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

项目总结 肌萎缩侧索硬化症(ALS)是一种破坏性的神经退行性疾病，影响运动神经元 是快速发展的，而且都是致命的。在90%-95%的病例中，肌萎缩侧索硬化症是零星的，没有明显的相关风险 各种因素。另外5%-10%的肌萎缩侧索硬化症是遗传的。肌萎缩侧索硬化症最常见的遗传原因是 GGGGCC六核苷酸重复序列扩增(HRE)发现于基因第9号染色体开放阅读框72 (C9ORF72)，约占所有家族性ALS病例的一半，约占散发性ALS病例的8%。 ALS“)。这种HRE被认为是由重复RNA和 RNA结合蛋白的封存，或通过以下途径生产二肽重复蛋白(Dprs) 重复关联的非8月翻译(RANT)。利用遗传筛选寻找GGGGCC介导的修饰物 对于果蝇的毒性，我们已经确定p62/SQSTM1是C9ORF72 ALS的一个强烈的遗传修饰。此外 作为一种遗传修饰物，p62阳性聚集体是C9-ALS和p62/SQSTM1的关键病理 突变很少被证明会导致患者的肌萎缩侧索硬化。此外，p62在蛋白质中起着关键作用。 动态平衡(蛋白质平衡)和蛋白质降解，这与发病机制密切相关 肌萎缩侧索硬化症。虽然p62似乎是一个关键的遗传靶点，但它在ALS发病机制中的作用尚不清楚。 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dprs是否 或GGGGCC RNA通过选择性地导致神经退行性变和蛋白质稳态缺陷 使每一个DPR降级。接下来，我将确定p62调节ggggcc介导的机制。 用分子和遗传工具确定果蝇神经退行性变中的哪种蛋白质 在表达果蝇的GGGGCC中，信号通路参与了p62介导的毒性作用。最后，我会 使用CRISPR产生新的p62等位基因，以评估p62在运动神经元退行性变中的作用。 这些等位基因将被用来研究p62和GGGGCC表达之间的相互作用以及其他 以深入了解肌萎缩侧索硬化症的遗传基础。

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.