Nck-associated protein 1, also known as Nap1, is a component of the WAVE1/2 complex that interacts with activated Rac1 [202]. NCKAP1 is located on chromosome 2q32.1. A de novo LGD mutation of NCKAP1 has been identified in ASD probands [203]. Subsequently, several de novo or maternally inherited mutations in NCKAP1 are found in multiple WES studies on ASD probands [95,161,204]. Recently, two studies on Chinese and Caucasian ASD cohorts found more de novo LGD mutations in NCKAP1, which suggests that NCKAP1 is a strong candidate gene for ASD [115,205]. Nap1 does not contain any known functional motif [206] (Figure 2C). Human Nap1 is extensively expressed in multiple tissues except peripheral blood leukocytes, with highest expression detected in the brain, heart, and skeletal muscle [207]. The expression of human Nap1 is ubiquitously observed in all brain regions, with relatively higher levels in the cerebellum, hippocampus, and amygdala [207]. A study reported that human Nap1 is preferentially expressed in neuronal cells and may participate in neuronal apoptotic pathway [207]. Immunoblots from different embryonic ages indicate a pattern of developmentally increased Nap1 expression in the mouse cerebral cortex [208]. Yokota and colleagues found that knockdown of endogenous Nap1 leads to defective neuronal differentiation in mouse cortical neurons [208]. They then generated a Nap1 mutant mouse line in which the N-terminal 898 aa of Nap1 fused with a 1291 aa β-geo reporter is expressed instead of the full-length protein [208]. However, these Nap1 mutant mice are embryonic lethal from E8.5–E10.5 due to neural tube and neuronal differentiation defects [208]. Therefore, mouse models of Nap1 deficiency are still absent for the investigation of Nap1 on regulating neural behaviors.

Cytoplasmic FMR1 interacting protein 1 (Cyfip1), also named Shyc and Sra1, is a Rac1-interacting protein and a partner of the WAVE complex that regulates actin filament. CYFIP1 is located on chromosome 15q11.2. Several studies have reported that patients with 15q11.2 microdeletions and microduplications between breakpoints 1 and 2, which encompass several genes including CYFIP1, are diagnosed with neurodevelopmental disorders including ASD, ID, SCZ, attention-deficit/hyperactivity disorder (ADHD), and obsessive-compulsive disorder (OCD) [209,210,211,212]. A paternally inherited rare variant of CYFIP1 is found in an autistic patient with a de novo SHANK2 deletion [173]. Another paternally inherited rare variant of CYFIP1 is found in a study on high functioning ASD patients [213]. Additionally, SNPs in CYFIP1 were reported to correlate with ASD in two studies [214,215]. A study on CYFIP1 mRNA expression in human dorsolateral prefrontal cortex revealed higher expression of CYFIP1 mRNA in ASD and classical autism patients, and identified several common variants of CYFIP1 in these patients [216]. A recent study also demonstrated significant increase in CYFIP1 transcripts in the peripheral blood of ASD patients [217]. All these clinical findings reveal that altered CYFIP1 dosage may contribute to the pathology of ASD. Cyfip1 has been shown to interact with Rac1, Fragile X mental retardation 1 (FMRP), and eukaryotic translation initiation factor 4E (EIF4E) [218,219] (Figure 2C). Cyfip1 is widely expressed in multiple tissues but not liver during development and is highly enriched in the hippocampus, cerebral cortex, cerebellum, olfactory bulb, and lateral septum in the adult brain [220]. Levels of Cyfip1 are high in the cortex and cerebellum at the stages of postnatal development, peaked at P23 and slightly decreased afterwards [221].

Four different strategies have been used for generation of Cyfip1 KO mice. However, because Cyfip1 is very important for early embryonic development, none produce homozygous Cyfip1 KO mice. Therefore, Cyfip1 HET mice are used for studying behavioral and neural phenotypes in these studies. The first mouse model was generated by mutagenesis with a gene trap vector inserted into intron 1 of Cyfip1. There are no Cyfip1 KO embryos in breeding [83]. Cyfip1 HET mice display normal learning and memory abilities in several memory tests, but show more rapid extinction in inhibitory avoidance test, a test for hippocampus-dependent memory [83] (Table 2; Supplementary Table S1). These results indicate that the accuracy of memory processing in HET mice is much poorer. Cyfip1 HET mice show increased mGluR-dependent LTD and abnormal presynaptic function in hippocampal slices [83,222]. The second one is generated by deleting exons 4–6 of Cyfip1. Inbreeded from Cyfip1^HET mice, fertilized Cyfip1 KO oocytes are detectable in blastocyst stage [223]. The KO embryos can survive until E8.5, but become lethal due to complete developmental failure [223]. The Cyfip1^HET mice show absence of interest for social cues and deficits in motor learning [84] (Table 2; Supplementary Table S1). The third one is generated by deleting exon 5 of Cyfip1, and the Cyfip1 homozygous KO embryos die before E9.5 [85]. Moreover, the difference between the maternal (m−/p+) and paternal (m+/p−) deficiency of Cyfip1 was investigated. Interestingly, Cyfip1 m−/p+ mice only display hypoactivity, whereas Cyfip1 m+/p− mice display increased freezing in cued fear conditioning and abnormal transitions in zero-maze test [85] (Table 2; Supplementary Table S1). Both Cyfip1 m-/p+ mice and Cyfip1 m+/p- mice show reduced field EPSC and increased PPF in hippocampal CA1 region, and enhanced mGluR-dependent LTD is observed in Cyfip1 m+/p- mice hippocampal CA1 neurons [85]. The fourth one is generated by inserting a gene trap cassette between exon 12 and 13 of Cyfip1. Cyfip1^+/- mice display impaired motor coordination, deficient sensory processing/novelty seeking behavior, reduced PPI, and decreased sensory motor gating [86] (Table 2; Supplementary Table S1), recapitulating some ASD and SCZ-like behavioral phenotypes. Cyfip1^+/- mice show reduced spontaneous neuronal activity and presynaptic function in cortical slices [86]. Besides the whole-body mutant mice, one Cyfip1 cKO mouse model (Cyfip1^NEX cKO mice) has been generated, in which exon 4–6 of Cyfip1 was deleted in forebrain excitatory neurons by NEX-Cre. These mice are viable until adulthood with no obvious abnormalities [224]. Cyfip1^NEX cKO mice show increased mIPSC amplitude in hippocampal CA1 neurons and display similar deficits in dendrite morphology and spine maturation to those described in Cyfip1 haploinsufficient models [224]. There is also a Cyfip1 mutant rat model generated by CRISPR/Cas9 technology, in which a 4 bp heterozygous deletion is introduced in exon 7 of Cyfip1, causing premature stop of the protein [87]. These Cyfip1 haploinsufficient rats exhibit normal learning ability during all behavioral tests, but show deficits in behavioral flexibility [87] (Table 2; Supplementary Table S1). As increased transcript levels of CYFIP1 is found in some ASD patients, two transgenic (Tg) Cyfip1 mouse lines (Tg line 1 and Tg line 2) were generated by overexpressing human CYFIP1. mRNA of human CYFIP1 was increased in the cortex and hippocampus of both Tg lines; Cyfip1 protein is also increased in the two brain regions of Tg line 1 and the hippocampus of Tg line 2, but not in the cortex of Tg line 2 [88]. Behaviorally, Tg line 2 mice display subtle defects of spatial learning memory and obvious increased fear response in contextual and cued fear conditioning test, whereas Tg line 1 mice show normal spatial learning memory and increased freezing only to the tone in the novel context during fear conditioning test [88] (Table 2; Supplementary Table S1). However, both Tg lines show no deficits in core ASD-related behaviors such as social and repetitive behaviors [88] (Table 2; Supplementary Table S1). Together, Cyfip1 deficient mice or rats recapitulate core features of ASD, whereas Cyfip1 Tg mice may not be appropriate ASD models.

As increased mGluR activation was caused by Cyfip1 deficiency, mGluR1 inhibitor LY367385 and mGluR5 antagonist MPEP (2-Methyl-6-(phenylethynyl) pyridine) were used together. Indeed, these mGluR blockers normalized the mGluR-LTD to control levels in hippocampal slices of Cyfip1 HET mice [83] (Table 3). However, the effect of these two inhibitors on behaviors has not been investigated. For non-pharmacological therapeutic approaches, motor training increased the number of newly formed dendritic spines in both WT and Cyfip1^HET mice, and the motor learning deficits in Cyfip1^HET mice can be alleviated by the behavioral training in early development but not in adult [84] (Table 3).

P21 activated kinase 2 (Pak2), activated by Rac1 and Cdc42, is a member of the group I PAK family that belongs to a family of serine/threonine kinases. PAK2 is located on chromosome 3q29, which is classified as one of six strong autism risk loci [225]. A study on six patients of 3q29 microdeletion syndrome identified a ~1.5 Mb microdeletion, which includes entire PAK2. Two of these patients displayed autistic features [226]. Another study on two patients of 3q29 microdeletion syndrome with common ID, a history of autism, and other psychiatric symptoms also reported the same length deletion [227]. In a subsequent study, 11 of 44 patients with 3q29 microdeletion syndrome were found to display diverse neurodevelopmental disorders including autism [228]. Moreover, one de novo copy-number deletion containing PAK2 was found in patients with ASD from the Simons Simplex Collection, and one de novo nonsense mutation and two inherited missense mutations were also found in PAK2 in 914 Han Chinese patients with ASD [89]. All these four studies reveal that PAK2 is a strong candidate for ASD. Pak2 has several recognized domains including two proline-rich regions and an AID (Autoinhibitory Domain) overlapping the PBD (p21-binding domain) in the N-terminal region, a kinase domain at the C-terminus, an acidic region, and a PIX (Pak-interacting exchange factor) binding site [229,230,231] (Figure 2C). Pak2 is ubiquitously expressed in multiple tissues [232]. Human PAK2 shows high expression levels in the brain during the fetal period and low levels after birth. Mouse Pak2 is also down-regulated at the postnatal development in the cortex [89]. Loss of Pak2 leads to embryonic lethality at ~E8 [232,233,234]. Therefore, a recent study used Pak2^+/- mice to investigate the behavioral and neural functional changes caused by Pak2 haploinsufficiency. This study revealed that Pak2^+/- mice display repetitive and stereotyped behaviors, impaired social interaction, social avoidance, reduced social preference index, and disruptive social memory [89] (Table 2; Supplementary Table S1). Pak2^+/- mice also exhibit decreased LTP in hippocampal CA1 region neurons [89]. Mechanistically, phosphorylation of both LIMK1, a major downstream target of group I PAKs, and its substrate cofilin were markedly decreased, suggesting abnormal LIMK1/cofilin-mediated actin polymerization in adult Pak2^+/- mice cortex [89]. To normalize endogenous p-cofilin levels in the cortex and hippocampus, a p-cofilin peptide was intravenously injected into adult Pak2^+/- mice as a substrate to compete with endogenous p-cofilin for phosphatases. As a result, this peptide moderately improved social behaviors but not repetitive behaviors in adult Pak2^+/- mice [89] (Table 3).

Inositol 1,4,5-trisphosphate receptor type 1, also known as IP₃R1, is a RhoA effector. IP₃R1 is a member of IP₃Rs, which are Ca²⁺ release channels on the endoplasmic reticulum. ITPR1 is located on chromosome 3p26.1. Three de novo missense variants in ITPR1 are found in ASD probands from WES studies [95,115,161]. In addition, a study on autism risk genes in probands from the Autism Clinical and Genetic Resources in China (ACGC) identified one maternally and one paternally inherited missense variant of ITPR1 [118], and another variant is found in patients with NDDs [235]. IP₃R1 has three main domains including a large N-terminal IP₃-binding domain, a short C-terminal hydrophobic domain, and an intervening regulatory domain [236] (Figure 2C). The expression of IP₃R1 is increased during embryogenesis [237]. Moreover, IP₃R1 is predominately expressed in the nervous system [238,239] and is expressed in a wide range of brain regions including the cerebellum, cerebral cortex, hippocampus, olfactory bulb, globus pallidus, and striatum [238]. It was reported that homozygous IP₃R1-deficient mice mostly die during the embryonic stage, and the born mice have severe ataxia and seizures and die at around P21 [240]. IP₃R1^+/- mice also show deficits in motor coordination [90] (Table 2; Supplementary Table S1). To study the neural function of IP₃R1, several IP₃R1 brain cKO mouse lines have been generated. A study reported that L7-Cre;Itpr1^flox/flox mice, in which Itpr1 is deleted in Purkinje cells, exhibit cerebellar ataxia at around 6 weeks and severe ataxia at 8 weeks after birth [91] (Table 2; Supplementary Table S1). L7-Cre;Itpr1^flox/flox mice could survive to adulthood, but display abnormal motor learning ability [91] (Table 2; Supplementary Table S1). In another study, three cKO lines were used, in which Itpr1 deletion was restricted to the cerebral cortex and hippocampus (Emx1-Cre;Itpr1^flox/flox mice), the cerebellum and brainstem (Wnt1-Cre;Itpr1^flox/flox mice), and the caudate putamen and globus pallidus (Gpr88-Cre;Itpr1^flox/flox mice) [92]. The Emx1-Cre;Itpr1^flox/flox mice and Gpr88-Cre;Itpr1^flox/flox mice were born normally and showed normal growth patterns until adulthood, without apparent dyskinesia like total Itpr1^-/- mice. However, Wnt1-Cre;Itpr1^flox/flox mice began to show ataxia at around P9, and exhibited dyskinesia from 2 weeks after birth [92] (Table 2; Supplementary Table S1). Wnt1-Cre;Itpr1^flox/flox mice show abnormal cerebellar Purkinje cell (PC) firing patterns, due to altered PC activity [92]. Itpr1 deficiency induces a series of abnormal electrophysiological features, including failed LTD in the PCs [241], and excessive LTP induction, attenuated depotentiation and LTP suppression, and altered presynaptic activity in hippocampal CA1 neurons [242,243]. However, typical ASD-related behaviors such as social and repetitive behaviors have not been investigated using these mice.

As enhanced PC activity and dystonia were observed in Wnt1-Cre;Itpr1^flox/flox mice, pharmacological inactivation of cerebellar activity by AMPA receptor antagonist (CNQX) infusion was examined for therapeutic effects. Indeed, CNQX ameliorates the dyskinesia in these mice [92] (Table 3). Furthermore, dystonic movements were completely absent in Wnt1-Cre;Itpr1^flox/flox mice with genetic deletion of cerebellar PCs, achieved by mating Wnt1-Cre;Itpr1^flox/flox mice with Lurcher mice (GluD2^LC/+) to kill most of PCs by a mutation of the delta 2 glutamate receptor (GluD2) [92] (Table 3).

Protein kinase C alpha (PKC-α) is a member of lipid-sensitive serine/threonine protein kinases that regulate various cellular functions including proliferation, migration, adhesion, differentiation, and apoptosis. PKC-α appears to be a common downstream effector of RhoA, Cdc42, Rac1. PRKCA is located on chromosome 17q24.2. In two WES studies, three de novo missense variants in PRKCA were reported in ASD probands [95,161]. PKC-α has a variable regulatory domain at the N-terminus consisting of a C1 domain and a C2 domain, which function as the binding sites of diacylglycerol (DAG) and Ca²⁺, respectively. PKC-α also has a highly conserved kinase domain at the C-terminus comprised by a smaller ATP-binding loop and a substrate-binding site [244,245] (Figure 2C). PKC-α is ubiquitously expressed in all tissues, including the heart, adrenal gland, testis, lung, kidney, spleen, and liver, and is also widespread in various regions of the brain [246]. PKC-α is enriched in both neuronal and glial cultured cells [246]. A PKC-α KO mouse line has been generated, which appears to be normal with regard to external characteristics, viability, and fertility [247]. However, ASD-related behavioral analysis of these mice has not been reported.

The Rac1 effector WAS protein family member 1 (WASF1), also known as WAVE-1/Scar1, is a member of the WASP-family. WASF1 is located on chromosome 6q21. Using exome sequencing and whole-genome sequencing, three de novo truncated mutations of WASF1 were reported in five unrelated individuals, all of whom presented ID with autistic features and seizures [248]. This is the only study so far that has reported the relationship between WASF1 and ASD. WAVE-1 is composed by a Scar homology domain, a basic domain, a proline-rich region, a WASF homology (WH) domain, and an acidic domain [249] (Figure 2C). In human tissues, the expression of WAVE-1 is restricted to the brain [250]. Mouse study reveals that WAVE-1 shows high expression in the hippocampus, cortex, hypothalamus, amygdala, and cerebellum [93,251,252]. Two mouse lines of homozygous Wave1 deletion are reported to be either postnatal lethal [251] or reduced in body size of offspring [93]. Behavioral analysis using Wave1 KO mice (WAVE-1 null mice) generated by the second strategy showed hypoactivity, impaired motor coordination and balance, reduced anxiety levels, and defected spatial, nonspatial, and emotional learning and memory in the mice [93] (Table 2; Supplementary Table S1).