, but NMDAR function in their brains shows oppo- also improved by rapamycin (Tsai et al., 2012), further suggest- site changes: NMDAR hypofunction with exons 6 and 7 deletion ing that rapamycin may be useful in reversing core symptoms and NMDAR hyperfunction with exon 7 deletion. Although of autism. further details remain to be explored, this is another example

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suggesting that NMDAR function in a normal range is important, excitatory synaptic transmission, which is mainly mediated by and that deviations in either directions can lead to similar behav- AMPAR receptors, can be determined by the regulators of the ioral abnormalities. Therefore, individuals with mutations in the synaptic trafficking and stabilization of AMPA receptors, and reg- same gene may have to be carefully diagnosed, for example by ulated by the signaling pathways in the downstream of NMDA high-through sequencing, in order to receive proper treatment. receptors, mGluRs, and monoamine receptors. Another core mechanism could be the E-I balance, which is determined by CORE MECHANISMS UNDERLYING ASD the relative amounts of excitatory and inhibitory synaptic trans- Given the diverse genetic variations underlying the development missions, and, together with the excitability of postsynaptic neu- of ASD, one obvious challenge in understanding how ASD devel- rons, determines firing patterns of postsynaptic neurons and, ops is the wide range of mechanisms associated with it. This subsequently, network activities across the brain. Establishing diversity poses a serious additional problem in treating ASD: these core mechanisms, if any, would require rigorous and time- a single medication is likely to cover only a small fraction of consuming verifications using a range of approaches, including individuals with ASD, or a limited spectrum of ASD symptoms. mouse genetics, electrophysiology, and behavior. A related well-known example is the selective effect of risperidone. Risperidone, a dopamine antagonist, is an antipsychotic INTEGRATING THREE ASPECTS OF ASD RESEARCH: HUMAN mainly used to treat schizophrenia and bipolar disorder, and GENETICS, MOUSE MODELS, AND POTENTIAL TREATMENTS it is currently one of the few FDA-approved medications for An important starting point for ASD research using mouse mod- autism. The drug mainly ameliorates irritability, hyperactivity, els would be to select best possible genetic variations that can and repetitive and restricted behaviors, but is largely ineffective provide us decent insights into the underlying mechanisms and against social withdrawal and language deficits of autistic indi- potential treatments. Luckily, a large number of ASD-related viduals (McPheeters et al., 2011). Similarly, risperidone rescues papers are being published each year (i.e., ∼2500 papers in 2012 repetitive grooming and hyperactivity, but not social deficits, in when “autism” was used as a search key word in PubMed). These Cntnap2−/− mice (Penagarikano et al., 2011). Another example is publications, which use diverse genetic and genomic approaches the demonstration that CDPPB rescues only social interaction in and often large size samples, have identified overlapping genes Shank2-deficient mice but fails to rescue impaired pup retrieval, and mutations, which are likely to have greater influences on repetitive jumping, hyperactivity, and anxiety-like behavior (Won the development of ASD. Characterization of transgenic mouse et al., 2012). The fact that some medications reverse only selec- lines that carry these frequent genetic variations would help us tive symptoms/phenotypes of ASD, however, may provide an efficiently obtain ASD mechanisms with a greater impact. opportunity to further explore detailed mechanisms underly- The synaptic and circuit mechanisms derived from ASD ing particular aspects of ASD etiology. This would, in principle, mouse model researches would provide clues to the ways to res- allow us to dissect and study synaptic or circuit mechanisms cue synaptic/circuit phenotypes and ASD-like behaviors in mice. that are specifically associated with certain aspects of ASD, such Given that there is no FDA-approved treatment for social deficits as impaired social interaction, impaired social communication, in ASD as of now, these rescue results will only be useful in repetitive behavior, restricted interests, intellectual disability, anx- supporting that the candidate mechanisms are indeed causing iety, and hyperactivity. the ASD-like phenotypes in mice. Importantly, however, some of A possible solution to the apparent diversity of ASD-related these mechanism-based rescues may serve as the basis for clinical mechanisms is to identify “core” mechanisms that cover a trials. Eventually, some of the clinically verified medications may large fraction of genetic variations, or a broader spectrum of return to basic ASD research and be used to identify additional ASD symptoms. The concept of core mechanisms is based on ASD mouse models with similar or novel underlying mecha- the assumption that a fraction of ASD-related proteins may nisms, which will help us understand a bigger picture, where act together and converge on a common pathway. A possi- many synaptic and circuit mechanisms act together and converge blecoremechanismcouldbeexcitatorysynaptictransmission. into more comprehensive mechanisms. Excitatory synaptic development can be regulated by a number of factors including synaptic adhesion molecules, synaptic ACKNOWLEDGMENTS scaffolding proteins, and actin-regulatory proteins. In addition, This study was supported by the Institute for Basic Science (IBS).

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