【作者】 王中平；

【导师】 李瑞锡； 彭裕文；

【作者基本信息】 复旦大学 ， 人体解剖与组织胚胎学， 2010， 博士

【摘要】 自闭症(autism)是一种广泛性神经发育障碍性疾病,一般于3岁前发病,主要表现出语言交流艰难、社会交往障碍、重复刻板行为三大核心临床症状。此外,autism患者多有个显著头部发育特征,即2-5岁时脑过度增长,大于同龄健康儿童。目前,autism的发病原因尚不清楚,普遍认为是遗传因素和环境因素共同作用的结果,如妊娠早期丙戊酸盐(Valproate, VPA)暴露会大大增加子代罹患autism的风险。Wnts蛋白是一类参与神经系统发育的重要信号分子,在经典Wnt信号通路中,Wnts蛋白与细胞表面卷曲蛋白受体(Frizzled, Frz)结合而激活下游的散乱蛋白(Dishevelled, Dvl),随后抑制GSK-3β、Axin和APC等分子复合体的形成,从而减少胞浆内β-catenin的降解。β-catenin蓄积并进入细胞核内,与TCF/LEF转录因子结合、诱导相关基因转录,产生一系列生物学效应。据报道,多种神经精神性疾病,如autism、精神分裂和抑郁症等,都可能与Wnt信号通路异常有关。有资料显示wnt2基因的突变与autism的发病有显著的相关性；动物实验表明,敲除了Wnt通路中dvl-1基因的小鼠表现出类似autism的社会行为障碍,也强烈提示Wnt信号通路与autism的发病有关。研究表明,表观遗传机制参与了autism的发病。DNA甲基化修饰是一种重要的表观遗传现象,是后天性的基因修饰过程,是基因对外界环境因素发生应答的重要机制,一般的规律是：甲基化可抑制基因的表达,而去甲基化则可表现出基因活跃表达。在一定的遗传易感性基础上,环境因素通过改变表观遗传信息如基因组甲基化模式而影响特定基因的表达,进而影响脑的发育,导致autism的发生。那么,autism患者脑内是否存在Wnt信号通路及表观遗传修饰异常?Wnt信号通路活性变化与表观遗传机制异常是否存在联系?目前尚不清楚。针对这这些问题,本课题集中研究autism动物模型及培养神经元中Wnt/β-catenin通路相关基因的甲基化修饰及其与该通路活性变化的关系,以探讨autism发病的相关分子机制。我们主要进行了以下三方面的工作：一、采用Wistar大鼠,在妊娠第12.5天时,腹腔注射(intraperitoneal injecting, ip) VPA制作子代autism大鼠模型,并运用热板致痛法、倾斜板实验、Morris水迷宫及Nissl染色等方法对模型大鼠进行行为和形态学检测。结果显示,与正常对照组比较,autism模型组大鼠脑发育异常、睁眼时间推迟、方向趋向性机能及伤害性知觉下降、游泳能力差、重复刻板动作增加、学习记忆和空间探索能力差。这些表现与人类autism患者的某些特征极为相似,表明用VPA注射法较成功地制作出了autism大鼠模型。二、Wnt/β-catenin信号通路相关基因甲基化修饰的研究。运用甲基化特异性PCR (Methylation specific PCR, MSP)和硫转化DNA测序技术(Bisulfite DNA sequencing, BDS)检测VPA模型大鼠额叶皮层和海马脑区及培养神经元Wnt通路信号蛋白Wnt1、Wnt2、WIF-1及DKK1等相关基因启动区的甲基化修饰。MSP结果显示,在VPA模型大鼠额叶皮层和海马脑区及VPA处理培养神经元中,Wnt通路刺激性信号蛋白Wnt1和Wnt2基因启动子区去甲基化水平显著高于对照组(P<0.001),Wnt通路抑制性信号蛋白WIF-1和DKK1基因启动子区去甲基化水平与对照组比较未见显著性差异(P>0.05)。BDS结果显示,wnt1基因启动子区从-723到-328共395bp的片断所含26 CpG双核苷酸中,VPA autism模型组大鼠额叶皮层和海马组织的去甲基化数目显著高于对照组(P< 0.01-0.001); wnt2基因启动子区从-400到-150共250bp的片断所含21 CpG双核苷酸中,VPA autism模型组大鼠额叶皮层和海马组织的去甲基化数目显著高于对照组(P< 0.01), BDS与MSP的结果相一致。Western blot显示,VPA处理对重要表观遗传修饰因子DNMT3b (DNA methyltransferase 3b)的表达无影响。上述结果表明VPA处理具有诱导Wnt/β-catenin信号通路相关基因的去甲基化作用。三、为揭示Wnt/β-catenin通路相关基因甲基化修饰对该通路活性的影响,应用实时定量PCR, Western blot及免疫荧光染色技术深入研究autism模型大鼠及原代培养神经元Wnt/β-catenin信号通路的活性变化,以及其对神经元生长的作用。实时定量PCR及Western blot结果显示,在VPA模型大鼠额叶皮层和海马脑区及VPA处理培养神经元中,wnt1和wnt2 mRNA及蛋白表达显著高于对照组,并且与VPA剂量呈现正比关系,与启动子区甲基化水平呈现正相关关系(wnt1:R2=0.9621, P< 0.01; wnt2:R2= 0.7805, P<0.05)。免疫荧光组织化学、Western blot及实时定量PCR显示,Wnt/p-catenin通路效应分子β-catenin及其靶基因在VPA自闭模型组及神经元中含量升高,表明Wnt/β-catenin通路活性上调；VPA处理促进神经元生长,表现为突起数目和总长度显著高于对照组；而Wnt/β-catenin通路抑制剂能显著抑制VPA诱导的神经元生长。以上结果表明,Wnt/β-catenin通路相关基因甲基化修饰导致Wnt/β-catenin通路活性上调,VPA促进神经元生长可能由于该通路上调所致。综上所述,我们的工作提示：环境因素(如VPA暴露)通过表观遗传效应诱导脑组织Wnt/β-catenin通路相关基因甲基化修饰改变,使脑内Wnt信号增强,引起神经元过快生长,患者脑组织体积增加,影响神经元正常发育和联系,阻碍了正常的神经网络形成,从而引起社会交往、情绪和语言等行为方面的异常,最终导致autism的发病。更多还原

【Abstract】 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.更多还原