环境污染物协同易感基因增加自闭症风险的内在分子机制

The prevelance of autism specturm disorders (ASD) has been reported to increase dramatically in the past several decades. Though genetic factors such as gene mutations and copy number variants have been considered as main underlying causes for ASD, these factors alone can only explain about 20% of syndromic and non-syndromic ASD. Recent research findings indicated that environmental factors (e.g., pollutants) might also play an important role in the etiology of ASD. Currently, it is unknown whether environmental and genetic factors will interact with each other and play a causative role in ASD outbreak. Given the fact that mid-pregnancy, corresponding to the neural progenitor proliferation phase of fetal brain, was one of the most sensitive stages for ASD induction in experimental models, yet aberrant cell cycle play a causative role in many neural disorder related diseases, we thus hypothesize that the increased ASD incidence could result from a combined risk of environmental pollutants and susceptible genes that was mediated through aberrant cell cycle of neural progenitor cells. To test our hypothesis, we propose to use the embryonic zebrafish model, which allows mimicking the fetal exposure scenario as in humans while simultaneously provides an easy platform for experimental manipulation and in vitro observation. We will also take the advantage of genetic manipulation tools such as microinjection of morpholino or mRNA into single cell stage zebrafish embryos to down or up regulate targeting susceptible genes. Effects of environmental pollutants on ASD development in zebrafish will be performed through waterborne exposure of environmental relevant doses at early developmental stages. The specific objectives of our proposal are to 1) observe whether environmental pollutants in coupled with susceptible genes will pay a synergic role in ASD induction; 2) identify early targets of environmental pollutants and/or susceptible genes induced ASD; and 3) explore the molecular mechanism for combined risk of ASD from environmental pollutants and susceptible genes. The findings of our research will not only reveal the underlying molecular mechanisms responsible for environmental pollutants and/or susceptible genes induced ASD, but also help unravel the underlying causes for increased prevalence, pathogenic complexity, and clinical heterogeneity associated with ASD.

自闭症是一种神经发育紊乱疾病，尽管遗传因素被认为是其主因，但仅从基因变异很难解释该病近年来为何呈现出爆炸式的增长。一些最新研究表明环境污染可能是诱发自闭症的另一主因，但其是否与基因变异协同作用而增加自闭症的风险尚属未知。鉴于自闭症的敏感窗口正好是神经前体细胞的增殖期，而细胞周期异常又是神经系统疾病的发病机制之一，我们提出环境污染物可能协同易感基因变异导致神经前体细胞分裂周期异常而增加自闭症发病风险的假说。本项目将以斑马鱼胚胎为研究材料，通过显微注射、基因人为调控、污染物水体暴露、自闭症症状综合评价、深度测序及其分析等方法来：1）察看环境风险污染物和易感基因是否在引发自闭症上存在叠加效应；2）寻找环境污染物和易感基因导致自闭症的早期作用靶细胞；3）探索环境污染物和易感基因引发自闭症的相同的或不同的内在分子机制。研究结果可以用来诠释环境污染物是否会协同易感基因增加自闭症的发病风险及其内在机制。

自闭症是一种神经发育紊乱疾病，环境污染可诱发自闭症发生。鉴于自闭症的敏感窗口正好是神经前体细胞的增殖期，而细胞周期异常又是神经系统疾病的发病机制之一，我们提出环境污染物可能协同易感基因变异导致神经前体细胞分裂周期异常而增加自闭症发病风险的假说。以斑马鱼胚胎为研究材料，通过显微注射、基因人为调控、污染物水体暴露、自闭症症状综合评价、深度测序及其分析等方法来环境污染物是否会协同易感基因增加自闭症的发病风险及其内在机制。本课题主要有以下六个方面的研究成果，①以模式生物斑马鱼为实验动物研究九种环境污染物短期暴露对胚胎早期发育的神经毒性和ASD行为效应。发现，BPA和TBBPA等具有较高的自闭症风险。②VPA暴露斑马鱼可产生ASD样表型，包括仔鱼大头表型，胚胎/幼虫运动行为异常活跃，以及ASD样社会行为增加。同时脑区细胞增殖、新生成熟神经元比例、神经干细胞增殖均增加，可能是ASD发生的原因。③BPA与kctd13或者chd8下调均可以协同引起受试仔鱼头部变大，且引起ASD样的神经行为变化，头部细胞增生增加，新生神经元比例增加和神经干细胞数量增加。④BPA慢性暴露可引起斑马鱼神经毒性和自闭症较高发生风险，主要包括自闭症样神经行为学改变、头部尺寸变大、神经细胞表达异常及自闭症易感基因表达改变等。脑部ASD基因chd8表达下调，调激活了wnt信号通路，上调下游mag、mpz、lamb1a、mmp13和mmp9基因表达。⑤BPA类似物BPF和BPS亚急性暴露增加斑马鱼自闭症风险，引起受试仔鱼头部尺寸变大；自主运动和仔鱼行为运动改变；增加细胞增生，新生成熟神经元和增殖神经干细胞的表达；上调钾离子通道和钙离子通道的基因。⑥使用CRISPR-Cas9基因编辑技术构建筛选出ASD基因KCTD13突变体。与野生型斑马鱼相比，KCTD13斑马鱼突变体24hpf自主运动频率降低、120hpf运动活力降低、暗光环境滞留时间增加、增加鱼群分散、两次间歇性闪光敏感度上升、镜面攻击次数减少、社会交往次数减少等。这些特点符合自闭症行为学特征。这些研究结果可以用来诠释环境污染物是否会协同易感基因增加自闭症的发病风险及其内在机制。