Role of p62 in Protein Aggregation and Neurodegeneration in ALS

描述（由申请人提供）：含有突变型铜锌超氧化物歧化酶（SOD1）的蛋白聚集体是家族性肌萎缩性侧索硬化症（ALS, Lou Gehrig's disease）的标志，ALS是一种与年龄相关的神经退行性疾病。野生型（WT）和突变型SOD1蛋白之间的构象差异已经通过多种生物物理技术在体外研究中得到证实，但体内聚集过程的细节仍有待确定。目前还不清楚蛋白质聚集体是有害还是有益。P62/Sequestome 1（本文简称P62）是一种多功能蛋白，参与了两种主要的蛋白质降解机制，泛素-蛋白酶体系统（UPS）和自噬-溶酶体途径。我们最近报道了内源性小鼠p62水平在G93A SOD1转基因小鼠发病前显著升高。此外，p62在G93A SOD1转基因小鼠脊髓运动神经元蛋白聚集体中与突变体SOD1和泛素共定位。我们已经与奥本大学的Marie Wooten博士建立了合作关系，她是研究p62信号级联的主要科学家，并将为该研究提供p62敲除小鼠。该合作项目的长期目标是了解p62在ALS相关SOD1突变和ALS运动神经元变性引起的蛋白质聚集中的作用。该项目的创新之处在于p62在ALS病因学中的作用在很大程度上是不确定的。该项目要验证的中心假设是p62可以识别错误折叠的突变体SOD1，并且p62可以通过将这些错误折叠的蛋白质穿梭到泛素-蛋白酶体系统（UPS）和/或自噬来改善突变体SOD1诱导的毒性。我们的初步研究表明，p62特异性识别突变体SOD1，而p62的泛素关联（UBA）结构域对这种相互作用不是必需的。我们的研究结果还表明，p62促进突变SOD1聚集体样包涵体的形成，但对WT SOD1影响不大。最近其他研究小组的结果表明，p62可以直接结合自噬效应蛋白Atg8/LC3，并且在G93A SOD1转基因小鼠中自噬的形成增加。这些数据有力地支持了中心假设。我们设计了三个特定的目标来确定p62识别突变体SOD1的机制，这种相互作用激活的下游途径，以及p62在神经元变性中的功能作用。目的1是绘制识别SOD1突变体并与之相互作用的p62结构域。目的2是确定p62是否以及如何介导突变体SOD1诱导的自噬激活。目的3是在p62 KO小鼠体内研究p62如何影响蛋白聚集和ALS疾病进展。这项研究的结果将为p62在ALS蛋白聚集和神经变性中的作用提供宝贵的见解，这将有助于更好地了解ALS的病因，并有可能发现新的治疗靶点。肯塔基大学的Zhu博士和奥本大学的Wooten博士的合作项目旨在了解p62在突变SOD1诱导的肌萎缩性侧索硬化症（ALS）（一种与年龄相关的神经退行性疾病）中蛋白聚集和运动神经元变性中的作用。该项目的创新之处在于p62在ALS病因学中的作用在很大程度上是不确定的。P62是一种多功能蛋白，参与了两种主要的蛋白质降解机制，泛素-蛋白酶体系统（UPS）和自噬-溶酶体途径。该项目要验证的中心假设是p62可以识别错误折叠的突变体SOD1， p62可以通过将错误折叠的蛋白质穿梭到UPS和/或自噬中来改善突变体SOD1诱导的毒性。我们已经获得了大量的初步数据，有力地支持了这些假设。三个特定的目标旨在进一步表征p62在ALS中蛋白质稳态（降解和聚集）和神经元存活中的作用的分子细节。目的1是绘制识别SOD1突变体并与之相互作用的p62结构域。目的2是确定p62是否以及如何介导突变体SOD1诱导的自噬激活。目的3是在p62 KO小鼠体内研究p62如何影响蛋白聚集和ALS疾病进展。这项研究的结果将为p62在ALS蛋白聚集和神经变性中的作用提供宝贵的见解，这将有助于更好地了解ALS的病因，并有可能发现新的治疗靶点。

DESCRIPTION (provided by applicant): Protein aggregates containing mutant copper-zinc superoxide dismutase (SOD1) are a hallmark of familial amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), an age-related neurodegenerative disease. Conformational differences between wild-type (WT) and mutant SOD1 proteins have been demonstrated by many in vitro studies using a variety of biophysical techniques, but in vivo details of the aggregation process remain to be defined. It is also unclear whether protein aggregates are toxic or beneficial. P62/Sequestome 1 (referred as p62 in this proposal) is a multifunctional protein involved in both of the two major protein degradation mechanisms, ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway. We recently reported that the endogenous mouse p62 levels significantly increased prior to the disease onset in G93A SOD1 transgenic mice. In addition, p62 was co-localized with mutant SOD1 and ubiquitin in the protein aggregates in spinal motor neurons in G93A SOD1 transgenic mice. We have established collaboration with Dr. Marie Wooten at Auburn University who is the leading scientist studying p62 in signaling cascades and who will provide the p62 knockout mice for the study. The long term goal of this collaborative project is to understand the role of p62 in protein aggregation caused by ALS-linked SOD1 mutants and motor neuron degeneration in ALS. The innovative aspect of this project is that the role of p62 in the ALS etiology is largely undefined. The central hypothesis to be tested in the project is that p62 can recognize misfolded mutant SOD1 and that p62 can ameliorate the mutant SOD1 induced toxicity by shuttling such misfolded proteins to the ubiquitin-proteasome system (UPS) and/or autophagy. Our preliminary studies demonstrate that p62 specifically recognizes mutant SOD1 and the ubiquitin-association (UBA) domain of p62 is not essential to such interaction. Our results also show that p62 enhances the formation of aggresome-like inclusion of mutant SOD1 but had little effect on WT SOD1. Recent results from other groups showed that p62 can directly bind to autophagy effector protein Atg8/LC3 and that the formation of autophagy was increased in the G93A SOD1 transgenic mice. These data strongly support the central hypothesis. Three specific aims are designed to determine the mechanisms by which p62 recognizes mutant SOD1, the downstream pathways activated by such interaction, and the functional role of p62 in neuronal degeneration. Aim 1 is to map the domains of p62 essential for recognizing and interacting with mutant SOD1. Aim 2 is to determine whether and how p62 mediates the autophagy activation induced by mutant SOD1. Aim 3 is to study how p62 influences protein aggregation and ALS disease progression in vivo using p62 KO mice. The results from this study will provide invaluable insights into the role of p62 in protein aggregation and neurodegeneration in ALS, which will result in better understanding of ALS etiology and potential discovery of new therapeutic target for ALS treatment. Project Narrative This collaborative project between Dr. Zhu at University of Kentucky and Dr. Wooten at Auburn University is to understand the role of p62 in mutant SOD1 induced protein aggregation and motor neuron degeneration in amyotrophic lateral sclerosis (ALS), an age-related neurodegenerative disease. The innovative aspect of this project is that the role of p62 in the ALS etiology is largely undefined. P62 is a multifunctional protein involved in both of the two major protein degradation mechanisms, ubiquitin-proteasome system (UPS) and autophagy- lysosome pathway. The central hypotheses to be tested in the project are that p62 can recognize misfolded mutant SOD1 and that p62 can ameliorate the mutant SOD1 induced toxicity by shuttling such misfolded proteins to UPS and/or autophagy. We have obtained substantial amount of preliminary data that strongly support these hypotheses. Three specific aims are designed to further characterize the molecular details regarding the role of p62 in protein homeostasis (degradation and aggregation) and neuronal survival in ALS. Aim 1 is to map the domains of p62 essential for recognizing and interacting with mutant SOD1. Aim 2 is to determine whether and how p62 mediates the autophagy activation induced by mutant SOD1. Aim 3 is to study how p62 influences protein aggregation and ALS disease progression in vivo using p62 KO mice. The results from this study will provide invaluable insights into the role of p62 in protein aggregation and neurodegeneration in ALS, which will result in better understanding of ALS etiology and potential discovery of new therapeutic target for ALS treatment.