Genetic mechanisms underlying synaptic pathology in Autism Spectrum Disorder(ASD)

HE Zheng-xiao ,LI Yong-yi,2 ,DING Xue-jiao ,JIANG Lin-yan ,ZHU Ming-wei ,TANG Ming-xi ,TANG Ya-Ping,2,4,5

1.Guangzhou Institute of Pediatrics,Guangzhou Women and Children’s Medical Center,Guangzhou Medical University,Guangzhou 510623,China;2.Guangdong Provincial Key Laboratory of Brain Function and Disease,Zhongshan School of Medicine,Sun Yat-sen University,Guangzhou 510080,China;3.Department of Pathology,the Affiliated Hospital of Southwest Medical University,Luzhou 646000,China;4.Department of Neurobiology,Southwest Medical University Luzhou 646000,China;5.VA None-Clinical Research Program,New Orleans,LA 70112,USA

【Abstract】Autism spectrum disorder(ASD)is a neuronal developmental disorder that is characterized by defects both in social interaction and verbal communication,and is often accompanied by restricted interest,and repetitive and stereotyped behaviors.The prevalence of ASD is approximately 0.8%～1.2%in China.Although its etiology remains unclear in most cases,over 1 000 genes or genomic loci have been linked to its pathogenic origin,indicating a strong genetic influence,as well as complicated pathogenic mechanisms.Here,we reviewed recent findings in the possible genetic effects on synaptic pathophysiology of ASD,and a particular focus was put on variants in genes that are related to synaptic morphology or functions,such as FMR1, NRXN,NLGN,SHANK and MeCP2.The synaptic pathology caused by these genetic defects may substantially contribute to the pathogenesis of ASD.This review outlines several lines of evidence that have been recently reported to support the hypothesis that a genetic defect may lead to a synaptic pathology that may underlie the pathogenesis of ASD.

【Key words】Autism spectrum disorders(ASD)Synapse Synapse elimination Synaptic transmission Synaptic plasticity

1 Introduction

Autism spectrum disorder(ASD)is a group of neurodevelopmental disorders that are characterized by a cluster of clinical core symptoms including impaired social interaction and communication,and repetitive and stereotyped behaviors.The term "autism" is first introduced by Prof Leo Kanner in 1943 at Johns Hopkins University in USA.Dr.Kanner followed up almost 30 years for 11 children who were featured by repetitive,stereotypical attention to objects and considered as ASD at the ages from 2 to 8[1].In 1944,Dr.Hans Asperger identified a group of children who showed maladaptation to their social environment and displayed certain repetitive and restricted behaviors[2].There is a significant clinical heterogeneity in ASD.In addition to the main core symptoms,a large portion of patients may show some mental comorbidities[3-4]such as intellectual disability[5],epilepsy[6],anxiety[7],attention deficit hyperactivity disorder[8]and sleep disorders[9]etc.or others such as gastrointestinal disorders[9].Except for some symptom-based medications,no actually effective medicines are currently available for ASD.Although FDA in USA approved for the clinical uses of Risperidone and Aripiprazole,those two choices are not really effective for any core symptoms of ASD[10-11].Currently,behavioral interventions,especially at the earlier stage,are the major usefully therapeutic approaches world-widely[12-13].

The global prevalence of ASD is approximately 0.8%～1.2%[14-16].In China,we still do not have a national-wide epidemiological data basis so far[17].A recent multi-center study has suggested that the prevalence of ASD in children within the ages between 6 and 12 years in China is about 0.6%～0.8%[18],while a local study in younger kids revealed a prevalence rate of 2.6%[19].Also,there was a report to claim that the prevalence of ASD in China is very close to that in western countries,that is about 1%[20].Nevertheless,the prevalence of ASD in most areas in this world is significantly increased during the past 30 years[21-22],due to whatever reasons,such as changes in the environmental exposures,changes in humanity culture,growing public awareness of the disorder,changes in ASD diagnostic criteria,and increasing services for ASD,etc.[23].However,there is evidence showing that the changes in environments may most dominantly attribute to this increased prevalence[22,24].

The pathogenic origin of ASD is still not clear.Over the past 50 years,one milestone in ASD studies is the establishment of the notion that the etiology of ASD is at least partially genetic/genomic oriented[14,25].The first lines of evidence came from twin studies,in which it was found that the concordance rate for ASD in monozygotic twins (80%～90%) was much higher than that in dizygotic twins (0%～10%)[26-28].However,due to the small sample size,short follow-up period,and inconsistent diagnostic criteria,those studies did not receive enough attention.Recently,large-scale cohort studies on multiple populations have further supported the findings that the incident rate of ASD in monozygotic twins is much higher than that of dizygotic twins[29-30],and further confirmed the strong influence of genetic components[29,31].To date,over 1 000 genes or genomic loci have been associated with ASD.Many of these genes are important players in molecular and cellular functions such as chromatin remodeling[32],mRNA transcription and translation[33],protein modification[34]，cellular stress[35]etc,or neuronal function such as neural migration[36],axonal guidance[37],dendritic growth[38],synapse formation[39],neurogenesis[40]and synaptic transmission[41],among which,synaptic dysfunction seems one of the most important mechanisms in the pathogenesis of ASD[42-44].

Synapses in the brain are the neural structure specifically for the functional connections between neurons,via which neural information is transmitted.The pathogenic events for ASD may occur during the late developmental stage,including prenatal and perinatal stages[45],a critical period for the development of synapses.Many genes that are associated with ASD,such as FMR1,NRXN,NLGN,SHANKandMeCP2,are actually important players in synapse formation,synaptogenesis,synaptic transmission,synaptic plasticity etc.[44,46-47],implying that synaptic dysfunction may lie on the core of the pathogenesis.In this review,we will provide an overview of the genetic mechanisms that underlie synaptic dysfunctions often observed in ASD,with a hope to strengthen the research on this aspect.

2 Genetic Factors

2.1 Copy number variants(CNVs)

Following the broad uses genome-wide analysis advanced,it has been generally accepted now that CNVs play an essential role in the human genome polymorphism,biodiversity and diseases.In 2007,Sebat et al.for the first time systematically reported that de novo CNVs were associated with ASD,in which over 10%ASD kids harbor CNVs,while in control subjects,only less than 1% has a similar genotype[48].Up to date,numerous studies have revealed that an overall frequency of CNVs in ASD patients range from 8%to 21%,which is significantly higher than that in controls[49-51].CNVs are generally classified into the recurrent and nonrecurrent types.Recurrent CNVs have standard sizes and breakpoints caused by non-allelic homologous recombination(NAHR)between low copy repeats(LCRs),while nonrecurrent CNVs are usually caused by nonhomologous end joining (NHEJ),fork stalling and template switching(FoSTeS),or microhomology-mediated breakinduced replication.The most common recurrent CNVs associated with ASD are microdeletions and microduplications of approximately 600 kb in the 16p11.2 region,[52].In addition,CNVs at 15q11-q13,7q11.23,16p11.2,22q11.2,and 1q21.1 were also linked to ASD[53].ASD patients with 16p11.2 microdeletions showed a significant deficit in functional connectivity and long-range integration in the prefrontal and parietotemporal region of the brain[54].TheUBE3Agene,located at 15q11-q13,encodes a translational ubiquitination protease E3A,and this molecule regulates protein homeostasis and synaptic plasticity.Evidence shows that the duplications ofUBE3Acan cause ASD-like symptoms[55].

2.2 Single nucleotide polymorphisms(SNPs)

In addition to CNVs,SNPs are also obtained a wide attention for their possible implication in ASD etiology.Although results from numerous studies have indicated a significant association of SNPs in over 1 000 genes or loci with ASD[46,56-57],controversial results are also often reported.Since ASD patients have a high heterogeneity in clinical symptoms,many GWAS studies were seeking to establish a connection between a specific clinical symptom and genetic variants[58-59].Prof Hu et al.screened 18 significant SNPs by dividing all ASD individuals into four subtype groups.When all patients were combined into the same cohort for analysis,however,no definitive association between the SNPs and ASD was found.The recent two studies also suggested that SNPs might make a minor contribution to ASD etiology[59-60].The cohorts tested so far in autism studies are much smaller than in other common diseases such as epilepsy or schizophrenia.Therefore,it may be necessary to expand the sample size to further look for the association of SNPs with ASD[61-64].

2.3 Chromosomal abnormalities

It has been estimated that the incident rate of chromosomal aberrations,including deletions,duplications,inversions,translocations in ASD patients is approximately 2%～5%[65-66].Most chromosomal structural abnormalities are rare,and their causative role in ASD is unknown[67].Among them,the most common chromosomal abnormality is 15q11-q13 duplication of maternal origin[68].It’s variable in size and presents in approximately 1%～3% of children with ASD.Many genes associated with ASD,such asgabra5,GABRB3,UBE3A,HERC2,SNRPNandCYFIP1,are located in this chromosomal region[69-71].Moreover,sex chromosomal abnormalities were identified in syndrome ASD patients,such as Turner syndrome,Klinefelter syndrome,XXX syndrome and XYY syndrome,which are consistent with the notion that many genes located on the sex chromosomes may play an essential role in brain structure,function and development[72].

3 ASD Risk Genes and Synaptopathology

Synapse formation and maturation are critical for the development of neural circuits in the brain,and synaptic dysfunctions are thought to be underlying the pathogenesis of neurodevelopmental disorders.It has been suggested that ASD-like symptoms may be attributed to deficits in synaptic function,and thus the role of those synaptic function-related genes in the pathogenesis of ASD has received an extensive attention.Mutations in those synaptic function-related genes,such as FMR1,NRXN,NLGN,SHANKandMeCP2,have been clearly fund to be harmful for synapse formation,maturation,or synaptic plasticity,and many others[43].Thus,abnormal synaptic development and synaptic dysfunction may be particularly important in triggering pathogenic cascade for ASD.

3.1 FMRP gene

Fragile X Syndrome (FXS) is the most common cause for inherited intelligent deficit (ID),accounting for approximately 1%～2% of all ID patients.Also,FXS is the most common single-gene disorder causing ASD,with approximately 60%～74% of FXS patients meeting the diagnostic criteria for ASD.The direct cause of FXS mutation is the inactivation of the FMR1 gene.At the same time,about 2%～5% of all ASD are diagnosed with a test of the FMR1 gene mutations[73].

The FMR1 gene cords for the Fragile mental retardation protein (FMRP) that acts as a translational repressor of mRNA binding.In normal cells,FMRP tightly regulates mRNA stability and transport and translation[74].In recent years,hundreds of possible FMRP target mRNAs have been identified through high-throughput approaches,with a considerable number of mRNAs encoding pre-and post-synaptic proteins associated with ASD,including NMDARs,mGluR5,PSD-95,SHANK1-3,HOMER 1,NEUREXIN and others[75].The dysfunction of FMRP in promoting translational activities is considered as a critical cause of ASD-associated synaptic and cognitive deficits.An increased local translation ofNLGNmRNAs and targeting of nlgn1 andnlgn3to the postsynaptic membrane was found in FMR1 KO mice[76-79].In FXS mouse models,mGluR5 in the CA1 region of the hippocampus was found to be increased binding to short Homer 1a and reduced binding to the long Homer isoform,resulting in mGluR5 being significantly more mobile post-synaptically[80].

3.2 SHANK genes

Shank (also known as pro sap) is a major scaffolding protein localized at PSD of excitatory synapses in the brain.The Shank family consists of three major isoforms (Shank1,Shank2 and Shank3),and they share the following similar domains:an N-terminal ankyrin repeat,an Src homology 3 (SH3) domain,and a PSD-95/discs large/ZO-1 (PDZ) domain,an extended proline-rich region,and a sterileαMotif (SAM) domain.The function of Shank family is linked to cell adhesion molecules,actin remodeling,and they enhance synaptic plasticity,maintain synaptic ecology,induce dendritic spines,induce excitatory synapse formation,and is essential for excitatory glutamatergic synapses.

Shank1is encoded by theSHANK1gene,which is located on chromosome 19q13.33.Sato et al.identified deletions in the Shank1gene in two multiplex ASD families[81].Hung et al.found that Shank1knockout mice(PDZ domain suppression that disrupts all Shank1isoforms) showed an reduced locomotor function and impaired memory for fear conditioning,whereas increased spatial memory in the maze test[82].Wohr et al.showed that Shank1knockout mice showed reduced ultrasonic articulation levels and odor marker behavior,indicating an inability to learn social experience,in consistent with the phenotype of ASD[83].These studies all suggest that Shank1may play an important role in the pathogenesis of ASD.

Shank2 is encoded by theSHANK2gene,which is located on chromosome 11q13.3-11q13.4.Shank2 variants include deletions,missense mutations,truncations,and changes in the promoter region.Leblond et al.found de novoshank2gene deletions (truncations)in three individuals with ASD[84].He also reported a de novo Shank2 deletion in one ASD patient carrying a paternally inherited deletion of the synaptic translation repressor CYFIP1.Recently,novel Shank2 knockout models in mice (shank2△7 -/ -and shank2 E6-7 -/-) have been generated.Both strains of mice showed a lack of social interaction,increased hyperactivity,self-grooming and anxiety[85].At the biochemical level,the mutant mice showed a reduced synaptic transmission,reduced frequency of mEPSCs,and an increased NMDA/AMPA ratio etc.All these results suggest that Shank2 is involved in the pathological process of ASD[86].

Shank3is encoded by theSHANK3gene,which is located on chromosome 22q13.3.Shank3was first identified in the 22q13.3 deletion syndrome,also known as Phelan McDermid syndrome (PMS),characterized by global developmental delay.More than 50%of PMS patients have autistic-like behavior[87].The first ASD case with a de novo deletion mutation in Shank3was identified by Durand et al.in 2007.Since then,many ASD cases carrying with Shank3mutations were reported.Shank3Neurons harboring Shank3mutation showed reduced PSD proteins,including GKAP,HOMER1b/C,GluA1 subunit of AMPAR,and NR2A subunit of NMDAR[39,88].By compared with shank3ab heterozygotes,Yang et al.showed that shank3ab heterozygotes have more severe reductions in synaptic transmission and LTP in hippocampal CA1[89].Subtle morphological changes in dendritic spines have also been observed upon shank3ab mutations,manifesting with reduced levels of homer1b/C,GKAP,and GluA1 in addition to activity-dependent redistribution of GluA1(containing AMPA receptors) in primary hippocampal neurons.In summary,studies using Shank3mutant murine models have provided with us with evidence in further understanding of the neurobiological basis for ASD.

3.3 NLGNs and NRXNs

NLGNs are a class of cell adhesion factors that are located in the postsynaptic membrane of neurons.This group of protein belongs to the type I transmembrane proteins,which mainly consist of large extracellular domains (containing one acetylcholinesterase homology domain,EF-hand type motif,and O-glycosylation domain),a single-pass transmembrane domain,and intracellular domains.There are 5 NLGNs proteins,namelyNLGN1,NLGN2,NLGN3,NLGN4andNLGN4y.NLGN1is exclusively located in excitatory postsynaptic membranes[90],andNLGN2is mainly located on the inhibitory postsynaptic membrane[91].NLGN3andNLGN4are found in both types of postsynaptic membranes[92-93].NLGNs bind to axonal proteins (neurexins) of the presynaptic membrane and play an essential role in synapse development and function,regulating the balance between excitatory and inhibitory synapses.Nlgns gene variants were first reported to be associated with ASD by Jamain et al[94],who found the r45lc missense mutation in theNlgn3gene and the c.1186inst frameshift mutation in theNlgn4gene on the X chromosome in two Swedish ASD families.By direct sequencing,Xu et al.identified four rare missense mutations,namely,p.g426s (Nlgn3),p.g84r (Nlgn4),p.q162k (Nlgn4) and p.a283t (Nlgn4),in 318 Chinese Han ASD patients,and these four missense mutations were all located in the acetylcholinesterase homology domain of Nlgns,a crucial structure for binding to axonal proteins to trigger synaptic activity[95].Several Nlgns mutant mouse models were used to clarify the role of the Nlgns gene family in the pathogenesis of ASD.Abnormalities in excitatory or inhibitory synaptic neurotransmitter transmission were found in almost all those models,and several studies identified an altered synaptic density and morphology in the hippocampus[96].The abnormal behavior of Nlgns mutant mice resembles typical symptoms of ASD.for example,Blundell et al.found thatNlgn1knockout mice exhibited decreased social behavior,impaired spatial learning and memory,and repetitive stereotypical grooming behaviors[96-97].At the same time,synapse number,synapse length,synaptic connections,dendritic spine density of excitatory synapses were significantly increased in the hippocampus.In consistent,electrophysiological studies revealed a significant increase in excitatory postsynaptic potential (EPSP) and inhibitory postsynaptic potential (IPSP) ratios (E/I) and impaired LTP[97-99].Similar phenotypes were described in other mouse models ofNlgn2,Nlgn3,andNlgn4[100].

Genes encoding neurexins includeNrxn1,Nrxn2,andNrxn3.Axonal proteins are a class of highly polymorphic neuronal surface proteins involved in critical physiological processes such as synapse formation and signaling[101].In a cohort study of 116 Caucasian children with ASD,J Yan et al discovered five missense mutations,including a predicted splice mutation[102].It has been concluded that mutations and abnormalities of structural genes in theNrxn1may increase susceptibility to ASD[61].Moreover,such mutations and structural abnormalities occur at different frequencies in different human populations.Nrxn1was also associated with ASD in an association study with genome-wide SNPs in a large group of samples.Although without any social impairments or deficits in learning,theNrxn1null animal model exhibits repetitive grooming behavior.The electronic signals transmitted by synapses in the hippocampus are weaker than normal controls[96,103].All these results point to a special role of these genes in the pathogenesis of ASD.

3.4 Synaptic elimination

In addition to being associated with abnormalities in synaptogenesis,synaptic neurotransmission,and synaptic plasticity,several ASD risk genes (includingMEF2C,FMR1,dlg4andpcdh10) are involved in synaptic pruning.Therefore they will also be reviewed in this article as a whole[104].

Synapse formation initially predominates during prenatal brain development,while excessive synapse formation beyond the required level for normal physiological function may lead to a pathological condition.During subsequent developmental stages,synapses closely related to normal physiological function are retained,while excess synapses are trimmed and eliminated,leading to reduced synapse number.The maintenance of cognition and expected behavior is mainly dependent on the precise formation of mature neuronal circuits.Therefore,proper synaptic pruning is essential to maintain precise neural circuits.

Loss of MEF2C in mice results in a significant increase in the number of excitatory synapses,whereas neuronal expression of the hyper-activated form of MEF2C leads to a decrease number of excitatory postsynaptic sites.MEF2 and FMRP cooperatively regulate the expression of pro-adhesin-10(pcdh10),and activation of MEF2 leads to PSD95 ubiquitination by the ubiquitin E3 ligase Murine Double Minute-2 (Mdm2),which is then delivered by pcdh10 to the proteasome for degradation,enabling synaptic pruning function[105-107].All these results indicated that a deficit in synaptic pruning may underlie the cellular and neuronal mechanism of ASD.

4 Conclusions

With the advance of gene sequencing technology,more and more researchers pay much attention to the genetic role in the pathogenesis of ASD.An increasing number of ASD risk genes are being identified,suggesting that the genetic mechanisms are more and more important.However,because of the complexity of ASD inheritance,other than a simple monogenic mode of inheritance,there are multiple genes with complex effects,incomplete penetrance,making it very challenging to define an exact role of a genetic component in the pathogenesis of ASD.Even though,it seems very promising to focus on the genes that are synaptic function related,in our attempts to further understanding the possible genetic and neuronal mechanism.Further elucidation of the underlying synaptic pathology,as well as advances in gene therapy and targeted agents to modulate these processes,may lead to promising new therapies for the treatment of ASD.