常染色体隐性遗传小脑性共济失调新的致病基因CAX的功能研究

Autosomal recessive cerebellar ataxias (ARCAs) are a group of neurodegenerative disorders that are characterized by complex clinical and genetic heterogeneity. We previously identified a novel ARCAs candidate gene, CAX, through exome sequencing and copy number variation (CNV) analysis in a two-generation ARCA family. CAX encodeing protein belongs to Kelch-like (KLHL) subfamily, which is implicated in ubiquitination by forming Cullin-RING E3 ligases complex and involved in the pathogenesis of several hereditary neurologic disorders. However, the function of this newly identified protein remaines unknown. In our previous work, we identified that CAX was highly expressed in cerebellum and interacted with Cullin3. The mutant residue PNNL was predicted to have a functional impact by SIFT and PolyPhen-2. Moreover, the over-expression of CAX caused abnormal axon morphology in cerebellum granule cell. Taken together, these results demonstrate that CAX may be a putative substrate adaptor for Cul3-RL that functions as a neurological specific ubiquitin ligase, and play an important role in the pathogenesis of CAX-associated ARCA. In this research, we intend to clarify whether the mutatants impact its subcellular localization, protein interaction, abnormal neuronal growth and dysfuncrion of ubiquitin-proteasome system. In addition, mass spectrum screening assay will used to seek its interacting proteins, CAX knockout mouse model will set-up to detect its behavior, histochemistry and pathophysiology alterations. All these might help us to understand the physiological function of CAX and reveal the association of CAX mutation and ARCA, and provide new clues of pathologenesis of ARCAs.

CAX基因是本课题组在前期工作中发现并克隆的常染色体隐性遗传小脑性共济失调新的候选致病基因，属于KLHL基因家族，功能尚不清楚，但该家族中多种基因均发现与神经系统遗传病相关，且参与形成Cul3-RING泛素连接酶复合体，调节重要底物蛋白降解的功能。前期工作中我们已证实该蛋白在小脑中高表达；功能预测提示在家系内发现的错义突变PNNX具有致病性；CAX与Cullin3存在相互作用，过表达CAX可抑制小脑颗粒细胞的轴突生长；提示CAX基因具有重要的生理功能并参与了遗传性共济失调的致病过程。本课题拟在此基础上，在细胞水平研究突变对CAX蛋白亚细胞定位、泛素蛋白酶体通路功能、神经元生长、蛋白相互作用等的影响；同时筛选和鉴定CAX蛋白的互作蛋白，进一步了解其生理功能；建立CAX基因敲除/敲入小鼠模型，从行为学、组织化学和病理生理学等多角度探讨CAX基因与ARCA致病性的关系，为揭示其发病机制研究提供新的线索。

前期工作中，我们在一个近亲婚配的ARCAs家系中发现并克隆了CAX基因序列变异(c.191 C>T/ p.P64L)。CAX蛋白属于KLHL家族成员，并在小脑中高表达，与其它同家族成员类似，CAX可与CULLIN3相互作用，构成CRLs泛素连接酶复合体。研究发现，CAX蛋白主要在细胞质中表达，它通过BTB和BACK结构域与CULLIN3相互结合构成CRLs复合体，并特异性识别底物HNRNPA2B1，可对底物的泛素蛋白酶体降解过程进行调控。P64L序列变异位于BTB结构域，研究表明该突变影响了CAX蛋白稳定性，导致CAX蛋白降解速度变快，该点突变还抑制了CAX对其底物HNRNPA2B1的泛素化降解作用。在CAX基因敲除（CAX-/-）小鼠和点突变敲入（CAXP60L/P60L）小鼠中，CAX-/-和CAXP60L/P60L小鼠小脑组织中CAX蛋白表达量较同窝对照明显下降， HNRNPA2B1的泛素化降解过程受到抑制，导致HNRNPA2B1异常蓄积。行为学实验证实，与野生型小鼠相比，CAX-/-和CAXP60L/P60L小鼠均出现运动平衡功能受损的表型，且CAX-/-和CAXP60L/P60L小鼠小脑浦肯野细胞密度明显减少。以上研究结果证实CAX很可能是ARCAs新的致病基因。