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宣布竞争性修订申请的恢复法案资金可用性”的回应。“我们要求对我们的NIH资助R21 AG 032567进行竞争性补充，标题为“p62在ALS中蛋白质聚集和神经变性中的作用”。“含有突变型铜锌超氧化物歧化酶（SOD 1）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（以下简称p62）是一种多功能蛋白，参与泛素-蛋白酶体系统（UPS）和自噬-溶酶体途径两种主要的蛋白质降解机制。在亲本R21项目中待测试的中心假设是p62可以识别错误折叠的突变体SOD 1，并且p62可以通过将这种错误折叠的蛋白质穿梭于UPS和/或自噬来改善突变体SOD 1诱导的毒性。我们以前报道过，p62与突变型SOD 1共定位于G93 A SOD 1转基因小鼠脊髓运动神经元的蛋白聚集体中。免疫共沉淀实验表明，p62特异性识别突变体SOD 1，但不是WT蛋白。当时设计了三个具体目标来检验这一假设。目的1是定位p62识别突变SOD 1并与之相互作用所必需的结构域。目的2是确定p62是否以及如何介导突变SOD 1诱导的自噬激活。目的3是使用p62 KO小鼠研究p62如何影响体内蛋白质聚集和ALS疾病进展。我们已经基本实现了目标1和2，目前正在进行目标3中提出的实验。 这一竞争性修订是建立在R21项目取得的进展和新的初步数据的基础上的，这些数据表明HDAC 6可能是p62和突变SOD 1之间的衔接蛋白。此外，HDAC 6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项目来测试一个新的假设，即HDAC 6是介导突变体SOD 1-p62相互作用的衔接蛋白，调节突变体SOD 1的自噬降解，并介导突变体SOD 1诱导的应激反应。提出了三个新的具体目标来检验这一假设。目的1是剖析HDAC 6作为突变SOD 1和p62之间的衔接子的详细机制。 目的2是确定HDAC 6在突变SOD 1的聚集和自噬降解中的作用。 目的3研究HDAC 6和p62在突变型SOD 1诱导的应激反应中的意义。这项研究的结果将为HDAC 6和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病因学，并可能发现ALS治疗的新靶点。 与《美国复苏与再投资法案》（ARRA）的相关性：该申请一旦获得资助，将创建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ARRA所预期的经济。 公共卫生相关性：肌萎缩侧索硬化症（Amyotrophic lateral sclerosis，ALS）是一种以运动神经元优先死亡为特征的与年龄相关的神经退行性疾病。编码铜锌超氧化物歧化酶（SOD 1）的基因突变与家族性ALS病例的一个子集有关。含有突变SOD 1的蛋白质聚集体是家族性ALS的标志。 P62/Sequestome 1（以下简称p62）是一种多功能蛋白，参与泛素-蛋白酶体系统（UPS）和自噬-溶酶体途径两种主要的蛋白质降解机制。亲本R21是为了测试p62可以识别错误折叠的突变体SOD 1并通过将这些错误折叠的蛋白穿梭到UPS和/或自噬来改善突变体SOD 1诱导的毒性的假设。我们已经基本上完成了R21中提出的目标1和2，目前正在进行目标3中提出的实验。基于这些进展和新的初步结果，我们建议测试一个名为组蛋白去乙酰化酶6（HDAC 6）的衔接蛋白在突变体SOD 1蛋白降解、聚集和神经毒性中的意义。在扩展项目中待检验的具体假设是HDAC 6是介导突变体SOD 1-p62相互作用的衔接蛋白，调节突变体SOD 1的自噬降解，并介导突变体SOD 1诱导的应激反应。 这项研究的结果将为HDAC 6和p62在ALS蛋白聚集和神经变性中的作用提供新的见解，这将有助于更好地了解ALS病因学，并可能发现ALS治疗的新靶点。此外，这项申请一旦获得资助，将创造两个新的科研人员职位。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《美国复苏与再投资法案》所预期的那样。

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.