类泛素化通路UX的E1激活酶突变导致新型小脑共济失调的机制研究

The mutations in a novel gene UX-E1, an ubiquitin-like modifier activating enzyme, were determined through exome-sequencing in an autosomal recessive cerebellar ataxia cohort. The understanding of biological function of UX cascade remains largely elusive, although UX is present in nearly all eukaryotic organisms with the exception of fungi. We observed RNAi knockdown of UX-E1, UX, or UX-E2 exhibits abnormal wing positioning, climbing and flight defects, short life-span and abnormal NMJ morphology, which perfectly mimics the neurodegenerative phenotypes of the patients. Overexpression of human and drosophila UX-E1 effectively rescued the neurodegeneration, while mutant UX-E1 failed. To date, only two targets were reported in HEK293 and MIN6 pancreatic cell lines and distinct UX conjugates were found in the mouse brain tissue. We intend to identify UX targets in the central nervous system of knock-in mice with 6×His tag insertion upstream of UX gene through SILAC quantitative proteomics, an ideal platform for measuring dynamic post-translation modification changes. UX targets with high credibility will be validated by Co-IP, in vitro conjugation assay, subcellular fractionation, and in vivo fly targets rescue experiments. High-throughput screens of 350 deficiency Drosophila strains and 3200 chemical compounds have been performed, that may suggest proteins and molecular pathways that contribute to the disease process and thus provide novel targets for therapeutic interventions. Subsequent functional study will be conducted in the suitable cell lines and UX Drosophila model. Our study will point out UX targets and molecular pathways involved in the pathogenesis of the disease, elucidate the underlying molecular mechanism how mutations in UX-E1 caused the autosomal recessive cerebellar ataxia.

外显子组测序在一常染色体隐性小脑共济失调家系发现一新致病基因UX-E1，为UX类泛素化通路激活酶。UX从人到植物均保守，但其生物学功能有待研究。我们发现UX-E1及UX和UX-E2敲低果蝇均表现出翅膀、爬行、飞行、寿命及神经肌肉接头等方面神经变性，可模拟患者表型。过表达野生型UX-E1显著拯救神经变性，而突变型无法有效拯救。目前只在HEK293与MIN6胰岛细胞中鉴定出两个UX靶基因，且小鼠脑组织中存在特异的UX结合蛋白条带。我们拟利用SILAC这一理想的翻译后修饰定量蛋白质组平台，在UX基因前加6×His标签小鼠中鉴定神经系统中高可信UX靶基因；利用果蝇这一大规模筛选优秀模型，对350种缺陷型果蝇、3200种化合物进行筛选，找到影响神经退行表型的modifiers。基于以上，我们将体内外证明UX通路及靶基因发挥的生物学功能，阐明UX-E1突变导致小脑共济失调的致病机制并发现特异治疗靶点。

UX是一种类泛素化修饰，在多细胞生物中高度保守，由E1激活酶（UX-E1）、E2结合酶（UX-E2）和E3连接酶（UX-E3）组成。最近研究发现UX、UX-E1和UX-E2突变会导致小头畸形、精神发育迟滞和运动障碍等，但其致病机制尚未阐明。小头畸形患者大脑尺寸减少，神经系统发育障碍。人类大脑中的神经元细胞均由神经干细胞分化产生。神经干细胞增殖缺陷，导致神经干细胞数量异常，引起小头畸形。本研究利用果蝇模型研究UX类泛素化通路是如何引起小头畸形。果蝇幼虫神经母细胞（neuroblasts，NBs）通过不对称有丝分裂完成其自我更新和分化，是研究神经干细胞生物学和小头畸形潜在机制的理想平台。果蝇胚胎早期的13次核分裂快速同步化进行，是研究有丝分裂的经典模型。在果蝇幼虫NBs中敲低UX类泛素化水平，NBs数量明显减少，引起幼虫中枢神经系统显著减小，表现出小头表型。敲低UX类泛素化水平NBs显示出一系列异常表型，包括：有丝分裂指数统计分析表明正在进行有丝分裂的细胞增多；FUCCI果蝇模型显示G2和M期的细胞比例增多，有丝分裂进程紊乱；NBs不对称分裂异常，aPKC的极性分布消失、纺锤体组装异常。在合胞体胚胎中敲低UX类泛素化水平，引起胚胎早期发育停滞，合胞体胚盘细胞核区域性丢失，核分裂后期和末期出现染色体桥，造成染色体不稳定。敲低UX类泛素化水平影响纺锤体组装和核分裂同步化过程。利用time-lapse实时动态追踪染色体运动，合胞体胚胎中敲低UX-E3类泛素化水平后，分裂间期时间缩短，胚胎加速进入有丝分裂期。CDK1是G2 / M转换的关键调控分子，激活CDK1是进入有丝分裂必不可少的步骤。敲低UX类泛素水平，CDK1的抑制性磷酸化减少，CDK1的活性升高；过表达UX类泛素化水平，CDK1的抑制性磷酸化升高，CDK1的活性降低。以上结果表明UX类泛素化通路的果蝇模型模拟患者的小头畸形，影响NBs自我更新，引起有丝分裂进程紊乱。我们的研究揭示了UX化类泛素通路新的生物学功能，即引起细胞周期紊乱从而导致小头畸形。