SHANK3基因表达下调对神经干细胞发育的功能基因组学影响

The SHANK3 gene encodes one of the most important components in the synaptic complex. Genetic variants in SHANK3 have been strongly associated with the pathogenesis and neuropathology of autism spectrum disorders(ASDs), but the mechanism underlying this association has not been fully investigated, due to the lack of appropriate disease models for ASDs. We previously developed a neural stem cell based model to knockdown NRXN1 and NLGN4X and investigate the functional effects by examining cellular and molecular phenotypes. In the current proposal, we will develop functional genomics strategy by combining expertise on bioinformatics, genomics and neurobiology: 1) to model the cellular phenotypes of SHANK3 gene knockdown in neural stem cells derived from human induced Pluripotent Stem Cells (hiPSCs) by RNA interfere techniques.2) to investigate the disturbed pattern of gene expression network and protein network in the development time-course using RNA-Seq techniques. 3) to explore the mechanism of molecular pathophysiology from upstream (single gene variant) to downstream (cellular phenotypes) from the perspective of genomics. The combination of high-throughput genomics (next-generation sequencing) and the use of neural stem cell models could contribute to the process of modeling neurodevelopment in a high-throughput fashion. Successful completion of the study will enable exploration of the molecular mechanism of cellular phenotype abnormalities resulting from SHANK3 dysfunction, and contribute to improving related disease research.

SHANK3是突触前后复合体的核心物质和功能基础，SHANK3基因变异被确认为自闭症谱系障碍等神经精神疾病易感因素，但该基因变异增强相关疾病易感性的分子机制尚不明确。本项目拟在申请人既往关于突触结构基因NRXN1和NLGN4X的研究基础上，结合生物信息学和神经生物学研究方法，发展功能基因组学研究策略：1）靶向下调神经干细胞发育模型中SHANK3基因表达水平，模拟单基因缺失效应，2）动态监测发育过程中继发于SHANK3单基因变异的差异表达基因网络、蛋白网络和分子通路变异，3）阐释“基因变异”至“神经细胞结构与功能发育异常”的基因组学机制。本研究采用人类神经干细胞模型有利于模拟人源性神经细胞动态发育过程，转录组测序结合生物信息学分析方法具有高通量、全局性研究效能，其结果有助于明确人源性SHANK3基因变异影响神经细胞结构与功能发育的全局性动态分子机制，推动神经精神疾病分子病理机制研究进展。

SHANK3表达定位于神经元兴奋性突触后，是突触后核心骨架蛋白。突触后离子通道受体NMDAR, AMPAR，代谢型离子通道受体mGluR及cortactin等骨架蛋白通过直接或间接的方式锚定于SHANK3不同结构域上，可见SHANK3是神经元突触前后复合体的核心物质和功能基础。自闭症谱系障碍疾病与遗传密切相关，大量研究表明SHANK3基因变异被确认为自闭症谱系障碍等神经精神疾病易感因素，但该基因变异增强相关疾病易感性的分子机制尚不明确。本项目在申请人既往关于突触结构基因NRXN1和NLGN4X的研究基础上，结合生物信息学和神经生物学研究方法，发展功能基因组学研究策略：1）建立了人源性诱导多能干细胞神经发育模型，基于该模型，建立了shSHANK3基因敲减慢病毒感染系统，2）在神经发育不同时间点，动态监测继发于SHANK3表达下调的神经元形态复杂度发育异常：神经元轴突长度、树突分支数目、长度及神经元胞体、生长锥等参数；动态监测继发于SHANK3表达下调的神经元功能发育异常；3）基于高通量测序深度挖掘继发于SHANK3表达下调的神经元形态与功能发育异常的潜在分子效应机制。基于上述研究发现： SHANK3表达下调导致发育早期神经元轴突和树突长度减小，树突分支数量减少，神经元胞体和生长锥面积减小，神经元钠通道阈值显著提高；hippo signaling、central nervous system development等信号通路发育异常可能是上述形态功能发育异常的潜在分子通路效应机制。本研究不仅提示继发于SHANK3表达异常导致发育终末期神经元形态功能异常，也提示SHANK3异常表达导致神经细胞发育早期系统性发育异常。上述发现部分提示了自闭症谱系障碍等神经发育疾病诊疗策略关注早期干预的科学性和必要性。