Role of p62 in Protein Aggregation and Neurodegeneration in ALS

描述(由申请人提供)：本竞争性修订申请是对NOT-OD-09-058“NIH宣布恢复法资金可用于竞争性修订申请”的回应。我们要求对我们的NIH拨款R21AG032567进行竞争性补充，题为“p62在ALS中的蛋白质聚集和神经变性中的作用”。含有突变的铜锌超氧化物歧化酶(SOD1)的蛋白质聚集体是家族性肌萎缩侧索硬化症(ALS，Lou Gehrig病)的标志，这是一种与年龄相关的神经退行性疾病。P62/Sequestosome 1是一种多功能蛋白，参与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。要在父R21项目中检验的中心假设是，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UPS和/或自噬来改善突变的SOD1引起的毒性。我们曾报道在G93A SOD1转基因小鼠脊髓运动神经元的蛋白聚集体中，p62与突变型SOD1共定位。免疫共沉淀实验表明，p62能特异性识别突变体SOD1，但不能识别WT蛋白。当时设计了三个具体的目标来检验这一假设。目的1是定位p62的结构域，这些结构域对于识别突变的SOD1并与之相互作用是必不可少的。目的2确定p62是否以及如何介导突变型SOD1诱导的自噬激活。目的3是利用p62 KO小鼠研究p62对体内蛋白聚集和ALS疾病进展的影响。我们已经基本实现了目标1和目标2，目前正在进行目标3中提出的实验。这一竞争性修订是基于R21项目中取得的进展和新的初步数据，表明HDAC6可能是p62和突变体SOD1之间的适配蛋白。此外，HDAC6已被证明在自噬激活和应激反应中发挥关键作用。因此，我们建议扩展亲本R21项目来检验一个新的假设，即HDAC6是一个介导突变体SOD1-p62相互作用的适配蛋白，调节突变体SOD1的自噬降解，并介导突变体SOD1诱导的胁迫反应。为了检验这一假设，本文提出了三个新的具体目标。目的1是剖析HDAC6如何作为突变体SOD1和p62之间的接头的详细机制。目的2确定HDAC6在突变体SOD1聚集和自噬降解中的作用。目的3研究HDAC6和p62在突变型SOD1诱导的应激反应中的意义。这项研究的结果将为了解HDAC6和p62在ALS蛋白聚集和神经退变中的作用提供宝贵的见解，这将有助于更好地理解ALS的病因，并可能发现ALS治疗的新靶点。与美国复苏与再投资法案(ARRA)相关：一旦获得资金，这项申请将创造两个新的科学研究人员职位。为拟议的研究购买必要的设备、用品和试剂也将刺激经济，正如ARRA所希望的那样。 公共卫生相关性：肌萎缩侧索硬化症(ALS，Lou Gehrig病)是一种以运动神经元优先死亡为特征的年龄相关性神经退行性疾病。编码铜锌超氧化物歧化酶(SOD1)的基因突变与家族性肌萎缩侧索硬化症病例的子集有关。含有突变SOD1的蛋白质聚集体是家族性肌萎缩侧索硬化症的标志。P62/Sequestome 1是一种多功能蛋白，参与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。亲本R21将检验P62可以识别错误折叠的突变体SOD1的假设，并通过将这些错误折叠的蛋白质穿梭到UPS和/或自噬来改善突变体SOD1的毒性。我们已经基本实现了亲本R21中提出的目标1和目标2，目前正在进行目标3中提出的实验。根据研究进展和新的初步结果，我们建议测试一个名为组蛋白脱乙酰酶6(HDAC6)的接头蛋白在突变的SOD1蛋白降解、聚集和神经毒性中的意义。在扩展项目中要检验的具体假设是，HDAC6是一个适配蛋白，介导突变的SOD1-p62相互作用，调节突变的SOD1的自噬降解，并介导突变的SOD1诱导的应激反应。本研究结果将对HDAC6和p62在ALS蛋白聚集和神经退行性变中的作用提供新的见解，这将有助于更好地理解ALS的病因，并可能发现ALS治疗的新靶点。此外，这项申请一旦获得资金，将创造两个新的科学研究人员职位。为拟议的研究购买必要的设备、用品和试剂也将刺激经济，正如美国复苏和再投资法案所希望的那样。

DESCRIPTION (provided by applicant): This Competitive Revision application is in response to NOT-OD-09-058 "NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications." We request competitive supplement to our NIH grant R21AG032567 entitled "Role of p62 in Protein Aggregation and Neurodegeneration in ALS." 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. P62/Sequestosome 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. The central hypothesis to be tested in the parent R21 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 UPS and/or autophagy. We previously reported that p62 was co-localized with mutant SOD1 in the protein aggregates in spinal motor neurons in G93A SOD1 transgenic mice. Co-immunoprecipitation experiments showed that p62 specifically recognized mutant SOD1, but not the WT protein. Three specific aims were designed to test the hypothesis at the time. Aim 1 was to map the domains of p62 essential for recognizing and interacting with mutant SOD1. Aim 2 was to determine whether and how p62 mediated the autophagy activation induced by mutant SOD1. Aim 3 was to study how p62 would influence protein aggregation and ALS disease progression in vivo using p62 KO mice. We have largely accomplished the Aims 1 and 2 and are currently performing experiments proposed in Aim 3. This Competitive Revision is built on the progress made in the R21 project and the new preliminary data suggesting that HDAC6 is likely an adaptor protein between p62 and mutant SOD1. In addition, HDAC6 has been shown to play a critical role in autophagy activation and stress response. We thus propose to expand the parent R21 project to test a new hypothesis that HDAC6 is an adaptor protein mediating mutant SOD1-p62 interaction, regulate the autophagic degradation of mutant SOD1, and mediate the stress response induced by mutant SOD1. Three new specific aims are proposed to test this hypothesis. Aim 1 is to dissect the detailed mechanism how HDAC6 functions as an adaptor between mutant SOD1 and p62. Aim 2 is to determine the role of HDAC6 in aggregation and autophagic degradation of mutant SOD1. Aim 3 is to study the significance of HDAC6 and p62 in mutant SOD1 induced stress response. Results from this study will provide invaluable insights into the role of HDAC6 and 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. Relevance to the American Recovery & Reinvestment Act (ARRA): This application will create two new scientific researcher positions as soon as it is funded. Purchase of necessary equipment, supplies and reagents for the proposed research will also stimulate the economy as intended by the ARRA. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease) is an age-related neurodegenerative disorder characteristic of the preferential motor neurons death. Mutations in the gene encoding copper-zinc superoxide dismutase (SOD1) have been linked to a subset of familial ALS cases. Protein aggregates containing mutant SOD1 are a hallmark of familial ALS. 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. The parent R21 was to test the hypothesis that p62 could recognize misfolded mutant SOD1 and ameliorate the mutant SOD1 induced toxicity by shuttling such misfolded proteins to the UPS and/or autophagy. We have largely accomplished the Aims 1 and 2 proposed in the parent R21 and are currently performing experiments proposed in Aim 3. Based on the progress and new preliminary results, we propose to test the significance of an adaptor protein named histone deacetylase 6 (HDAC6) in mutant SOD1 protein degradation, aggregation and neurotoxicity. The specific hypothesis to be tested in the expanded project is that HDAC6 is an adaptor protein mediating mutant SOD1-p62 interaction, regulates the autophagic degradation of mutant SOD1, and mediates the stress response induced by mutant SOD1. Results from this study will provide new insights into the role of HDAC6 and 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. Moreover, this application will create two new scientific researcher positions as soon as it is funded. Purchase of necessary equipment, supplies and reagents for the proposed research will also stimulate the economy as intended by the American Recovery & Reinvestment Act.