PAK2在神经发育障碍疾病中的机制研究

Neurodevelopmental retardation, the common clinical manifestation shared by intellectual disability (ID), autism spectrum disorders (ASD), epileptic encephalopathy (EE), and schizophrenia (SCZ), displays a vital impact on children's health. Large samples-based linkage analysis indicate that a 1.6Mb deletion in chromosome 3 (3q29 microdeletion), encompassing serine/threonine protein kinase PAK2 gene, is related to mental retardation or microcephaly; however, the role of PAK2 in 3q29 microdeletion is not well-established. Through whole Trio-based exome sequencing, we identified a de novo nonsense mutation (C1435T) in PAK2 in one proband with ASD. In addition, another three missense mutations in PAK2 were identified from previous case-control studies. Overexpression of human PAK2 with the nonsense mutation in several cell lines consistently reduced the level of phosphorylation of c-Abl and expression of MAPKAP5, but had no effect in cell apoptosis pathway. As c-Abl and MAPKAP5 are key regulators in cytoskeleton remodeling and F-actin polymerization, our preliminary works suggest that PAK2 may affect function in cytoskeleton and cell adhesion instead of apoptotic pathway to regulate neural development. Given the fact that developmental delays and intellectual retardation are the common clinical symptoms of autism and mental retardation, we hypothesized that PAK2 may be involve in the pathogenesis of these diseases by regulating brain development. In this application, we will establish both PAK2 nonsense mutation cell model and mouse model by CRISPRs/Cas9 system. By employing the genetic disrupted cell line and animal model, we will investigate whether PAK2 could participate in the regulation of brain development by controlling the cytoskeleton and focal adhesion pathway, and determine whether the disruption of PAK2 gene could result in brain malformation and dysfunction through the above mechanisms at molecular, neuronal, and behavioral level. The implement of this project will provide a foundation for the pathogenesis of these neurodevelopmental disorders.

神经发育障碍是智力缺陷（ID）、自闭症谱系障碍（ASD）、癫痫、精神分裂症共有的病理基础，严重影响儿童健康。GWAS分析显示染色体3q29区缺失PAK2与ID相关，但PAK2影响神经发育和智力水平的机制尚不清楚。通过全外显子和大规模ASD样品扫描分析，我们在ASD患者中鉴定出PAK2新生无义突变（C1435T）和3个错义突变，提示PAK2可能是ID和ASD的共病基因。在细胞中过表达PAK2-C1435T可降低c-Abl磷酸化(参与细胞骨架重排)水平和MAPKAP5(调节F-肌动蛋白聚合)表达，但不影响细胞凋亡。据此，我们推测PAK2可能通过影响细胞骨架和粘附等调控神经发育，参与疾病进程。在本项目中我们拟运用CRISPRs系统建立PAK2-C1435T细胞和小鼠模型，从分子生化、形态结构、行为学等层次确定PAK2在神经发育障碍疾病中的调控机制，以期为此类疾病发病的分子机制提供新证据。

自闭症谱系障碍（简称自闭症）是一类复杂的神经发育障碍性疾病的统称，具有很高的遗传性和异质性。在数以百计的自闭症基因中，只有少数基因的致病机制得以阐述，这使自闭症遗传学研究和临床诊断治疗之间存在巨大鸿沟。PAK2 蛋白是丝氨酸/苏氨酸蛋白激酶家族成员，参与细胞骨架重排等重要的生物学过程。然而，目前尚缺乏PAK2参与神经发育和相关行为的证据。本项目通过对自闭症患儿进行大规模测序分析后发现了一个位于PAK2激酶结构域的新生无义突变位点（C1435T）。在小鼠中， Pak2 基因缺失会导致神经元突触密度降低，突触可塑性受损以及自闭症样行为。在分子机制方面，我们发现调控肌动蛋白的关键分子LIMK1和Cofilin磷酸化水平以及这些分子调控的肌动蛋白聚合水平在PAK2杂合小鼠海马和皮层中显著下降。PAK2缺失还可广泛地干扰多个与自闭症相关的功能网路，提示PAK2/LIMK1/CFL调控的突触细胞骨架是自闭症相关基因突变后共同影响的通路。综上，本项目从分子、形态、行为和遗传等多个水平确定了PAK2在脑发育中的重要功能，并阐明了其在自闭症发病中的内在分子机制，这为自闭症的精准诊断和治疗提供了实验依据。

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