The role of p62/SQSTM1 in the pathogenesis of motor neuron disease

项目摘要 肌萎缩侧索硬化症（ALS）是一种影响运动神经元的破坏性神经退行性疾病， 是迅速发展的，而且是致命的在90-95%的病例中，ALS是散发性的，没有明确的相关风险 因素另外5-10%的ALS是遗传的。ALS最常见的遗传原因是， 在基因9号染色体开放阅读框72中发现GGGGCC六核苷酸重复扩增（HRE） （C9 orf 72），占ALS的所有家族性病例的约一半和约散发性病例的约8%（称为“C9-C9 orf 72”）。 ALS”）。这种HRE被假设是由功能获得引起的，通过重复RNA的表达， 螯合RNA结合蛋白，或通过产生二肽重复蛋白（DPR）， 重复相关非AUG翻译（RANT）。使用基因筛选GGGGCC介导的 在果蝇中的毒性，我们已经鉴定了p62/SQSTM 1作为c9 orf 72 ALS的强遗传修饰剂。此外 作为一种遗传修饰剂，p62阳性聚集体是C9-ALS的关键病理，而p62/SQSTM 1 突变很少被证明会导致患者的ALS。此外，p62在蛋白质合成中起着关键作用。 体内平衡（蛋白质平衡）和蛋白质降解，这与发病机制密切相关 的ALS。虽然p62似乎是一个关键的遗传靶点，但其在ALS发病机制中的作用知之甚少。 该项目将确定1）C9-ALS中蛋白质稳态被破坏的机制和2） p62在C9-ALS发病机制中的作用。首先，我将使用新的转基因品系来确定DPR是否 或GGGGCC RNA负责神经变性和蛋白质稳态缺陷， 每个DPR都在降级。接下来，我将确定p62调节GGGGCC介导的细胞凋亡的机制。 使用分子和遗传工具在果蝇神经变性，以确定哪些蛋白质稳态 和信号通路导致表达GGGGCC的果蝇中p62介导的毒性。最后，我将 使用CRISPR产生p62的新等位基因，以评估p62在运动神经元变性中的作用。 这些等位基因将用于研究p62和GGGGCC表达之间的相互作用以及其他基因表达之间的相互作用。 ALS致病基因，以便深入了解ALS的遗传基础。

PROJECT SUMMARY Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease affecting motor neurons that is rapidly progressive and uniformly fatal. In 90-95% of cases, ALS is sporadic with no clearly associated risk factors. The other 5-10% of ALS is inherited with a genetic cause. The most common inherited cause of ALS, a GGGGCC hexanucleotide repeat expansion (HRE) found in the gene chromosome 9 open reading frame 72 (C9orf72), accounts for about half of all familial cases and about 8% of sporadic cases of ALS (called “C9- ALS”). This HRE is hypothesized to be caused by a gain-of-function either by expression of repeat RNA and sequestration of RNA-binding proteins, or through the production of dipeptide repeat proteins (DPRs) through repeat-associated non-AUG translation (RANT). Using a genetic screen for modifiers of GGGGCC-mediated toxicity in Drosophila, we have identified p62/SQSTM1 as a strong genetic modifier of c9orf72 ALS. In addition to being a genetic modifier, p62-positive aggregates are a key pathology in C9-ALS, and p62/SQSTM1 mutations have rarely been shown to cause ALS in patients. Furthermore, p62 plays a key role in protein homeostasis (proteostasis) and protein degradation, which have been strongly implicated in the pathogenesis of ALS. Although p62 appears to be a key genetic target, its role in ALS pathogenesis is poorly understood. This project will determine 1) the mechanism by which protein homeostasis is disrupted in C9-ALS and 2) the role that p62 plays in C9-ALS pathogenesis. First, I will use novel transgenic lines to determine whether DPRs or GGGGCC RNA are responsible for neurodegeneration and protein homeostasis defects by selectively degrading each DPR. Next, I will determine the mechanism by which p62 regulates GGGGCC-mediated neurodegeneration using molecular and genetic tools in Drosophila to determine which protein homeostatic and signaling pathways contribute to p62-mediated toxicity in GGGGCC expressing Drosophila. Lastly, I will use CRISPR to generate novel alleles of p62 in order to assess the role of p62 in motor neuron degeneration. These alleles will be used to study the interactions between p62 and GGGGCC expression as well as other ALS-causing genes in order to provide insight into the genetic basis of ALS.