Neurexins (NRXN) are a family of synaptic adhesion proteins that are located on the presynaptic membrane and bind to their postsynaptic counterpart, NLGNs. The NRXN family consists of three genes (NRXN1, NRXN2, and NRXN3), each of them generating a long mRNA encoding α-NRXN and a short mRNA encoding β-NRXN from two independent promoters. The intracellular domains of α-NRXNs and β-NRXNs are identical, whereas the extracellular domains are different. Specifically, the extracellular domains of α-NRXNs contain six laminin, nectin, and sex-hormone binding globulin (LNS) domains and three epidermal growth factor (EGF) domains, which form three repeated LNS (A)-EGF-LNS (B) structure. However, β-NRXNs have no EGF domain and only one LNS domain (Tabuchi and Sudhof, 2002). NRXN1, NRXN2, and NRXN3 are located on chromosomes 2p16.3, 11q13, and 14q31, respectively. α-NRXN triple knockout mice had reduced synaptic Ca²⁺ channel function, which causes impaired spontaneous and evoked neurotransmitter release (Missler et al., 2003).

Rare copy number variations and/or point mutations in NRXN genes have been repeatedly reported to be associated with ASDs (Table 1). Friedman and Luiselli (2008) first reported a 320 kb de novo heterozygous deletion of the NRXN-1α promoter and exons 1–5 in a boy with cognitive impairment, autistic features and physical dysmorphism. Later, the Autism Genome Project Consortium et al. (2007) identified a de novo heterozygous deletion that eliminated several NRXN1 exons, including 1α and 1β, in two affected female siblings in one ASDs family. Several other studies have also reported deletions in NRXN1 in ASDs patients (Kim et al., 2008; Marshall et al., 2008; Morrow et al., 2008; Glessner et al., 2009; Pinto et al., 2010; Bremer et al., 2011; Levy et al., 2011). To date, no homozygous deletions in NRXN1 have been found, which may suggest that the dosage of NRXN1 is very important for neurological development. P300P, an NRXN1 common variant, was associated with ASDs in a Chinese ASDs patient. In addition to being associated with ASDs, NRXN1 deletions have also been reported in other psychiatric conditions, such as schizophrenia, bipolar disorder, attention deficit hyperactivity disorder, and Tourette syndrome (International Schizophrenia Consortium, 2008; Walsh et al., 2008; Guilmatre et al., 2009; Zhang et al., 2009; Sundaram et al., 2010; Lionel et al., 2011).

A truncated mutation of NRXN2 inherited from a father with severe language delay and a family history of schizophrenia was identified by Gauthier et al in an ASDs patient (Gauthier et al., 2011). NRXN3 deletions have also been found in four ASD individuals: one was a de novo mutation, two were inherited from a non-affected mother or father, and one was inherited from a father with subclinical autism (Vaags et al., 2012).

NRXN animal models have provided evidence supporting the role of NRXN in ASDs pathology. NRXN1α KO mice showed a defect in excitatory synaptic strength, with a decrease in miniature excitatory postsynaptic current frequency and in the input–output relation of evoked postsynaptic potentials (Etherton et al., 2009). Behavioral studies have shown that NRXN-1 deficient mice display decreased prepulse inhibition and increased grooming behaviors but no obvious changes in social behaviors or spatial learning (Etherton et al., 2009). Studies in an α-NRXN triple KO mice with all three α-NRXNs (Nrxn1α/2α/3α) deleted have shown that α-NRXNs were not required for synapse formation but were essential for Ca²⁺-triggered neurotransmitter release (Missler et al., 2003).

Contactin associated protein-2 (CNTNAP2, also known as Caspr2) is a member of the NRXN superfamily and is involved in neuron–glia interactions and clustering K⁺ channels in myelinated axons. Strauss et al. (2006) identified a homozygous mutation of CNTNAP2 in Amish children with PDD, seizures, and language regression (Strauss et al., 2006; Table 1). Bakkaloglu et al. (2008) found 13 rare non-synonymous variants unique to ASDs patients, which suggests that ASDs patients carry more CNTNAP2 rare variants. Several other studies have also found other common polymorphisms of CNTNAP2 that are associated with ASDs (Alarcón et al., 2008; Arking et al., 2008; Sampath et al., 2013). Interestingly, Alarcón et al. (2008) found that CNTNAP2 provided a strong male affection bias in ASDs.

CNTNAP2 KO mice exhibited deficits in the core ASDs behavioral domains, such as stereotypic motor movements, behavioral inflexibility, communication, and social behavior abnormalities (Peñagarikano et al., 2011).

Neuroligins (NLGN) are a different type of synaptic cell adhesion proteins that are located in the postsynaptic membrane. NLGNs bind to their adhesive counterpart NRXNs and play an important role in synapse formation and function (Figure 1). The human NLGN family includes five NLGN genes (NLGN1, 2, 3, 4, 4Y), which are localized at 3q26 (NLGN1), 17p13 (NLGN2), Xq13 (NLGN3), Xp22.3 (NLGN4), and Yq11.2 (NLGN4Y). NLGNs contain a large extracellular domain that shares sequence homology with acetylcholinesterase and that is necessary for β-NRXN binding and synaptogenic activity, two EF-hand motifs that bind to Ca²⁺, an O-glycosylation region, a transmembrane domain, and a cytoplasmic C-terminal tail that contains a PSD-95/Dlg/ZO-1 (PDZ) interaction site (Dean and Dresbach, 2006; Figure 1). NLGNs-1, -3, and -4 localize mainly to glutamate synaptic sites, whereas NLGN-2 localizes primarily to gamma-aminobutyric acid (GABA) synapses (Missler et al., 2003; Figure 1). In vitro and in vivo, NLGN-1 overexpression increases excitatory synaptic responses and potentiates synaptic NMDA receptor (NMDAR)/AMPAR ratios. In contrast, NLGN-2 overexpression increases inhibitory synaptic responses. Accordingly, the inhibition of NLGN-1 expression selectively decreases the NMDAR/AMPAR ratio, whereas the deletion of NLGN-2 selectively decreases inhibitory synaptic responses. Furthermore, NLGN-1 expression selectively increases the maturation but not initiation of excitatory synapse formation in adult-born neurons (Chubykin et al., 2007; Schnell et al., 2012).

The earliest report regarding the potential association of NLGN genes and ASDs is Jamain et al. (2003; Table 1). In one ASDs multiplex family, the group found three ASDs siblings carrying one frameshift mutation (1186insT) of NLGN4 that was inherited from the non-affected mother, creating a stop codon that led to a premature termination of the protein. In another family, a R451C transition in NLGN3 that changed a highly conserved arginine residue into cysteine within the esterase domain was identified in two affected siblings. This point mutation was inherited from the non-affected mother. A study in a large French family found a 2 bp deletion (1253delAG) that resulted in a premature stop codon in the middle of the sequence of the normal NLGN4 gene (Laumonnier et al., 2004). In this family, 10 males had non-specific X-linked mental retardation, two had autism, and one had pervasive developmental disorder. All affected patients had the same frameshift mutation. Missense changes in NLGN4 were also found in Portuguese ASDs families (Yan et al., 2005). A study in a small Finnish autism sample did not find any functional mutation of NLGN1, NLGN3, NLGN4, or NLNG4Y, although three common variants (rs1488545 in NLGN1, DXS7132 in NLGN3, and DXS996 in NLGN4) that showed minor association with ASDs were found. Despite the evidence that NLGN is associated with ASDs, several studies have failed to find associations between rare mutations and common variants and ASDs (Talebizadeh et al., 2004; Vincent et al., 2004; Gauthier et al., 2005; Blasi et al., 2006; Wermter et al., 2008; Liu et al., 2013).

Several NLGN mutant mouse models have been developed to investigate the role of NLGN mutations in ASDs. Nlgn3 R451C knock-in mice, which corresponded to the human non-synonymous SNP (R451C) in NLGN3 found in ASDs patients (Jamain et al., 2003), showed social interaction deficits and increased spatial memory and an electrophysiological phenotype consisting of increased inhibitory synaptic transmission in the somatosensory cortex (Tabuchi et al., 2007; Etherton et al., 2011a). Furthermore, researchers found that both NLGN-3 R451C-knockin and NLGN-3 knockout mutations in mice showed impairment in tonic endocannabinoid signaling (Földy et al., 2013). Another NLGN3 mutation, R704C, was introduced into mouse NLGN-3 by homologous recombination, and electrophysiological and morphological studies have shown that although the NLGN-3 R704C mutation did not significantly alter synapse formation, it dramatically impaired synapse function. Moreover, the R704C mutation caused a major and selective decrease in AMPA receptor-mediated synaptic transmission in pyramidal neurons of the hippocampus, without similarly changing NMDA or GABA receptor-mediated synaptic transmission and without detectably altering presynaptic neurotransmitter release (Etherton et al., 2011b). Mice lacking the human NLGN4 (NLGN4-KOs) ortholog exhibited highly selective deficits in reciprocal social interactions and communication reminiscent of ASDs (Jamain et al., 2008).

Shank family proteins, also known as ProSAP, are synaptic scaffold proteins that bind NLGN-NRXN and NMDAR complexes at the postsynaptic density (PSD) of excitatory glutamatergic synapses (Figure 1). There are three genes that encode Shank proteins (SHANK1, SHANK2, and SHANK3). All Shank proteins are expressed in the brain but exhibit different patterns. Shank1 is expressed in most parts of the brain, except for the striatum, and it is highly expressed in the cortex and hippocampus. Shank2 and Shank3 are also present in the cortex and hippocampus. Shank2 is mostly absent from the thalamus and striatum, whereas Shank3 appears to be predominantly expressed in those regions. In the cerebellum, Shank2 is restricted to Purkinje cells, whereas Shank3 is restricted to granule cells (Sheng and Kim, 2000). SHANK directly or indirectly binds to NLGNs in the PSD. In vitro and in vivo studies highlight the important role of Shank3 for synaptic function. SHANK3 functions as a scaffolding protein in spine morphogenesis and synaptic plasticity. Knockdown of Shank3 in cultured hippocampal neurons leads to a reduced number and increased length of dendritic spines. When overexpressed in cultured hippocampal neurons, Shank3 promotes the maturation and enlargement of dendritic spines (Betancur et al., 2009). Knock-down of Shank3 in hippocampal neurons decreases spiny density, whereas transfection of Shank3 in aspiny neurons induces the formation of dendritic spines with functional synapses (Betancur et al., 2009).

Shank contains multiple domains for protein–protein interaction, including ankyrin repeats (binding to α-fodrin to link to the actin cytoskeleton and calpain/calmodulin-mediated Ca²⁺ signaling), an SH3 domain (binding to glutamate receptor-interacting protein to link with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors to the postsynaptic scaffold), a PDZ domain (binding different molecules within the PSD, including GKAP, to allow Shank to attach to PSD-95), a proline-rich region (which contains sites for Homer and cortactin), and a sterile alpha motif domain (which is involved in the polymerization of Shank molecules; Lim et al., 1999; Yoo et al., 2014; Figure 1).

SHANK3 was the first gene in the SHANK family reported to be associated with ASDs (Table 1). The SHANK3 gene is located on chromosome 22q13.3 within the critical region of 22q13.3 deletion syndrome (also known as Phelan-McDermid syndrome, PMS). 22q13.3 deletion syndrome is characterized by neonatal hypotonia, global developmental delay, absent or severely delayed speech, autistic behaviors, and intellectual disability (Phelan, 2008). The size of the deleted segments varied widely in individuals with this syndrome, but deletions of SHANK3 were present in nearly all cases (Wilson et al., 2003, 2008; Dhar et al., 2010). Three ASDs families were observed to carry alterations of 22q and/or the SHANK3 gene (Durand et al., 2007). In one family, an individual carried a de novo deletion of 22q13, in which the deletion breakpoint was located in intron 8 of SHANK3 and a 142 kb of the terminal 22q13 was removed. In the second family, two affected siblings were heterozygous for an insertion of a guanine nucleotide in exon 21, and the mutation was a de novo mutation. In the third family, a terminal 22q deletion was identified in a girl with autism who exhibited severe language delay. A 22qter partial trisomy in her brother who had Asperger syndrome was also identified, although the boy demonstrated precocious language development and fluent speech. These unbalanced cytogenetic abnormalities were inherited from a paternal translocation, t(14;22) (p11.2;q13.33). This finding suggests a gene dosage effect of SHANK3. A study of SHANK3 variants by screening SHANK’s exon sequence in 400 ASDs families found 10 novel non-synonymous variants in ASDs (Moessner et al., 2007). Among these mutations, one was a de novo mutation, and the other nine were all inherited from one unaffected parent. Rare functional mutations of SHANK3 have been identified in two ASDs families. One was a de novo deletion at an intronic donor splice site, and one was a missense mutation inherited from an epileptic father (Gauthier et al., 2009). Moreover, three deleterious variants (one 6-amino acid deletion upstream of the SH3 domain, one missense variant in the PDZ domain, and one insertion/deletion of a repeated 10 bp GC sequence located 9-bp downstream from the 3′ end of exon 11) were found in Japanese ASDs families (Waga et al., 2011). It has been reported that 2.3% of ASDs patients carry deleterious mutations in SHANK3 (Boccuto et al., 2013).

Several studies have identified de novo deleterious mutations of SHANK2 in ASDs (Berkel et al., 2010; Pinto et al., 2010; Leblond et al., 2012; Table 1). In one study, a de novo nonsense mutation, seven rare inherited changes and additional variants specific to ASDs were identified by sequencing SHANK2 in 396 ASDs patients. In a separate study, SHANK2 was sequenced in 455 patients. When combining the results of these two studies, a significant enrichment of variants affecting conserved amino acids in 29 of 851 (3.4%) patients and 16 of 1090 (1.5%) controls was observed. In neuronal cell cultures, the variants identified in patients were associated with reduced synaptic density at dendrites compared with the variants only detected in controls (Leblond et al., 2012).

The SHANK1 gene rare mutation has also been associated with ASDs (Sato et al., 2012; Table 1). An inherited deletion of 63.8 kb encompassing SHANK1 and the CLEC11A gene was found in a multigenerational family. In this family, four males carrying this deletion showed high-functioning autism or a broader autism phenotype, whereas the two females carrying the same deletion were not affected by ASDs (Sato et al., 2012). SHANK1 deletions may be associated with high-functioning autism in males (Guilmatre et al., 2013).

Genetic mouse models resembling different SHANK mutations have been created to investigate the role of Shank in synapse conformation and function and its contribution to autistic pathology. Shank1 mutant mice (with a deletion of exons 14 and 15, which includes most of the PDZ region, resulted in the knockout of all detectable Shank1 protein in these animals) showed decreased movement in the open field and deficits in motor learning and contextual fear conditioning. Although these animals did not show apparent repetitive behaviors and seemed to have normal levels of social interaction, they showed a general deficit in social communicative behaviors by both ultrasonic vocalizations and urine-based communicative behaviors (Hung et al., 2008; Silverman et al., 2011; Wöhr et al., 2011). Shank2 mutant mice, mimicking the human microdeletion of exons 6 and 7, which targeted the PDZ domain and knocked out of all Shank2 isoforms, also showed alterations in behavior and synaptic plasticity (Schmeisser et al., 2012; Won et al., 2012). Shank3 exons 4–9 KO mice (resulting in the loss of the longest two isoforms of Shank3) were used to mimic human SHANK3 mutation by several research groups. Although the deficits in social interaction in these models were not consistent (Bozdagi et al., 2010; Peça et al., 2011; Yang et al., 2012), all of the models showed a repetitive self-grooming phenotype (Wang et al., 2011; Yang et al., 2012).

Postsynaptic density protein 95 (PSD-95; also known as DLG4, SAP90), is a member of the membrane-associated guanylate kinase family of synaptic molecules and serves as a major functional bridge interconnecting the NRXN-NLGN-SHANK pathway (Figure 1). PSD-95 contains three PDZ domains, a single interior SH3 domain, and a COOH-terminal guanylate kinase domain. The cytoplasmic domains of all three NLGNs bind to the third PDZ domain of PSD-95, whereas NMDA2 receptors and K⁺channels bind to the first and second PDZ domains. PSD-95 is localized at excitatory synapses and has been implicated in promoting synapse stability. PSD-95 makes synaptic contacts more stable in older neurons than in younger neurons.

PSD-95 knockout mice exhibit reduced AMPAR function and a decreased frequency of AMPAR-mediated miniature EPSCs, suggesting that PSD-95 may regulate synaptic maturation through postsynaptic AMPA-type glutamate receptors (GluARs).

Thus far, no rare point mutation or CNV or common variants have been reported to be associated with ASDs, but PSD-95 deletion (Dlg4^-/-) mice have been shown to exhibit a complex range of behavioral and molecular abnormalities relevant to ASDs (Table 1). Dlg4^-/- mice showed increased repetitive behaviors, abnormal communication and social behaviors, impaired motor coordination, increased stress reactivity, and anxiety-related responses. Dlg4^-/- mice also had subtle dysmorphology of amygdala dendritic spines and altered forebrain expression of various synaptic genes (Feyder et al., 2010).

The synapsins are a family of presynaptic phosphoproteins that account for 9% of the vesicle protein and can regulateneurotransmitter release and neurite outgrowth (Rosahl et al., 1995). Synapsins contain a mosaic of conserved (A–C, E) and individual domains (D, F–J). They have three family members in mammals (synapsin 1, synapsin 2, and synapsin 3), which locate on chromosomes Xp11.23, 3p25.2, and 22q12.3, respectively. Cultured neurons from synapsin 1,2,3(–/–) triple knock-out mice exhibit severely dispersed synaptic vesicles and considerably reduced synaptic vesicles number (Fornasiero et al., 2012).

Mutations in synapsin 1 (Q555X, A51G, A550T, and T567A) were found in a large French-Canadian family with epilepsy and ASDs (Fassio et al., 2011; Table 1). Furthermore, the nonsense Q555X mutation can reduce the phosphorylation caused by CaMKII and Mapk/Erk, which regulate synaptic vesicles trafficking and neurite outgrowth. The missense mutation of A550T and T567A can impair the targeting to nerve terminals (Fassio et al., 2011).

Synapsin 2 has also been identified as an autism predisposing gene. In a study involving 190 individuals with ASDs, researchers found one nonsense mutation (p.A94fs199X) and two missense mutations of synapsin 2 (p.Y236S and p.G464R; Corradi et al., 2014; Table 1).

Synapsin knockout mice may be identified as a useful experimental model of ASDs and epilepsy. Researchers found that synapsin knockout mice exhibit social novelty abnormality and avoidance behavior in social approach which are reminiscent of ASDs. Specifically, synapsin 2 deletion mice display deficits in short-term social recognition and increased repetitive self-grooming behavior. Synapsin 1 and synapsin 3 deletion mice display an impaired social transmission of food preference. Synapsin 1and synapsin 2 deletion mice display a decreased environmental interest (Greco et al., 2013). The results demonstrate an involvement of synapsins in the development of the behavioral traits of ASDs.

Gephyrin is a key scaffold molecule of the postsynaptic membrane at inhibitory synapses (Figure 1). It contains three domains, G domain in N-terminal, E-domain in C-terminal, and a large linker domain of the two. Gephyrin can interact with glycine receptor and alpha and beta subunits of the GABA_A receptor to mediate inhibition. NLGN 2 can bind to protein gephyrin through a conserved cytoplasmic motif and activate collybistin. NLGN 2, gephyrin, and collybistin complexes are sufficient to inhibitory neurotransmitter receptors clustering. Deletion of NLGN 2 in mice leads to a loss of recruitment of gephyrin at perisomatic but not dendritic sites (Poulopoulos et al., 2009; Table 1). Gephyrin-deficient mice die early postnatally and display loss of postsynaptic GABA(A) receptor and glycine receptors clustering, whereas glutamate receptor subunits were normally localized (Kneussel et al., 1999; Grosskreutz et al., 2003).

Exonic microdeletions in gephyrin gene have been reported a correlation with neurodevelopmental disorders including ASDs (Lionel et al., 2013). In one family, the proband with ASDs has a 357 kb de novo deletion in gephyrin and exhibits limited movement, slow motor development, and language delay. The second family has a 319 kb paternally inherited deletion in gephyrin and exhibits mild global developmental delay in early life, social difficulties, and repetitive behaviors. The third family has a 273 kb de novo deletion in gephyrin gene and exhibits developmental delay, cyclical seizures, and behavioral issues including anxiety, obsessive compulsive disorders, tics, and impulsive behaviors (Lionel et al., 2013).

Cadherins (CDHs) are a family of glycosylated transmembrane proteins that mediate cell–cell adhesion, neuronal migration, spine morphology, synapse formation, and synaptic remodeling (Redies et al., 2012). Because the function of CDHs is dependent on the presence of Ca²⁺, they are named for the Ca²⁺-dependent cell adhesion molecule family. The CDH family is classified into classical CDHs, desmosomal cadherins, and protocadherins (PCDHs). Genome-wide association studies on a cohort of 4305 autistic subjects have shown that common variants between the CDH9 and CDH10 genes on chromosome 5p14.1 are associated with autism (Wang et al., 2009). Similarly, recurrent larger genomic deletions in 16q23 in CDH13 was also observed in ASDs patients in 1124 ASDs families participating in genome-wide analyses (Sanders et al., 2011; Table 1). Furthermore, after the detection of 14 SNPs of protocadherin α in DNA samples of 3211 individuals with autism, 5 SNPs were showed significantly associated with autism (Anitha et al., 2013). In addition, CNVs in PCDH9 and homozygous deletions in PCDH10 have also been reported in ASDs (Betancur et al., 2009; Table 1).

In situ hybridization analysis in the embryonic and postnatal mouse demonstrated that CDH8 expression is restricted to specific developing gray matter structures. Later, a study using the PPL statistical framework identified that CDH8 is expressed in the developing human cortex of ASDs family, which implicates CDH8 in susceptibility to autism (Redies et al., 2012).

Thousand-and-one-amino acid 2 kinase (TAOK2), also known as TAO2, is a serine/threonine-protein kinase that is encoded by the TAOK2 gene in humans. It can activate mitogen-activated protein kinase (MAPK) pathways to regulate gene transcription. TAOK2 interact with semaphorin 3A receptor neuropilin 1, which regulates basal dendrite arborization. In addition, TAOK2 can be phosphorylated and activated by Sema3A. In cultured cortical neurons, TAOK2 downregulation can decrease JNK phosphorylation and cause its inactivation. Furthermore, basal dendrite formation in cortical neurons caused by TAOK2 downregulation can be rescued by active JNK1 overexpression. TAOK2 is involved in membrane blebbing, the DNA damage response, and the MAPK14/p38 MAPK stress-activated MAPK cascade. Recently, TAOK2 has been shown to play a role in basal dendrite formation (de Anda et al., 2012).

The TAOK2 gene is located in the 16p11.2 chromosomal region. Approximately 1% of autistic subjects have been shown to have a novel, recurrent microdeletion, a de novo deletion of 593 kb on chromosome 16p11.2, and a reciprocal microduplication on chromosome 16p11.2 (Weiss et al., 2008), suggesting that 16p11.2 or the TAOK2 gene is involved in susceptibility to ASDs (Table 1).

Contactins (CNTNs) are members of the immunoglobulin superfamily. They are glycosylphosphatidylinositol-anchored neuronal membrane proteins and play important roles in axon growth and guidance and synapse formation and plasticity.

Array-based comparative genomic hybridization identified a paternally inherited chromosome 3 copy number variation in three autistic subjects. Specifically, a deletion in two siblings and a duplication in an unrelated individual were detected. Furthermore, these variations were mediated by disruptions of CNTN4 (Roohi et al., 2009), suggesting that CNTN4 may be involved in ASDs (Table 1). Recently, a study conducted in a Chinese population also came to the same conclusion (Guo et al., 2012). Although the sample sizes of these two studies were small, a CNV analysis involving 2195 autistic subjects indicated that CNTN4 deletions and duplications are associated with ASDs (Betancur et al., 2009). In a cohort of ASDs subjects, a CNV in the CNTN5 gene was identified in one individual. In addition, a CNTN6 deletion has also been found in an autistic family (Zuko et al., 2013).

Cntn6 knockout mice exhibited slower learning in terms of equilibrium and vestibular senses (Zuko et al., 2013), indicating that Cntn6-deficiency leads to defects in motor coordination. Other characteristics of ASDs, such as social interaction and social communication, remain to be determined in these mice. Cntn5 knockout mice exhibited decreased susceptibility to audiogenic seizures and impaired hearing, which may be related to the impairment of sensory information integration reminiscent of ASDs (Zuko et al., 2013).

Gamma-aminobutyric acid is the major inhibitory neurotransmitter in the human brain and is synthesized from excitatory neurotransmitter glutamate via the action of glutamate decarboxylase (GAD) enzymes, which have two main isoforms, GAD65 and GAD67. There are two main types of GABA receptors, ionotropic GABA_A receptors and metabotropic GABA_B receptors. GABA_B receptors are localized at pre-, post-, or extrasynaptic sites as functional heterodimers, whereas GABA_A receptors are the major mediators of fast inhibitory neurotransmission in the mammalian brain.

There are three GABA_A receptor genes (GABRB3, GABRA5, and GABRG3) localized on the human chromosome 15q11–q13, a part of the genome which is involved with genome instability, gene expression, imprinting and recombination and is one of the most complex regions in the genome (Martin et al., 2000). Duplications of the 15q11–13 locus have been observed in ASDs in several studies (Bolton et al., 2001; Kwasnicka-Crawford et al., 2007; Depienne et al., 2009). Duplication of the region containing GABA_A receptor subunits may lead to excessive inhibitory neurotransmission due to gene dosage; however, an in vitro study using a human neuronal cell line carrying a maternal 15q duplication showed that this variant leads to reduced GABRB3 expression via impaired homologous pairing (Meguro-Horike et al., 2011), suggesting that 15q11–q13 genes are regulated epigenetically at the level of both inter- and intra-chromosomal associations and that chromosome imbalance disrupts the epigenetic regulation of genes in 15q11–q13.

Moreover, mouse models mimicking human 15q11–q13 duplication have exhibited features of autism, such as poor social interaction, behavioral inflexibility, and abnormal ultrasonic vocalizations (Nakatani et al., 2009).

GABA_B receptors play an important role in maintaining excitatory-inhibitory balance in brain. In autistic brain subjects, researchers have found that the expression of GABA_B receptor subunits GABA_B receptor 1 (GABBR1) and GABA_B receptor 2 (GABBR2) were significantly reduced (Fatemi et al., 2009). Furthermore, clinical trials show that the selective GABA_B receptor agonist STX209 (arbaclofen) has a potential to improve social function and behavior in patients with fragile X syndrome and was generally well-tolerated in ASDs individuals (Berry-Kravis et al., 2012; Erickson et al., 2014).

In vitro, STX209 (arbaclofen, R-baclofen) can correct the elevated basal protein synthesis in the hippocampus of Fmr1-knockout mice, an animal model of Fragile X syndrome. In vivo, acute administration of STX209 can decrease mRNA translation in the cortex of Fmr1-knockout mice. Furthermore, the chronic administration of STX209 in juvenile mice can improve the increased spine density in Fmr1-knockout mice (Henderson et al., 2012). Since ASDs individuals have something in common with Fragile X syndrome, this implies that GABA_B receptor agonist STX209 may also improve synaptic abnormalities in ASDs.

Consistent with the genetic evidence for the involvement of GABAergic genes in ASDs, the expression of GABAergic genes and related proteins have been reported to be reduced in the post-mortem ASDs brain. GAD65 and GAD67 proteins were reduced in the cerebellum and parietal cortex (Fatemi et al., 2002), GAD67 mRNA was reduced in cerebellar Purkinje cells (Yip et al., 2007), and GABA_A receptor binding was reduced in the hippocampus (Blatt et al., 2001) and anterior and posterior cingulate cortices (Oblak et al., 2009, 2011).

Although technical difficulties still exist, researchers have attempted to measure GABA function in vivo, and these results support the presence of GABAergic defects in ASDs patients. Using proton magnetic resonance spectroscopy ([¹H]MRS; Harada et al., 2011) reported that GABA concentrations were reduced in the frontal cortex of ASDs children, whereas no differences were observed in the basal ganglia (Harada et al., 2011). Two studies using SPECT (Single Photon Emission Computed Tomography) found reductions in GABA_A receptors in both ASDs adults and children (Mori et al., 2012; Mendez et al., 2013).

In addition to the genes/proteins involved directly in GABA synthesis and transmission, many other factors exert indirect effects on GABA functioning through the regulation of gene expression, receptor trafficking, and downstream signaling pathways; therefore, GABAergic dysfunction could also be a downstream consequence of mutations in the genes involved in the increase or decrease of GABA transmission. CNTNAP2, which is a part of the NRXN family, has been associated with autism (Gregor et al., 2011; Stein et al., 2011). CNTNAP2 knockout mice showed specific deficits in inhibitory signaling, with reduced GAD1 expression and a reduced number of GABAergic interneurons (Pe Peñagarikano et al., 2011). Similar findings have been observed in another ASDs candidate gene model; CADPS2 knockout mice showed reduced cortical parvalbumin GABA interneurons and a reduced number of cerebellar Purkinje cells (Sadakata et al., 2007).

Glutamate is the major excitatory neurotransmitter in the human brain. Glutamate receptors (GluARs) are composed of ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs). Findings from genetic studies, post-mortem brain studies, animal models, and clinical drug trials have implicated a dysfunctional glutamatergic system in ASDs; however, hypo- and hyperfunction coexists in different forms of ASDs.

Ionotropic glutamate receptors are classified into NMDA (N-methyl-D-aspartate), AMPA (2-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), and kainate receptors based on structural, pharmacological, and physiological properties. iGluRs are tetramers encoded by 18 genes. NMDARs are obligate heteromers formed as tetramers from the co-assembly of GluN1, GluN2A-GluN2D, GluN3A, and GluN3B subunits. Each NMDAR channel contains a combination of two GluN1 and two GluN2A–GluN2D subunits or two GluN1 with one GluN2 and one GluN3 subunit. AMPARs are homo- or hetero-tetramers formed from the GluA1–GluA4 subunits and are Mg²⁺-insensitive. Kainate receptors are tetramers formed from combinations of the GluK1–GluK5 subunits.

Several genetic studies have reported that NMDARs genes are associated with ASDs. Two studies that sequenced ASDs patients identified rare disruptive mutations in the GluN2B (GRIN2B) gene (Tarabeux et al., 2011; O’Roak et al., 2012). Common polymorphisms in GRIN2B and GRIN2A have also been associated with ASDs (Barnby et al., 2005; Yoo et al., 2012). Interestingly, the NMDAR subunits have differential expression during development, with GluN2B expressed early in development, followed by GluN2A during later development and synapse maturation (Sanz-Clemente et al., 2013).

Ramanathan et al. (2004) identified a 19 mb deletion of chromosome 4q in an ASDs child, which included the AMPA 2 gene that encodes the glutamate receptor GluR2 sub-unit (Ramanathan et al., 2004). One study identified chromosome 6q21 as a candidate region for autism and found a functional SNP in glutamate receptor 6 (GluR6 or GRIK2) gene associated with ASDs (Jamain et al., 2002).

A post-mortem brain study also found that ASDs patients have specific abnormalities in AMPA receptors and glutamate transporters in the cerebellum (Purcell et al., 2001). The mRNA levels of excitatory amino acid transporter 1 and glutamate receptor AMPA1 (GluA1) were significantly increased in autism subjects, and AMPAR density was decreased in the ASDs cerebellum (Purcell et al., 2001).

Parvalbumin-selective NMDAR 1 knockout (NR1 KO) mice exhibited autism-like phenotypes compared with wild-type mice; the N1 ERP latency was delayed, sociability was reduced, and mating USVs were impaired (Saunders et al., 2013).

The administration of acute PCP and ketamine, NMDAR antagonists, has been shown to mimic the symptoms of autism in humans (Carlsson, 1998). Based on this phenomenon and neuroimaging and neuroanatomical studies, Carlsson (1998) proposed that infantile autism is a hypoglutamatergic disorder. Recently, both the use of an NMDAR agonist and antagonist has been reported in ASDs patients. Daily doses of D-cycloserine, an NMDAR glycine site partial agonist, significantly improved social withdrawal (Posey et al., 2004), and daily doses of amantadine (memantine), an NMDAR non-competitive antagonist, reduced some negative symptoms of autism, such as hyperactivity (King et al., 2001; Chez et al., 2007).

mGluRs are members of the group C family of G-protein-coupled receptors. mGluRs have seven transmembrane domains that span the cell membrane. Differently to iGluRs, they are not ion channels. There are eight different types of mGluRs, namely mGluR1 to mGluR8, which are divided into three groups, group 1, group 2, and group 3. mGluR1 and mGluR5 belong to group 1 family, mGluR2, mGluR3, and mGluR4 belong to group 2 family, and mGluR6, mGluR7, and mGluR8 belong to group 3 family. They can regulate neuronal excitability, learning, and memory.

A study using high-throughput multiplex sequencing revealed significant enrichment of rare functional variants in the mGluR pathway in non-syndromic autism cases. (Kelleher et al., 2012). Most recently, in a valproate-induced rat model of autism, the expressions of mGluR2/3 protein and mGluR2 mRNA were found significantly reduced. N-acetylcysteine (NAC) recued social interaction and anxiety-like behaviors of the VPA-exposed rats. In addition, these effects can be blocked by intra-amygdala infusion of mGluR2/3 antagonist LY341495 (Chen et al., 2014). These results indicate that the disruption of social interaction in VPA induced rats could be restored by NAC, which may depend on the activation of mGluR2/3.

A decrease in mGluR has been found in PTEN knockout mice showing autism-like behavioral deficits (Lugo et al., 2014). By reducing 50% of mGluR5 expression, several abnormalities of Fmr1 knockout mice can be rescued. For example, density of dendritic spines on cortical pyramidal neurons and basal protein synthesis in hippocampus are increased, inhibitory avoidance extinction and audiogenic seizures are improved (Dolen et al., 2007).