| Autism, a pervasive neurodevelopmental disorders, is belonged to one of autism spetrum diseases (ASDs) and characterized by abnormal social interactions, deficits in the communication and ritualistic-repetitive behaviors. Onsets of the typical clinical symptoms occur in infancy and are fully presented by age 3. Quite a number of autistic patients have a characteristic of brain overgrowth or macrocephaly in 2 to 5-year-old, compared with normal children. The exact etiological factors of autism are still unclear, and both the genetic and environmental are usually proposed to contribute to autism, for example, the prenatal exposure of valproate greatly increases susceptibility to ASDs in the offspring.Wnts proteins are very important signal molecules in the regulating of neurodevelopment. In the Wnt signaling pathway,β-catenin is generally phosphorylated in GSK-3β, Axin and adenomatous polyposis coli (APC) complex, thus entering the ubiquitin/proteasome degradation pathway. Wnts, together with Frizzled and LRP 5/6 forming a trimeric complex, activates the intracellular disheveled (Dvl). Activated Dvl inhibits GSK-3p and then leads to stabilization, accumulation and further translocation ofβ-catenin into nucleus to activate, together with TCF/LEF, the transcription of the target genes, resulting in series of biological consequences. Previous studies showed that a number of proteins involved in the Wnt signaling pathway demonstrated related phenotypes. The notion of wnt2 as an autism susceptibility gene was supported by screening wnt2 coding sequence for mutations in a large number of autistic probands; the dvl-1 knockout mouse displayed social interaction and sensorimotor gating abnormalities. Therefore, the constellation of Wnt pathway relative genes may contribute to a broad array of psychiatric and behavioral syndromes such as autism, schizophrenia and depressive disorder. It has been proposed that abnormal epigenetic modification is an underlying mechanism in the autism pathogenesis. DNA methylation, an important epigenetic modification, together with other epigenetic and genetic actions determines the levels of gene expression. Generally, methylation makes gene silent, while demethylation activates gene expression. Environmental factors may interact with genetic susceptibility through epigenetic mechanism such as DNA methylation modification to change gene expression to affect the brain development, increasing the likelihood of ASDs.Whether dysregulation of Wnt/β-catenin pathway and abnormal epigenetic modification coexist in the brain regions of autistic patients? What is the relationship between them? These problems remain still unknown. In this study, we focused on the relationship of the Wnt/β-catenin pathway related genes methylation modifications with the pathway activity in the autistic animal model and primary cultured neurons, hoping to explore the possible pathomechanism of autism. The works had been done as follows:In the first part of this present study, a kind of autistic animal model was obtained in the offspring of the female Wistar rat that received a single intraperitoneal injection of VPA at the 12.5th pregnancy day, and then the behavioral and morphological tests were performed. The results demonstrated that, compared to the control rats, the autistic ones had abnormal developmental brain, delayed timing of eye opening, lower geotaxis function, pain threshold, lower swimming performance, enhanced ritualistic-repetitive behaviors, lower memorial ability and lower spatial exploratory ability, which were similar to the symptoms in autistic patients. The results from this part showed that the rat model of autism was successfully established.In the second part, in order to test the methylation modifications of Wnt/β-catenin pathway related genes, methylation specific PCR (MSP) and bisulfite DNA sequencing (BDS) were used to investigate the methylation patterns in the promoter regions of wnt1, wnt2, WIF-1 and DKK1 in the prefrontal cortexes (PFC) and hippocampi (HC) of autistic rats and the primary cultured neurons exposed to VPA. MSP showed partial methylation in the promoter regions in both the VPA-treated and control groups. The changes of demethylation level were detectable specifically in the promoter regions of wntl and wnt2; the demethylation level was significantly higher in the VPA-exposed than the controls (P<0.001). However, no significant changes of demethylation level in the promoter regions of WIF-1 and DKK1 were seen in the VPA-exposed rats, compared with the controls (P> 0.05). Intriguingly, demethylation appeared only in the promoter regions of wntl and wnt2 upregulating canonical Wnt pathway, but not in those of WIF-1 and DKK1 inhibiting canonical Wnt pathway. After MSP assessments, to confirm the changes of wntl and wnt2, we further investigated the methylation status using bisulfite DNA sequencing, which could reveal the details of 26 CpG sites in the 395-bp fragment ranging from-723 to-328 (the transcription start site ATG defined as+1) in the promoter region of wntl as well as of 21 CpG sites in the 250-bp fragment from-400 to-150 in the promoter region of wnt2. From the results consistent with MSP ones, we found that the CpG islands were densely demethylated in both wntl and wnt2 in the prefrontal cortexes and hippocampi of the VPA-exposed rats. The ratio analysis of the unmethylated CpG sites from five independent experiments performed in duplicate indicated that the changed methylation status levels reached statistical significance (P < 0.01-0.001). Western blot showed that VPA had no effects on the protein expression of DNMT3b, an important epigenetic regulating molecule. From the above results, it is demonstrated that VPA induces demethylation modifications in the promoter regions of specific Wnt/β-catenin pathway related genes.In the third part, in order to study the effects of demethylation modifications on the activity of Wnt/β-catenin pathway, real-time quantitative RT-PCR, western blot and immunoflourescence were used to investigate the activity of Wnt/β-catenin pathway and its effects on neuronal growth. The results from quantitative real-time RT-PCR and western blot detection showed that the mRNA and protein expressions of both wntl and wnt2 were significantly increased in the PFC and HC of autistic rats and the VPA-exposed cultured neurons, compared to the controls. Furthermore, the mRNA and protein expression levels of both wntl and wnt2 were dose-dependent and directly correlated with the relative demethylation level in the promoter region (wntl: R2= 0.9621, P< 0.01; wnt2:R2= 0.7805, P< 0.05).β-Catenin, the key intracellular molecular effector, and the targeted genes of Wnt/β-catenin pathway were also enhanced in the autistic rats and the VPA-exposed cultured neurons, symbolizing the upregulation of Wnt/β-catenin pathway activity. VPA promoted the neurite morphological complexity, manifesting as significant increment of number of neurite branches and total neurite length in comparison with the controls. The promotion of neurite complexity induced by VPA could be suppressed by the inhibitor of Wnt/β-catenin pathway. These results demonstrated that VPA induced the upregulation of Wnt/β-catenin pathway through DNA demethylation on specific genes, and further promote the neuronal growth.In conclusion, we demonstrated that enviromental factors such as VPA induced methylation modifications on the specific genes of Wnt/β-catenin pathway and concomitant overexpressions of both mRNA and protein, thus resulting in the upregulation of Wnt/β-catenin pathway. The upregulation of Wnt/β-catenin pathway promotes neuronal growth and enlarge the brain, which affect the neuronal development and connections, resulting in behavioral, emotional and linguistical abnormality and ending with autism. Our study suggests an epigenetic action via which enviromental factors, when exposed in early pregnancy, could induce dysregulation of signaling pathway, further facilitating susceptibility to ASDs.
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