Autism throughout genetics: Perusal of the implication of ion channels

Abstract Background Autism spectrum disorder (ASD) comprises a group of neurodevelopmental psychiatric disorders characterized by deficits in social interactions, interpersonal communication, repetitive and stereotyped behaviors and may be associated with intellectual disabilities. The description of ASD as a synaptopathology highlights the importance of the synapse and the implication of ion channels in the etiology of these disorders. Methods A narrative and critical review of the relevant papers from 1982 to 2017 known by the authors was conducted. Results Genome‐wide linkages, association studies, and genetic analyses of patients with ASD have led to the identification of several candidate genes and mutations linked to ASD. Many of the candidate genes encode for proteins involved in neuronal development and regulation of synaptic function including ion channels and actors implicated in synapse formation. The involvement of ion channels in ASD is of great interest as they represent attractive therapeutic targets. In agreement with this view, recent findings have shown that drugs modulating ion channel function are effective for the treatment of certain types of patients with ASD. Conclusion This review describes the genetic aspects of ASD with a focus on genes encoding ion channels and highlights the therapeutic implications of ion channels in the treatment of ASD.

demonstrated that the prevalence of autism among siblings was 18 times higher than in the general population, suggesting the existence of a familial heritability factor. Also, the imbalance in the sex ratio among ASD cases, with four to five boys affected for each affected girl, has led to the suggestion of a segregation linked to a sex chromosome and the implication of genes whose variations are expressed on a sex-linked recessive mode (Lai, Lombardo, Auyeung, Chakrabarti, & Baron-Cohen, 2015;Ozonoff et al., 2011). The second argument in favor of genetic causes of ASD is based on the observation of a change in concordance rate between monozygotic and dizygotic twins, which was found to be 70%-90% in monozygotic twins compared with a lower rate of 0%-30% for dizygotic twins (Ronald & Hoekstra, 2014;Rosenberg et al., 2009). Thirdly, the existence of chromosomal aberrations detected in patients with ASD also points toward genetic causes (Vorstman et al., 2006). Finally, genome-wide association studies (GWAS) have led to the identification of numerous ASD susceptibility genes that are located on various chromosomes, especially 2q, 5p, 7q, 15q, 17q and on chromosome X .
In addition, some patients with ASD were found to have variations in syndromic Mendelian genes (e.g., FMR1 for fragile X syndrome, TSC1 and TSC2 for tuberous sclerosis, and MECP2 for Rett syndrome; Liu & Takumi, 2014). The identification of variations in neuroligin genes (NLGN4X and NLGN3X) in patients with ASD suggested that proteins involved in synapse formation and synaptic transmission play an important role in the etiology of ASD (Jamain et al., 2003). Similarly, rare variations have been detected in genes coding for ion channels (e.g., CACNA1 and CACNB2), as well as proteins involved in synaptic structure, gene transcription, and chromatin remodeling (e.g., NRXN1, CTTNBP2, CHD8, and SHANK3), indicating that altered synaptic plasticity and regulation of gene expression may also be involved in the etiology of ASD (Cross-Disorder Group of the Psychiatric Genomics Consortium, 2013;De Rubeis et al., 2014;Durand et al., 2007).
This review examines the genetic basis of ASD and highlights the involvement of ion channel dysfunctions in the causes of this disorder.
This expansion of knowledge is due to the advances in molecular technologies, which allowed detecting chromosomal rearrangements, copy number variations (CNVs), and candidate genes in patients with ASD.

| Chromosomal abnormalities and CNVs in ASD
Chromosomal rearrangements have been identified in 5% of individuals with ASD. These cytogenetic abnormalities are observed in chromosomes 5p15, 15q11-q13, 17p11, and 22q11.2 (Jacquemont et al., 2006;Sebat et al., 2007). Abnormalities affecting the 15q11-q13 region represent the most frequent variation associated with ASD accounting for close to 1% of all ASD cases (Badescu et al., 2016). Depending on the variation type and pattern of inheritance, this locus is associated with either Prader-Willi syndrome (PWS) or Angelman syndrome (AS) along with ASD (Badescu et al., 2016). This variation is based on whether the duplication affects the maternal or the paternal allele (Badescu et al., 2016). Besides structural rearrangements, other abnormalities of chromosomal numbers or aneuploidies are detected in ASD including trisomy 21, Turner syndrome (45, X), and Klinefelter syndrome (47, XXY; Devlin & Scherer, 2012).
Thanks to the comparative genomic hybridization (CGH) technique or SNPs array, CNVs were also found in multiple chromosomal regions at 1q21.1, 16p11.2, 17q12, and 22q11.2 (Jacquemont et al., 2006;Marshall et al., 2008;Matsunami et al., 2013;Pinto et al., 2010;Sebat et al., 2007). Further studies supported the association with ASD of two recurrent de novo CNVs at 16p11.2 (duplication and deletion) and 7q11.23 (duplication; Levy et al., 2011;Sanders Stephan et al., 2011). The chromosomal deletion found at 7q11.23 has been linked to William's syndrome, which includes intellectual disabilities, facial dysmorphic features, congenital heart defect, and transient hypercalcemia. The intellectual disabilities suggest that this chromosomal region may also contain genes associated with social behaviors . CNVs were found enriched in groups of genes implicated in cell signaling pathways that regulate neuronal development and cell proliferation along with a group of genes associated with the GTPase/Ras signaling pathway and neuronal plasticity . CNVs studies demonstrated also that there is alteration in the fragile X mental retardation protein (FMRP) in patients with ASD. In fact, fragile X syndrome (X-Fra) is associated with 1%-2% of ASD cases.
X-Fra syndrome is the second major cause of intellectual disability, which is caused by the expansion of CGG trinucleotide repeats in the FMR1 gene located on chromosome X and that encodes FMRP.
These data emphasized the role of CNVs in ASD, and further investigations in these regions have led to the identification of candidate genes in particular with whole-exome and whole-genome sequencing studies.  (De Rubeis et al., 2014;O'Roak et al., 2011). Three genes were found in ASD probands with two de novo variations in each of these genes: BRCA2, FAT1, and KCNMA1 (Neale et al., 2012). These studies also found significant enrichment of de novo variations in five ASD candidate genes including STXBP1, MEF2C, KIRREL3, RELN, and TUBA1A (Neale et al., 2012). Likewise, a region of chromosome 7q that includes the candidate genes RELN, FOXP2, WNT2, and CADPS2 has been implicated in ASD (Liu & Takumi, 2014). The extracellular glycoprotein RELN plays a key role in neuronal migration and cell interactions (Li et al., 2004). However, it appears that variations in RELN are insufficient to cause ASD, suggesting that secondary genetic or epigenetic factors are behind these cases of ASD (Li et al., 2004). FOXP2 is a crucial gene for language development. Variations affecting this gene have been detected in individuals who lack the ability of acquiring communication skills. However, evidence supporting the involvement of FOXP2 in ASD remains scattered (Toma et al., 2013). The WNT2 gene belongs to the large WNT gene family, which is highly expressed during development of the central nervous system and, therefore, it is not surprising that it could represent an ASD candidate gene (Kalkman, 2012;Li et al., 2004). Finally, the CADPS2 gene encodes a calcium (Ca 2+ )-binding protein and variations in this gene have been linked to patients with ASD and intellectual disability (Bonora et al., 2014). Others genes encoding synaptic proteins linked to ASD were also identified by NGS: They include the glutamate receptors (GRIK2, GRIA3), the cell adhesion molecule CNTNAP2, and the scaffolding protein SHANK3. SHANK3 is involved in (i) synapse formation and maturation, (ii) the link between neurotransmitter receptors and ion channels, and (iii) the interaction with scaffolding proteins and gene regulatory proteins (e.g., protein of chromatin remodeling CHD8; Anney et al., 2010;Cotney et al., 2015;De Rubeis et al., 2014;O'Roak et al., 2011). NRXN1, NLGN3/4X, and SHANK3 genes, which encode proteins involved in neuronal cell adhesion and in the regulation of synaptic transmission, are considered strong candidate loci for ASD (Weiss & Arking, 2009). Variations in those loci have also been detected in several patients with ASD O'Roak et al., 2011; Table 1).

| ASD candidate genes
These approaches have also identified variants in genes encoding ion channels. Here, we describe these variations and highlight the role of ion channels in ASD.

| Calcium signaling and voltage-gated Ca 2+ channels in ASD
Ca 2+ channels are present in many different cell types and they mediate Ca 2+ influx in response to stimuli which can be a response to (i) change in the membrane depolarization; known as voltage-gated channels or (ii) to a ligand-mediated activation (e.g., ryanodine receptor (RyR), inositol triphosphate receptor (IP3R) in the reticulum).
In the brain, the elevation of intracellular Ca 2+ concentration activates several signaling pathways that regulate important neuronal functions such as synaptogenesis, neuronal differentiation, and cell migration (Krey & Dolmetsch, 2007). Dysfunctions of these pathways are responsible for abnormalities observed in patients with ASD, which include an increased cell density, changes in neuronal size, dendritic and axonal branching alterations, as well as in neuronal connectivity (Krey & Dolmetsch, 2007). Voltage-gated Ca 2+ channels are devised in two categories: high-voltage-activated channels (HVA) and low-voltage-activated channels (LVA). HVA include L-type, the neuronal N-, P/Q-, and R-type. The low-voltage-activated Ca 2+ currents are represented by T-type channels. HVA are composed by a principal transmembrane subunit α1 (Cav α) associated with a disulfide-linked α2δ (Cav α2δ) dimer, an intracellular β subunit (Cav β), and a transmembrane γ subunit (Cav γ), while LVA channels are composed only by α1 subunit. Both of HVA and LVA channels control the passive flow of Ca 2+ across membranes. Therefore, alteration in their components leads to defective channel function that translates themselves into a variety of neurological disorders including hemiplegic migraine, episodic and spinocerebellar ataxia, epilepsy, and ASD (Bidaud, Mezghrani, Swayne, Monteil, & Lory, 2006;Breitenkamp, Matthes, & Herzig, 2015;Heyes et al., 2015;Parellada et al., 2014;Stary et al., 2008;Zamponi, 2016).
The Timothy syndrome (TS) is a channelopathy described to be associated with ASD. TS is a multisystem disorder characterized by autistic features, cardiac abnormalities (QT prolongation), defective immune response, and syndactyly (Splawski et al., 2004).
Variations affecting the gene encoding the pore-forming α 1 subunit of L-type voltage-gated Ca 2+ channels are associated with TS.   (2012) and Liu and Takumi (2014) TA B L E 1 (Continued) male patient affected with TS revealed a novel variation in CACNA1C gene (p.I1166T; Boczek et al., 2015). Electrophysiological analysis of HEK-293 cells expressing this gene variant showed a shift in the peak channel activation and a reduced current density (Boczek et al., 2015; Table 2). L-type channels are predominantly expressed in the heart and brain. They are localized at dendrites and cell bodies of mature neurons and regulate neuronal excitability and Ca 2+dependent signaling cascades involving cAMP-binding protein (CREB) and myocyte enhancer factor 2 (MEF2; Krey & Dolmetsch, 2007;Simms & Zamponi, 2014). CACNA1C plays a key role in the development and functionality of the central nervous system by modulating gamma-aminobutyric acid (GABA) transmission and influencing neuronal firing. In fact, mice with dysfunctional CACNA1C show defects in N-methyld-aspartate (NMDA) receptor activity leading to an NMDA-independent long-term potentiation in the CA1 region of the hippocampus that produces an acute decline in memory. These observations indicate that CACNA1C may play a role in NMDA receptor-dependent signaling and in synaptic plasticity in the hippocampus. In addition to ASD, SNPs in CACNA1C gene are linked to psychiatric disorders including schizophrenia and bipolar disorder Moosmang et al., 2005). In a large GWAS, two genes encoding the α 1 subunit of calcium channel (CACNA1C) and its regulatory β 2 subunit (CACNB2) were strongly linked to psychiatric disorders and ASD (Cross-Disorder Group of the Psychiatric Genomics Consortium, 2013).
In addition, three rare missense variations of CACNB2 (G167S, S197F, and F240L) were identified in families with ASD (Breitenkamp et al., 2014). Heterologous expression of these gene variants in HEK-293 cells followed by electrophysiological analysis showed remarkable changes in channel kinetics characterized by an increased sensitivity of voltage-dependent inactivation for both G167S and S197F variants. Unlike these variations, the third variation F240L showed a significant accelerated time-dependent inactivation (Breitenkamp et al., 2014; Tavassoli et al. (2014) subunit, respectively, was observed in patients with autistic manifestations (Smith et al., 2012). A chromosomal translocation of 2p:12p resulting in a deletion of both genes (CACNA1C and CACNA2D4) was detected in two ASD-affected individuals (Smith et al., 2012).
Furthermore, a whole-exome sequencing study identified de novo rare alleles in α 1 subunit loci CACNA1D and CACNA1E Pinggera et al., 2015). The α 1 subunit (Ca v 1.3) of L-type channels plays an important role in neuronal signaling and in brain function including memory and behavior (Pinggera et al., 2015). De novo variations in Ca v 1.3 subunit (CACNA1D) were identified in a cohort of patients affected with autism along with intellectual disability (Pinggera et al., 2015).  Table 2).
T-type voltage-gated Ca 2+ channels are known to play a key role in the cerebral cortex and in the thalamus (Simms & Zamponi, 2014 hypotonia, severe delay in expressive language, and facial dysmorphism associated with other clinical signs. However, this duplication was described by the author as "benign" because it was also found in the control individual (Yatsenko et al., 2012; Table 3).
The importance of defective regulation of intracellular Ca 2+ in the pathophysiology of ASD is further supported by the association between genes that encode plasma membrane Ca 2+ pumps and ASD.
In fact, three studies from different human populations reported an association between the ATP2B2 gene coding for the plasma membrane Ca 2+ ATPase and ASD phenotypes (Yang et al., 2013). It should be noted that ASD-associated genetic variations have been identified in genes encoding Ca 2+ channels and Ca 2+ transport pumps, as well as in genes encoding ion channels whose activities are under Ca 2+ modulation. To the best of our knowledge, until now, no association with other Ca 2+ channels such as ligand-gated Ca 2+ channels (RyR, IP3R) has been found, hampering the exploration of novel cellular pathways.

| Potassium (K + ) channels in ASD
K + channels are located in membranes of excitable and non-excitable cells and they assure K efflux out of cells. According to their structure and functions, K + channels are segregated into four categories: the voltage-gated channels, inwardly rectifying (Kir), tandem pore domain (K2P), and the ligand-gated (Kligand) channels (Kuang, Purhonen, & Hebert, 2015). They all share a pore-forming α subunit but different regulatory subunits are identified in each group. Ca 2+activated potassium channels (BK Ca ) are ligand-gated K + channels that participate to several cell functions such as the regulation of hormone and neurotransmitter releases (Kuang et al., 2015). In fact, BK Ca are abundantly distributed throughout the brain and are mainly localized at presynaptic terminals, where they partake in the adjustment of synaptic transmission and neuronal excitability (Kuang et al., 2015;Laumonnier et al., 2006). Laumonnier et al. (2006) Table 2). The regulatory β 4 subunit gene of BK Ca channel (KCNMB4) was classified as one of the three predictive genes in ASD as it was strongly associated with SNPs-ASD-associated in a large meta-analysis study (Skafidas et al., 2014; Table 3). On the other hand, a variation of KCNQ3 gene mapped to chromosome 8q24, encoding the voltage-gated potassium channel K v 7.3, has been linked to epilepsy. This locus was found disrupted as a consequence of a de novo chromosomal translocation in one patient with ASD. In addition, three patients with ASD shared a missense variation in KCNQ3. This variation could be described as a loss of function as identified by electrophysiological recordings in Xenopus laevis oocytes (Gilling et al., 2013; Table 2).
These findings established a link between ASD and potassium channels and highlight their physiological importance in neuronal functions.

| Sodium (Na + ) Channels in ASD
Voltage-gated Na + channels (Na v ) are essential for the initiation and propagation of action potentials in neuronal cells, muscles, and heart tissues. Na V channels are heteromeric complexes comprised of an α subunit (pore-forming) associated with one or more β regulatory subunits. We distinguish nine members of Na V channels (Na V 1.1 to Na V 1.9) that differ by their structure but also by their ligand-specific binding sites (toxins, drugs) which has led to their classification as critical drug targets (Bagal, Marron, Owen, Storer, & Swain, 2015).
Na v channels are primarily expressed in neurons and glial cells in the central and peripheral nervous system. Variations affecting the α subunit of Na + channels and their accessory β subunit are known to be responsible for Brugada syndrome, a cardiac disease (Weiss et al., 2003). In addition, several variations in SCN1A and SCN2A that encode Na v 1.1 and Na v 1.2, respectively, are associated with childhood epilepsy and ASD (Weiss et al., 2003). Variations in SCN1A and SCN2A were shown to cause familial hemiplegic migraine and to be implicated in severe seizure syndrome, epilepsy, and Dravet syndrome (Craig, de Menezes, & Saneto, 2012;Weiss et al., 2003).
It was shown that variations in SCN2A affect the calmodulin-binding site of the channel and reduce its affinity for Ca 2+ . This site is crucial for the binding between channel subunits and for connecting Na + channels to Ca 2+ signaling pathways (Weiss et al., 2003). Another study using array-comparative genome hybridization identified a de novo deletion in a chromosome 2 region (2q24.2-q24.3) that con-  Table 2). In addition, the α subunit 8 gene of a Na + channel was associated with ASD and identified by whole-genome sequencing in a family with ASD. In fact, a de novo heterozygous missense variation was found in SCN8A gene (p.N1768D), which alters a conserved residue of the channel. The biophysical consequences of this variation are an increase in Na + current and partial channel inactivation (Veeramah et al., 2012; Table 2). In a large study of consanguineous families with autism, a homozygous deletion of SCN7A gene was identified in one family, which is adjacent to SCN1A gene within the sodium channel gene cluster (SCN1A, SCN2A, SCN3A, and SCN9A) on chromosome 2 (Morrow et al., 2008; Table 3). This evidence supports the hypothesis that NKCC1 activity is associated with schizophrenia and ASD because these two conditions share the same genetic background (Merner et al., 2016 Kang andBarnes, 2013, andLaumonnier et al. (2006) TA B L E 3 (Continued) as a prospective drug by restoring the gradient and GABA inhibition and, thereby, considered as a potential ASD-therapeutic agent (Lemonnier et al., 2012; Table 3). Synaptic regulatory proteins mainly concern: glutamatergic (e.g., GRIN2B) and GABAergic (e.g., GABRA3 and GABRB3) neurotransmission, neuronal connection (e.g., CNTNAP2) and ion permeability (e.g., CACNA1, CACNA2D3, and SCN1A), as well as proteins directly involved in synapse formation such as neurexins (NRXNs) and Ca 2+ /calmodulin-dependent protein kinase 4 (CAMKIV). However, CYFIP1 and CAMKIV have been described as susceptibility genes in ASD and together combined with an altered activity of FMPR enhances the ASD risk (Waltes et al., 2014).

| Ion channels and dysfunctional pathways in ASD
Another pathway implicating calcium signaling and ASD is the Mammalian target of rapamycin (mTOR) pathway also known as the mechanistic target of rapamycin kinase. MTOR gene is a tumor suppressor that regulates calcium signaling and mitochondrial functions. mTOR controls cells growth, proliferation, and differentiation, involved in synapse plasticity, and inhibits autophagy by preventing protein degradation. Interestingly, mTOR is upstream regulated by several mediators such as growth factors signals (e.g., insulin) or in neurons by the brain-derived neurotrophic factor (BDNF) through the phosphoinositide-3-kinase (PI3K) activation the protein kinase B (Akt) and Ras to the extracellular signal-regulated kinase (Erk; Napoli et al., 2008;Schratt, Nigh, Chen, Hu, & Greenberg, 2004).
Both Erk and Akt act on the tuberous sclerosis complex (TSC1 and TSC2) by phosphorylating TSC2 inducing its dissociation of TSC1. TSC1 and TSC2 proteins act like GTPase proteins and downregulate a small GTPase Rheb (Ras homolog enriched in brain) protein via GAP protein through a mechanism that remains unknown (Ma & Blenis, 2009).
Rheb is a direct activator of mTOR complex by activating its regulatory associated protein (raptor; Ma & Blenis, 2009). Once the mTOR complex is activated, it phosphorylates a series of protein such as the S6 kinase 1 (S6K1), the eukaryotic translation initiation factor 4E-binding protein 1 (eIF-4BP1), and the carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD).
S6K1 and eIF-4BP1 are essential for protein synthesis and polypeptide translation in ribosomes and cell proliferation, while CAD is a key player in pyrimidine synthesis and so nucleotide synthesis (Ma & Blenis, 2009). In addition, the tuberous sclerosis complex can also be activated by AMPK, GSK3β, and p53 which leads the inhibition of mTOR pathway. Furthermore, variations in TSC1 and TSC2 genes have been associated with ASD (Devlin & Scherer, 2012). Also, variations in mTOR pathway repressors, such as for the neurofibromin 1 (NF1) gene NF1, cause neurofibromatosis type 1 syndrome as reported in 1% patients with ASD (Devlin & Scherer, 2012). The phosphatase and tension protein homolog (PTEN) is also known to downregulate mTOR pathway via both PI3K and AKT. Patients with ASD associated with cerebral malformation, like macrocephaly, have been found to carry variations in the PTEN gene in 7% of the cases (Devlin & Scherer, 2012;McBride et al., 2010; Figure 1).
Besides protein synthesis and translation that have been shown to be implicated in the process of ASD, the mechanism of protein degradation was also studied in ASD. Genetic studies indicate that ubiquitin-proteasome system is necessary for normal human cognitive function by regulating the synapse assembly and elimination (Mabb & Ehlers, 2010). The ubiquitin ligase enzyme Ube3A is a member of the E3 ubiquitin ligase family. The disruption of its activity leads to Angelman syndrome, while in turn the Angelman syndrome was described in ASD with CNVs and mutations in UBE3A gene (Greer et al., 2010). In Ube3A knockout mice, electrophysiological studies demonstrated an impaired long-term potentiation (LTP) in the hippocampus, which suggest that alteration of Ube3A results in the loss of neuronal plasticity. In fact, Ube3A increases transcription through the myocyte enhancer factor 2 (MEF2) complex and regulates synapse function by ubiquitinating and degrading the synaptic protein Arc (activity-regulated cytoskeleton-associated protein). The role of Arc is to decrease long-term potentiation by promoting the internalization of AMPA receptors, which are the mediators of the excitatory neurotransmission in the central nervous system (Greer et al., 2010). On another hand, a decrease in AMPAR expression at synapses has been observed in patients with fragile X syndrome.
This decrease is due to excessive mGluR5 signaling resulting in an increased Arc translation and consequently excessive AMPA receptors internalization (Dolen & Bear, 2008). In FMR1 knockout mice, injections of mGluR5 restore the AMPA receptors expression levels and prevent fragile X syndrome (Dolen et al., 2007).
Interestingly, it has been shown that an alteration of the inhibitory phosphorylation function of the Ca 2+ /calmodulin-dependent protein kinase II (CamKII) is coupled to an increase in AMPA receptors expressed at the synapse (Rose, Jin, & Craig, 2009 Figure 1).
Together, these studies emphasize the implication of ion channels in the pathophysiology of ASD and strengthen the hypothesis that pharmacological manipulation of ion channels function is a potential therapeutic target in ASD.

| Ion channels and drug therapy in ASD
Ion channels have always been considered as powerful drug targets for the treatment of a wide range of pathologies owing to their crucial role as regulators of cell excitability (Kaczorowski, McManus, Priest, & Garcia, 2008).
In 1884, cocaine was discovered as the first anesthetic drug (Vandam, 1987). Several decades later, cocaine was described as a Na + channel blocker (Kyle & Ilyin, 2007;Vandam, 1987). This observation led the chemists to the production of novel analogs of cocaine, all classified under the term of "caine" and constituting a novel family of anesthetics (e.g., benzocaine, lidocaine; Casale, Symeonidou, & Bartolo, 2017;Tremont-Lukats, Megeff, & Backonja, 2000). Thereafter, drug-mediated modulation of Na + channel properties was found to have other therapeutic functions such as anticonvulsants and antidepressants (e.g., carbamazepine) used in the treatment of neuropathic pain (Tremont-Lukats et al., 2000).
Valproic acid (VPA) is one of the most widely used anti-epileptic drugs for the treatment of tonico-clonic seizures that act by modulating Na + channel kinetics in neurons (Loscher, 2002). VPA is also used for the treatment of bipolar disorder, anxiety, and migraine (Loscher, 2002). Studies showed that the exposure to VPA during pregnancy induces neurobehavioral abnormalities similar to autism traits in both rodents and humans (Bertelsen et al., 2016;Choi et al., 2016;Mony, Lee, Dreyfus, DiCicco-Bloom, & Lee, 2016). In fact, VPA treatment of postnatal rats was shown to affect DNA synthesis and astrocyte proliferation and was associated with autistic behavior (Mony et al., 2016). A recent study showed that the phenotypic signs of ASD induced by VAP exposure in rats can be significantly improved or recovered by the administration of vitamin D in early stages of development (Du, Zhao, Duan, & Li, 2017). In addition, it has been demonstrated that persistent Na + current is responsible for hypoxia in neurons leading to neuronal damages (Faustino & Donnelly, 2006). In fact, the persistence of Na + currents leads to the increased activity of Na + /Ca 2+ exchangers in neurons, itself resulting in an increase in Ca 2+ cytoplasmic concentration (Faustino & Donnelly, 2006). In order to correct this situation, it has been proposed that an increase in Na + influx into cells prevents trauma in the nervous system (Ates et al., 2007). Some Na + channel blockers (e.g., phenytoin, riluzole) showed neuroprotective activity in experimental spinal cord injury studies, in neurobehavioral studies and tissue recovery (Ates et al., 2007). It is important to mention that gabapentin, the first known drug in the treatment of neuropathic pain, specifically binds to the α2δ1 subunit of N-type Ca 2+ channels and decreases the current (Zhu et al., 2017).
N-methyld-aspartate receptors (NMDAR) are well known to be associated with psychiatric disorders (Lakhan, Caro, & Hadzimichalis, 2013). With no surprise, they were also linked to ASD risk (Lee, Choi, & Kim, 2015). Their activation follows the binding of glutamate once the D-serine or glycine co-agonists engage the specific allosteric site of the receptor (Kim et al., 2005). These two ligands were used in a clinical study on patients with severe schizophrenia as antipsychotic agents and were able to correct some negative clinical aspects (Buchanan et al., 2007). In 1991, Haring et al. characterized an antibody, named B6B21, which showed a remarkable action in rat neurons by increasing long-term potentiation in CA1 pyramidal cells. This antibody has a high binding affinity for NMDA receptors.
As a matter of fact, the authors concluded that B6B21 acts in a similar way to glycine on the receptor (Haring, Stanton, Scheideler, & Moskal, 1991). From B6B21, derived a family of small peptides called glyxines (Santini et al., 2014). One of these peptides, named GLYX-13, was found to modulate NMDAR properties in a similar way to glycine. Treating ASD-affected rats with of GLYX-13 resulted in promising improvements of autistic signs. Thereafter, authors suggested that this antibody might be a potential treatment for patients affected by ASD (Santini et al., 2014). Moreover, d-cycloserine, which is a partial NMDAR glycine agonist, is known to have effects on the behavioral deficits observed in autism and schizophrenia (Posey et al., 2004).
In a recent clinical trial carried out on 20 patients with autism, it has been shown that D-cycloserine treatment alleviated the stereotyped behavior of these patients (Urbano et al., 2014). To go more into details, the administration of D-cycloserine during 8 weeks with different dosages showed to be effective on ASD manifestations in these patients without showing any side effects (Urbano et al., 2014). Additional studies will be required to determine the therapeutic effect of this drug in ASD.
Concerning another therapeutic target, the implication of the acetylcholine receptor in ASD was demonstrated for the first time F I G U R E 1 Synaptic signaling pathways associated with autism spectrum disorder (ASD). Alterations in the mechanistic target of rapamycin complex (mTOR) are considered risk factors for ASD. mTOR is activated by Rheb-GTP. Upstream of Rheb is the tuberous sclerosis complex (TSC1-TSC2). TSC2 contains a GTPase-activating protein (GAP) domain that converts Rheb from GTP-bound form to its inactive GDP-bound form. Several upstream signaling pathways ranging from PI3K-AKT, Ras-ERK, LKB1-AMPK and Wnt-GSK3β pathways, positively or negatively regulate mTOR signaling. (AMPK, AMP-activated protein kinase; ERK, extracellular signal-regulated kinase; GSK3β, glycogen synthase kinase 3β; and PI3K, phosphoinositide 3-kinase). The mTOR pathway is also regulated by the brain-derived neurotrophic factor (BDNF) which binds to the tropomyosin-related kinase B (TRKB). BDNF plays a key role in the development and the plasticity of the central nervous system and it is considered a risk factor for ASD because increased levels of BDNF concentration have been observed in the serum and brain of patients with ASD. PI3K is also regulated by the synaptic protein SHANK, which is associated with metabotropic glutamate receptors type 1 (mGluR1) via the neuronal scaffolding protein HOMER1. The mTOR complex is a key modulator of protein synthesis by direct phosphorylation of 4E-binding proteins (4E-BPs) and activation of the ribosomal subunit S6 kinase (S6Ks), which in turn phosphorylate translation initiation factors. Thus, mTOR blocks the activation of cell autophagy and promotes cell proliferation, growth, and differentiation. The activity of the proteasome is also regulated by neuronal activity. The expression of UBE3A is increased through the transcription factor MEF2 and regulates the degradation of ARC protein, which promotes the internalization of AMPA-R and regulates excitatory synapse development. Variations in the neuronal L-type Ca 2+ channel α subunit CACNA1C have been associated with Timothy syndrome and with ASD. In addition, Ca 2+ /calmodulin-dependent protein kinases are associated with components of the neuronal complex including the fragile X mental retardation protein (FMRP) and its protein interaction CYFIP1, which also consider candidate genes in ASD. UBE3A: ubiquitin-protein ligase E3A; MEF2: myocyte-specific enhancer factor 2; ARC: activity-regulated cytoskeleton-associated protein; AMPR: AMPA receptors; CYFIP1: cytoplasmic FMRP-interacting protein 1  Pandya, & Yakel, 2015). Due to their role and implication in several pathways (e.g., PI3K/Akt and Wnt), α7 nAChR is considered as powerful therapeutic candidates (Deutsch, Burket, Urbano, & Benson, 2015).

| CON CLUS IONS
A number of genetic studies came to classify autism as the most genetically complex disease. However, only a few numbers of contributing alleles or co-inherited alleles are found in ASD proving that additional epigenetic factors or environmental conditions may contribute to the clinical manifestation of this disorder.
The several Mendelian pathologies associated with autism, for example, fragile X syndrome, provide the strongest argument highlighting the genetic basis of autism. These ASD-associated pathologies are commonly confounded with autistic behaviors and therefore make it more difficult to carry out case studies that focus exclusively on ASD. The most promising genes identified so far include NLGN, SHANK, and SYNGAP1, which are involved in neurogenesis and synaptogenesis, suggesting that synaptic malfunction is a significant contributor to the etiology of ASD.
Among ASD-associated pathologies, Timothy syndrome linked channelopathies to ASD. In fact, recent studies implicate variations and mutations of genes encoding ion channels (Ca 2+ , K + , Na + , and Cl − channels) as a leading risk factor for ASD. Alterations of these channels highlight the complexity of the pathology that remains not fully understood. Interestingly, the wide implication of ion channels encoding genes in ASD may provide opportunities for pharmacological treatments of autistic patients because these channels represent powerful drug target. For instance, bumetanide is one of these potential therapeutic agents currently under evaluation. Some of ASD pathological conditions show an increase or decrease in ion channel activation/deactivation kinetics, suggesting that ion channel modulators may be therapeutic candidates for the treatment of ASD.
In conclusion, ASD is not a simple pathology and it is associated with a large spectrum of other diseases. Nevertheless, the genetic abnormalities so far indicate that defective neuronal function from the onset of neural development appears to be a leading cause in the manifestation of this syndrome.
Furthermore, defective regulation of ion flux through the cell membrane caused by altered kinetics of ion channels and transporters appears to cause an imbalance of excitation/inhibition in neural function that may lead to defective neuronal circuit formation and physiological response. Restoring ion dynamics to their physiological equilibrium may represent a promising therapeutic strategy for this devastating neurodevelopmental psychiatric disorder.

ACK N OWLED G M ENT
There are no acknowledgments to declare.

CO N FLI C T O F I NTE R E S T
All authors state that they have no conflict of interests.