中心体蛋白CDK5RAP2在大脑皮层发育中的作用和分子机理

CDK5RAP2 enoding a centrosomal protein, is one of seven genes leading to autosomal recessive primary microcephaly (MCPH),which is a neurodevelopmental disease characterized by reduced head circumference at birth with variable degree of mental retardation, resulting from deficient neuron production in the developing cerebral cortex. However, how the embryonic neurogenesis is dysregulated still remains unclear. Recent studies have shown that CDK5RAP2 is abundant in human and mouse brain, and required for centriole replication, spindle formation, cell cycle regulation, microtubule dynamics, ect, in cultured cells, suggesting CDK5RAP2 may play an important role in neural stem cell/ neural progenitor cell proliferation and neurogenesis regulation thereafter. However, there is no appropriate animal model to study it in vivo so far. .In this proposal, we will generate a tissue-specific knockout mouse line, in which, CDK5RAP2 is absent in neural stem cells/ neural progenitor cells, and utilize a series of in-vivo and in-vitro techniques, including immunohistochemistry,in utero electroporation, brain slice culture, high-resolution time-lapse imaging, neural stem cell culture and differentiation to investigate the role of CDK5RAP2 in neural stem cell/neural progenitor cell maintenance, proliferation, cell fate determination and corticogenesis. .The achievements from this proposal would not only provide a system for studies on the basic molecular mechanism of neurogenesis and cortical formation but also disclose pathological molecular signaling pathways of microcephaly, which may open an avenue to the therapeutic researches on microcephaly and other related neurodevelopmental diseases. ..

CDK5RAP2编码中心体蛋白，在细胞分裂和细胞周期调控中极为重要，该基因在神经系统中表达丰富，基因突变导致头小畸形症（microcephaly）。作为神经系统发育缺陷型疾病，患者表现为头围低于平均值3个标准差，并有认知障碍等大脑功能缺陷,多发现在新生儿中。目前，由于实验动物模型的缺乏，尽管CDK5RAP2的功能在细胞水平上研究较多，但在组织器官甚至生物整体水平上关于CDK5RAP2如何调控大脑皮层神经生成的分子机理仍不清楚。本课题计划应用转基因模式动物、细胞生物学和分子生物学相结合的方法，利用孕鼠宫体电转、时序荧光显微成像、干细胞培养及定向分化和免疫荧光染色等实验室现有的技术特长，从小鼠模型到人源细胞，结合体内和体外的研究体系，初步阐明中心体蛋白CDK5RAP2在大脑皮层发育中是如何调控神经生成的分子机理。

头小畸形症（MCPH）是一种神经系统发育型疾病，头小畸形患者表现为头围低于平均值3个标准差，同时伴有智力发育障碍、情绪失控、运动能力欠缺、并有认知障碍等大脑功能缺陷，严重影响患者的正常生活。CDK5RAP2编码中心体蛋白，该基因突变可以导致头小畸形症的发生。为了研究CDK5RAP2突变引起脑发育缺陷的病理机制，我们首先利用shRNA的技术在小鼠的大脑皮层中特异性敲减CDK5RAP2基因，发现CDK5RAP2的减少使得神经干细胞的增殖能力减弱，更倾向于分化，同时影响神经元发生的过程，最终使得神经元数量减少。然后我们利用Cre-Loxp技术，在小鼠的大脑皮层中条件性敲除CDK5RAP2后，得到了类似于头小畸形病人的表型，包括，头小，大脑皮层变薄，神经元数量减少。我们进一步的研究发现CDK5RAP2敲除后会引起神经干细胞在分裂后凋亡，从而神经干细胞的数量减少，神经元数量减少但是分层不受影响。本项目应用转基因模式动物、细胞生物学和分子生物学相结合的方法，利用孕鼠宫体电转、时序荧光显微成像、免疫荧光染色等技术，初步阐明了中心体蛋白CDK5RAP2在大脑皮层发育中是如何调控神经发生的机理。