Role of LRRK2 in idiopathic Parkinson's disease

LRRK2 在特发性帕金森病中的作用

• 批准号: 10224659
• 负责人: J Timothy Greenamyre
• 金额: $ 34.23万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10224659
• 关键词: Aftercare; Anatomy; Antibodies; Antioxidants; Autopsy; Binding; Biochemical; Biological Assay; Brain; Cells; Co-Immunoprecipitations; Consensus; Dissociation; Gene Proteins; Hippocampus (Brain); Human; Hydrogen Peroxide; Idiopathic Parkinson Disease; In Situ; In Vitro; Individual; LRRK2 gene; Ligation; Mediating; Microglia; Mitochondria; Mitochondrial DNA; Modeling; Mutation; Nerve Degeneration; Neuroglia; Neurons; Oxidative Stress; Parkinson Disease; Pathogenesis; Pathology; Phosphorylation; Phosphotransferases; Physiological; Play; Population; Post-Translational Protein Processing; Proteins; Rattus; Resolution; Risk Factors; Role; Rotenone; Signal Transduction; Specificity; Specimen; Western Blotting; alpha synuclein; base; brain tissue; case control; cell type; disease-causing mutation; dorsal motor nucleus; genomic locus; human tissue; immunocytochemistry; in vivo; inhibitor/antagonist; kinase inhibitor; knock-down; lymphoblast; mutant; mutation carrier; nigrostriatal system; novel; olfactory bulb; overexpression; oxidation; virtual

Mutations in LRRK2 cause familial Parkinson disease (PD) and, in some populations, may account for up to 40% of all cases3. The LRRK2 gene locus also contains a risk factor for `idiopathic' PD (iPD); however, the role of LRRK2 in typical iPD is not clear. Furthermore, the relationship of LRRK2 to other genes and proteins associated with PD, such as α-synuclein, is also relatively unexplored. While the mechanism(s) by which mutant LRRK2 causes neurodegeneration are not entirely certain, it is generally believed that disease-causing mutations may be associated with increased kinase activity, at least in situ in intact cells. However, a critical barrier to understanding the role of endogenous, wildtype LRRK2 in iPD is the absence of a practical, high-resolution assay for its activation state. We have developed and validated a pair of novel proximity ligation assays with excellent anatomical resolution that can rapidly provide information regarding activation state, cellular localization and physiological regulators of LRRK2. The assay is based on (i) S1292 phosphorylation and (ii) dissociation of 14-3-3 from LRRK2. Using this and other assays, we have preliminary evidence that (i) LRRK2 is activated in nigrostriatal neurons in iPD; (ii) sublethal concentrations of rotenone activate LRRK2; (iii) overexpression of α-synuclein in vivo activates LRRK2 in nigrostriatal neurons; (iv) LRRK2 activation is mediated by oxidative mechanisms. We now propose to examine the role of LRRK2 in iPD and its potential physiological interactions with α-synuclein and mitochondria. We will investigate the following questions: (1) What is the activation state of LRRK2 in human iPD brain tissue? (2) To what extent can endogenous LRRK2 activation be modeled in the rotenone rat? (3) Does!α-synuclein activate endogenous WT LRRK2? (4) Does oxidative stress activate LRRK2?

LRRK2 突变会导致家族性帕金森病 (PD)，在某些人群中，可能高达 40% 案例3. LRRK2 基因位点还包含“特发性”PD (iPD) 的危险因素；然而，LRRK2 的作用 典型的iPD尚不清楚。此外，LRRK2 与其他与 PD 相关的基因和蛋白质的关系，例如 α-突触核蛋白也相对未经探索。 LRRK2突变体引起神经退行性变的机制 并不完全确定，一般认为致病突变可能与激酶增加有关 活性，至少在完整细胞中的原位活性。然而，理解内源性野生型的作用的一个关键障碍 iPD 中的 LRRK2 缺乏对其激活状态的实用、高分辨率测定。 我们开发并验证了一对具有出色解剖分辨率的新型邻近连接检测方法，可以 快速提供有关 LRRK2 激活状态、细胞定位和生理调节因子的信息。这 测定基于 (i) S1292 磷酸化和 (ii) 14-3-3 从 LRRK2 的解离。使用这个和其他分析，我们 有初步证据表明 (i) LRRK2 在 iPD 的黑质纹状体神经元中被激活； (二) 亚致死浓度 鱼藤酮激活 LRRK2； (iii)体内α-突触核蛋白的过度表达激活黑质纹状体神经元中的LRRK2； （四） LRRK2 激活由氧化机制介导。我们现在建议研究 LRRK2 在 iPD 中的作用及其 与 α-突触核蛋白和线粒体的潜在生理相互作用。我们将调查以下问题：（1） 人iPD脑组织中LRRK2的激活状态如何？ (2) 内源性LRRK2激活到什么程度 以鱼藤酮大鼠为模型？ (3)α-突触核蛋白是否激活内源性WT LRRK2？ (4)氧化应激是否激活 LRRK2？