IntroductionAutism spectrum disorder (ASD) is a neurological developmental disability that is characterized by impaired social communication and interaction as well as restricted, repetitive patterns of behavior, interests, or activities [1]. It has been clinically known that ASD is accompanied by other neurodevelopmental disorders such as intellectual disability or attention-deficit hyperactivity disorder (ADHD). Additionally, epilepsy and aggression are comorbidities in around 25% of ASD cases [2]. Currently, autism is receiving increased attention worldwide. The estimated prevalence of ASD among 8-year-old children in the US has risen from around 1.1% in 2008 to 2.3% in 2018 [3].Researchers have identified one hundred potential genes that are significantly associated with autism [4], yet these genetic mutations cannot fully explain the pathogenic characteristics of autism. Another layer of complexity in “epigenetics” may make a significant contribution to the etiology of autism. Epigenetic modifications on DNA or histones can influence the transcriptional activation and silencing of genes. With the ongoing advancements in whole-exome and whole-genome sequencing data from autistic patients, mutations/dysregulation in genes that regulate DNA methylation mechanisms have been found to be closely linked to the pathogenesis of autism. Genetic studies have identified novel mutations in DNA methyltransferases and readers (DNMT3A, MECP2, MBD5) in autism pathogenesis [5,6,7]. These mutations can lead to loss or gain of function in epigenetic modifier enzymes. These genetic findings from patients provide substantial molecular evidence in support of the hypothesis that epigenetic dysfunction contributes to autism etiology. The most well-known Rett syndrome gene, MECP2, as a methylation-dependent transcriptional repressor, participates in regulating the expression of Dnmt1, TDP-43, and CREB genes [8, 9]. The dosage balance of MECP2 is crucial for the development of autism. Overexpression of MECP2 can lead to abnormal increases in excitatory neuronal dendritic spines, which in turn causes an imbalance between neuronal excitation and inhibition in neural circuits, triggering autism-like behavior [10]. Studies on transgenic (TG) mice with MECP2 duplication syndrome demonstrate that the medial prefrontal cortex (mPFC) plays a significant regulatory role in the social deficits of MECP2 duplication syndrome [11]. Functional MRI shows excessive excitability in the mPFC of Mecp2 TG mice, and specific deletion of Mecp2 in the mPFC can alleviate the social deficits in mice [12].Drug-induced autism is also considered one of the major etiologies of autism. One of the major exposure risk factors for drug usage during pregnancy is antiseizure drugs, and a recent clinical study has confirmed that valproic acid, the most established drug, increased the incidence of autism in offspring, while other drugs have failed to establish the link [13]. VPA treatment in rats and mice can reproduce most symptoms of autism, including social deficiency, repetitive behaviors, and accompanied with seizures [14]. Interestingly, epigenetic dysregulation, including histone methylation/acylation dysregulation and DNA methylation abnormality, is recognized as the major underlying mechanism in these autism animal models. Importantly, VPA induced reduced MECP2 expression through miR-132-BDNF regulatory loop during E12.5 day treatment [15], and the mechanistic study showed that convergence of VPA induced alteration with major epigenetic regulators, including MECP2. However, the hub gene and the downstream pathway regulated by both MECP2 and VPA remain elusive, limiting the targeted intervention based on the pathway affected by these two most established monogenic and environmentally induced autism models.GADD45A is a member of the GADD45 gene family, whose transcription levels increase under conditions of stress-induced growth arrest and treatment with DNA-damaging agents, responding to environmental stress. Recent research has found that in cell lines, the binding of GADD45A to DNA-RNA hybrids (R-loops) triggers the recruitment of TET1, mediating the demethylation of the tumor suppressor TCF21 promoter [16]. Genome analysis in embryonic stem cells has identified thousands of R-loop-dependent TET1 binding sites located on CpG islands, suggesting an important role in the regulation of methylation levels during neural development. Gadd45b, as a member of the same gene family as Gadd45a, has similar biological functions. Previous Studies, including our team, have found that the knockdown of Gadd45b leads to changes in adolescent social behavior and reduced expression of several genes associated with psychiatric disorders, including MECP2 and BDNF [17, 18]. These data suggest that Gadd45b plays an important role in the epigenetic regulation of complex social behavior, to some extent indicating the association of epigenetic-related genes with autism. A recent study found that the drug intervening Gadd45a-related pathway could rescue autism-like behaviors in BTBR mice [19]. However, whether dysregulation of GADD45A, the first reported member of the GADD45 family, is directly involved in the etiology of neurodevelopmental diseases, such as autism and epilepsy, and the underlying mechanism, remains unclear.In the current study, for the first time, through transcriptome data from the VPA-treated animal models and MECP2 mutant models, we have identified that GADD45A is a hub gene dysregulated in both the VPA-induced autism model and MECP2-related animal models. We have shown that deletion of Gadd45a leads to autistic traits and accompanying symptoms in the mouse model, including epilepsy phenotype. GADD45A is preferentially expressed in the excitatory neurons, and social deficits can be reversed when the GADD45A level is rescued in the excitatory neurons of mPFC. Through transcriptome and ChIP-Sequencing studies, we have also shown that GADD45A could recruit TET1 to regulate the promoter region in the downstream genes, specifically enriched on the ion channels. Finally, we have confirmed that KCNQ5 is a critical target regulated by GADD45A through an R-loop-dependent pathway, which is essential for maintaining the normal function of excitatory neurons. Therefore, our study uncovered a critical role of GADD45A/TET1-KCNQ5 methylation and R-loop dependent regulatory axis, which may represent a key molecular pathway mediating the pathogenesis of neurodevelopmental disease, including autism and epilepsy, among others.ResultsGADD45A functions as a potential downstream effector gene of the etiology of autism in multiple autism modelsCurrent understanding of the etiology of autism includes well-known factors such as prenatal exposure to VPA leading to autism in offspring [13], along with mutations in the MECP2 gene [20], and so on. Intriguingly, there were studies reporting that VPA exposure can suppress the expression of MECP2 [21,22,23]. In addition, we validated their conclusion by treating SH-SY5Y cells with VPA. We found that VPA treatment could attenuate the mRNA expression of MECP2 in vitro (Fig. 1A). This evidence indicated that there appears to be a relationship in the pathogenesis of autism between VPA-induced autism and MECP2-deficient autism, suggesting the potential shared downstream pathogenic molecular mechanisms. Therefore, in the GEO database, we collectively analyzed differential gene sets (GSE129241) in reprogrammed human neurons on post-induction day 1 treated with valproic acid to simulate the impact of VPA on early neural development, and differential gene sets (GSE230714) in reprogrammed human neurons of MECP2 knockout. We identified 385 genes that were commonly dysregulated between the two datasets. Moreover, the dosage balance of MECP2 is crucial for the development of autism. The gain-of-function of MECP2 represents the clinical causes of the MECP2 duplication patients. We analyzed the transcriptome data of transgenic MECP2 monkeys (GSE57974) and mice (GSE123372) in public databases, and identified 28 genes that were conserved and altered in the MECP2 overexpression models by intersecting the two different datasets. To identify the potential common pathogenic factors underlying seemingly different yet interconnected autism models, including the VPA-induced autism model and the autism model resulting from MECP2 dosage abnormalities, we reanalyzed the intersection genes from the previous two intersection sets and ultimately identified three shared differentially expressed candidate genes (Fig. 1B). Among them, GADD45A was of particular interest, as it has been reported to be epigenetically repressed by MECP2 in prostate cancer cell lines. [24]. Notably, GADD45A was the only gene that exhibited consistent downregulated alterations in both MECP2 transgenic monkey and mouse models compared with wild-type controls. In addition, dysregulation of GADD45B within the GADD45 family has been previously associated with autism-like behaviors [17]. Moreover, GADD45A expression was reported to be induced by valproic acid in vitro [25]. And our own data confirmed that VPA treatment could result in a time-dependent increase of GADD45A expression in SH-SY5Y cells (Fig. 1C). And we further utilized Western Blot to detect Mecp2 KO cortex in mice and found knockout of Mecp2 led to an increase of GADD45A expression (Fig. S1A), consistent with the result of GSE230714 in MECP2 knockout human neurons. Conversely, MECP2 duplication caused decreased expression of GADD45A in both transgenic monkey and mouse models, showing the opposite effect on GADD45A expression to that observed in MECP2 knockout and VPA exposure. Considering that MECP2 duplication syndrome (MDS) occurs predominantly in male patients [26], we next asked whether male autism patients display a similar dysregulated pattern of GADD45A. We recruited two independent cohorts of male children. Cohort 1 consisted of 9 children with ASD and 9 typically developing controls, and Cohort 2 included 7 children with ASD and 8 typically developing controls. Quantitative PCR analysis of peripheral blood samples revealed that GADD45A expression levels were significantly decreased in ASD patients in both cohorts (Fig. 1D, S1B). In Cohort 1, where multiple autism assessment scales (ABC, SRS, RBS-R, CBCL) were administered, we further examined the association between GADD45A expression and autism-related behavioral phenotypes. We found that two (ABC, SRS) of these scores were statistically negatively correlated with the relative expression levels of GADD45A, of which more SRS reflecting the more severity of social deficits in the autism spectrum (Fig. 1E, F, S1C, S1D). These results indicated that GADD45A dysregulation was closely relevant to the etiology of autism, at least in the social deficits module. In summary, through transcriptome mining in public databases, validation with our own patient samples, mouse samples, and cell experiments, as well as literature research, our findings suggested that GADD45A dysregulation is closely associated with autism-related molecular and behavioral phenotypes.Fig. 1: GADD45A dysregulation is associated with autism in multiple models and male patients.A The RT-qPCR detected MECP2 mRNA expression of SH-SY5Y treated with 1 mM VPA after 0 h, 24 h, 48 h, 72 h, 96 h. Three individual biological replications per group. One-way ANOVA followed by Dunnett’s multiple-comparison test. B The Venn diagram in the upper left panel shows the intersection of the differential gene sets of MECP2 knockout and VPA-treated neurons. The Venn diagram in the upper right panel shows the intersection of differentially expressed gene sets between MECP2 transgenic monkeys and Mecp2 transgenic mice in the cortex. The Venn diagram below shows the intersection of the common differentially expressed genes from the two groups above to identify shared differentially expressed genes among multiple autism models. Whether the affected gene is upregulated or downregulated compared with control in each dataset is annotated by using arrows (↑/↓) and consistent color coding (red for upregulation, blue for downregulation). C The RT-qPCR detected GADD45A mRNA expression of SH-SY5Y treated with 1 mM VPA after 0 h, 24 h, 48 h, 72 h, and 96 h. Three individual biological replications per group. One-way ANOVA followed by Dunnett’s multiple-comparison test. D The RT-qPCR results manifest that the GADD45A mRNA level in autistic boys (Cohort 1) decreased compared with the control group using mRNA from peripheral blood. n = 9, each group. Unpaired Student’s t-test. E, F Both the scores of the Aberrant Behavior Checklist Scores (ABC) and Social Responsiveness Scale Scores (SRS) in all participants were utilized to analyze the correlation with GADD45A gene expression. Spearman’s rho (rs) and p values presented in the figures were calculated by Spearman’s rank correlation coefficient. *p