孤独症相关基因CNTN4在神经元生长过程中的作用机制研究

Autism is a severe childhood neurodevelopmental disorder. The most recent epidemiological study demonstrated that 1 in 50 children has autism. In our previous genetic study, we identified 3 patients with copy number variations (CNVs) which disrupted CNTN4 gene. In addition, we revealed that CNTN4 secreted protein can promote neurite outgrowth. Subsequently, we identified two trans-membrane proteins associated with neurodevelopment (ITGB1 and RGMB) interacting with CNTN4 by Y2H. Additionally, we have established CNTN4 knockout mouse. Based on our previous studies, this proposal will further investigate the mechanism that how CNTN4 promotes neurite outgrouth from the following 3 aspects: (1) study on the involvement of CNTN4 in neurite outgrowth and its relationship with ITGB1 and/or RGMB interaction, and the signaling pathways they mediated; (2) functional study of autism-associated CNTN4 mutations and its impacts on neurite outgrowth and signal transduction; (3) study on CNTN4 knock-out mouse, figuring out how CNTN4 contribute to neurite outgrowth in vivo and evaluation the behavior phenotypes of CNTN4 knockout mouse. This proposal is very important for clarifying the roles of CNTN4 in neurodevelopment and the molecular pathogenesis of autism.

孤独症是一种严重影响儿童健康的神经发育障碍性疾病，发病率已高达1/50。前期研究我们发现3例孤独症患者携带了打断CNTN4基因的拷贝数变异。进一步，我们发现CNTN4分泌蛋白能够促进神经元的生长，而且利用酵母双杂交发现CNTN4可以和ITGB1以及RGMB这两个与神经发育密切相关的跨膜蛋白相互作用。另外，我们还构建了CNTN4基因敲除小鼠。基于这些研究基础，本项目将继续研究CNTN4参与神经元生长的作用机制，重点研究CNTN4对神经元生长的促进作用与ITGB1和RGMB介导的信号通路的关系。我们还将对CNTN4孤独症相关的突变体进行功能研究并重点研究其对神经元生长和信号传导的影响。最后利用构建的CNTN4基因敲除小鼠在体研究CNTN4对神经元生长和突触发生的影响并评估CNTN4敲除小鼠的行为学表型。本项目对阐明CNTN4如何参与神经发育及孤独症的分子发病机制具有重要意义。

CNTN4为重要的含有神经免疫球蛋白结构域的细胞粘附分子成员，参与多种神经发育疾病的发病风险，包括孤独症，精神分裂症等。本项目在前期遗传学及初步功能探索的基础上对CNTN4参与神经发育的机制及相关的神经元和小鼠表型进行进一步深入研究。主要研究结果如下：1）发现 CNTN4 OE增加树突复杂度，而KD则降低树突棘密度，并只对兴奋性突触有影响；2）孤独症相关的CNTN4错义突变不能发挥促进神经元生长和树突发育的功能，可能为丢失功能性突变；3）发现 CNTN4可能通过影响树突棘密度参与神经微环路的形成，并且这一机制可能是通过与APP 相互作用参与的；4）发现 CNTN4 KO小鼠出现多动，焦虑/抑郁降低，节律异常等一系列躁狂表型；KO小鼠前额叶皮层GluR1表达下降，额叶皮层mEPSC幅度和频率下降，兴奋/抑制性神经传递失衡。本项目顺利完成了预期研究内容，达到预期目标，研究结果为进一步阐明CNTN4参与神经发育的分子机制奠定重要基础，同时提供了一个研究躁狂相关表型分子机制的一个重要动物模型。