Toward understanding disease pathogenesis in the polyglutamine disorder Spinocere

描述（由申请人提供）：脊髓小脑性共济失调3型（SCA3）是一种进行性、致死性和不可治疗的神经退行性疾病，由ATXN3基因中编码多聚谷氨酰胺的CAG扩增引起。突变ataxin-3 （ATXN3）在大脑中的错误折叠和聚集被认为是这种疾病发病机制的核心。几种SCA3的细胞培养和动物模型概括了关键的疾病特征，并确定了可能导致突变ATXN3聚集和SCA3发病的几个因素。然而，现有的SCA3模型过表达高于生理浓度的突变蛋白。因此，在生理条件下体内影响ATXN3突变体聚集和SCA3发病机制的因素尚未被探索。本提案的目的是确定影响Atxn3聚集和SCA3发病机制的关键因素。核心假设是，突变Atxn3的蛋白水解裂解增加，极大地增强了其在体内的积累和聚集，异常的核定位导致神经元中有害的转录变化。为了探索疾病的发病机制，我们的实验室最近建立了第一个SCA3敲入小鼠模型，在内源性小鼠Atxn3位点插入82个CAG重复序列。敲入的初步结果揭示了Atxn3的年龄依赖性积累和聚集具有区域和亚细胞特异性。出乎意料的是，敲入结果显示海马体中有广泛的聚集，包括辐射层和托下的大量核外沉积物，海马体中高度活跃和突触丰富的区域。我的第一个具体目标是确定神经元兴奋如何影响突变Atxn3在体内的裂解、积累和聚集。我将使用电子显微镜、生化分析和敲入的原代神经元培养来确定生理表达条件下兴奋驱动突变体Atxn3切割和聚集的程度。然后，我将在敲入小鼠中诱导海马癫痫发作，以直接确定神经元活动对体内突变Atxn3切割和聚集的影响程度。我期望这一目标的结果表明，神经元活动的增加增强了突变Atxn3的切割和聚集，突变Atxn3片段优先积聚在神经元核中。由于一些研究已经确定突变体ATXN3的核定位是毒性的重要决定因素，我的第二个具体目标是确定突变体ATXN3表达诱导的SCA3敲入的异常转录变化。我将使用RNA-seq，一种灵敏的下一代测序方法，从SCA3敲入中识别疾病易感神经元的转录变化，这些神经元异常地积累突变的Atxn3，即小脑深部核。在对RNA- seq数据进行分析后，我将在不同年龄的敲入小鼠和SCA3人类脑组织中确认被预测在SCA3发病机制中重要的基因表达变化。我希望找出可能导致神经毒性的关键候选转录变化。总之，这些目标有望确定SCA3的关键致病因素。这一建议的结果可能有助于未来开发有效的SCA3治疗方法。关键词：神经退行性变，三联体重复扩张障碍，ataxin-3

DESCRIPTION (provided by applicant): Spinocerebellar ataxia type 3 (SCA3) is a progressive, fatal, and untreatable neurodegenerative disorder caused by a polyglutamine-encoding CAG expansion in the ATXN3 gene. Misfolding and aggregation of mutant ataxin-3 (ATXN3) in the brain is believed to be central to the pathogenesis of this disorder. Several cell-culture and animal models of SCA3 recapitulate key disease features and have identified several factors that might contribute to mutant ATXN3 aggregation and SCA3 pathogenesis. However, existing models of SCA3 overexpress the mutant protein above physiological concentrations. Consequently, the factors that influence mutant ATXN3 aggregation and SCA3 pathogenesis in vivo, under physiological conditions, have not been explored. The objective of this proposal is to identify key factors that influence Atxn3 aggregation and SCA3 pathogenesis. The central hypothesis is that increased proteolytic cleavage of mutant Atxn3 critically enhances its accumulation and aggregation in vivo, with aberrant nuclear localization leading to detrimental transcriptional changes in neurons. To explore disease pathogenesis, our laboratory recently generated the first knock-in mouse model of SCA3 with 82 CAG repeats inserted in the endogenous murine Atxn3 locus. Preliminary results in the knock-in reveal the age-dependent accumulation and aggregation of Atxn3 with regional and subcellular specificity. Unexpectedly, the knock-in revealed extensive aggregation in the hippocampus, including large extranuclear deposits in the stratum radiatum and subiculum, highly active and synapse-rich regions of the hippocampus. My first specific aim is determine how neuronal excitation affects mutant Atxn3 cleavage, accumulation, and aggregation in vivo. I will use electron microscopy, biochemical analyses, and primary neuronal cultures from the knock-in to determine the degree of excitation-driven mutant Atxn3 cleavage and aggregation under physiological expression conditions. I will then pharmacologically induce hippocampal seizures in knock-in mice to directly determine the extent to which neuronal activity affects mutant Atxn3 cleavage and aggregation in vivo. I expect the results of this aim to show that increased neuronal activity enhances mutant Atxn3 cleavage and aggregation, with mutant Atxn3 fragments preferentially accumulating in neuronal nuclei. Since several studies have determined that the nuclear localization of mutant ATXN3 is an important determinant of toxicity, my second specific aim is to identify aberrant transcriptional changes in the SCA3 knock-in induced by mutant Atxn3 expression. I will use RNA-seq, a sensitive next- generation sequencing method, to identify transcriptional changes in disease-susceptible neurons from the SCA3 knock-in that aberrantly accumulate mutant Atxn3, the deep cerebellar nuclei. After analysis of the RNA- seq data, I will then confirm gene expression changes predicted to be important in SCA3 pathogenesis in knock-in mice of different ages and SCA3 human brain tissue. I expect to identify key candidate transcriptional changes that might contribute to neurotoxicity. Together these aims are expected to identify key pathogenic factors in SCA3. The results of this proposal could help in the future development of effective therapies for SCA3. Keywords: Neurodegeneration, triplet repeat expansion disorder, ataxin-3