中心体蛋白CPAP在小鼠胚胎新皮层发育过程中的调控机制

CPAP (centrosomal P4.1-associated protein) associated with the ?-tubulin complex, was identified as centrosomal protein. The human full length CPAP protein of 1338 amino acid contains a 112-amino acid long microtubule-destabilizing motif PN2-3 and two C-terminal 14-3-3 binding sites. CPAP/CENPJ is localized at the centrosome during mitosis and it is concentrated at the mitotic spindle poles during pro-metaphase and metaphase. Mutations in CPAP gene lead to the autosomal recessive primary microcephaly (MCPH). MCPH is a rare genetic disease defined by a decrease in occipito-frontal head circumference at birth of greater than 3 standard deviations. Patients often have a broad spectrum of neurological problems, including mental retardation, focal or generalized seizures, hyperactivity and attention deficit disorder. The decrease in brain volume without major architectural abnormalities most likely stems from a primary defect in neurogenesis. Recently, it has been demonstrated that CPAP is a key regulator that controls centriole length, and cilia formation. The centrosome is critical for normal cell cycle regulation, cell division and migration, but why mutations in CPAP genes, encoding the centrosome proteins, generate such particular defects in human brains is still unclear. Due to the lack of mouse models has precluded investigation of the direct role CPAP gene plays in brain development. Thus, we hypothesize that CPAP gene has pivotal functions in neurogenesis, neuronal migration, and neuronal maturation that, when disrupted, leads to the structural and functional defects mentioned above. We will test this hypothesis using loss of function studies via gene knockdown by RNA interference (RNAi) of CPAP or generating conditional knockout mouse. Understanding the molecular pathogenesis leading to CPAP will grow our knowledge of brain development in both normal and pathological states, and begin to shed light on molecular pathways that may ultimately be effective therapeutic targets for MCPH and similar neurodevelopmental disorders.

CPAP/CENPJ基因编码中心体相关蛋白，参与调控中心体的复制和延长。该基因突变导致先天性头小畸型症。目前已知的导致先天性头小畸形症的七个基因中，所有基因均编码中心体相关蛋白。中心体是动物细胞中非常重要的细胞器之一，主要作为微管组织中心，调控细胞分裂和迁移。近几年，关于CPAP蛋白作用于中心体的细胞生物学机制有不断的进展，但由于缺乏基因敲除模式动物模型，关于CPAP基因突变导致先天性头小畸形症的分子致病机制仍不清楚。本研究将通过制备条件性CPAP/CENPJ基因敲除小鼠，并结合孕鼠电场转基因shRNAi技术和高分辨时序荧光成像追踪等方法，系统的研究CPAP基因缺陷导致大脑皮层发育畸形的致病机理。 此外，通过研究此单基因突变显著影响大脑皮层的大小与形态机制，将为深入理解大脑皮层的生物演化提供重要分子机制参考。

头小畸形是一类神经系统发育型疾病，头小畸形患者的头部尺寸仅为正常同龄人的三分之一，伴有智力发育障碍、情绪失控等症状，严重影响患者的正常生活。导致头小畸形的突变大都是编码中心体蛋白的基因。本项目关注一种导致头小畸形的中心体基因CPAP，又名CENPJ，它不仅可以调控中心粒的生长，也可以控制细胞骨架结构。为了探讨人类CENPJ突变引起脑发育缺陷的病理机制，我们首先利用Cre-Loxp的技术在小鼠的大脑皮层中特异性敲除Cenpj基因，获得了具有的头小、大脑皮层变薄、神经元数量减少的表型的小鼠模型，这与头小畸形病人的表型非常类似。我们研究发现Cenpj条件敲除小鼠的神经干细胞数量显著减少，增殖分裂能力明显降低，并伴随有大量的细胞凋亡现象，提示了其机制很可能是Cenpj的敲除使得细胞分裂受阻造成神经干细胞不能持续保持增殖活性，从而导致神经元群体的数量减少，皮层变薄，大脑皮层不能正常发育。最终，我们通过对小鼠皮层的RNA-Seq分析揭示了Cenpj调控干细胞增殖的信号通路。