LRRK2 and oxidative stress in Parkinson’s disease

LRRK2 与帕金森病的氧化应激

• 批准号: 10799999
• 负责人: J Timothy Greenamyre
• 金额: $ 55.65万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10799999
• 关键词: 4 hydroxynonenal; Accounting; Address; Alanine; Automobile Driving; Brain Pathology; Cells; Chloroquine; Complex; Consensus; Curcumin; Cysteine; Disease; Dopamine; Genetic; Genetic Risk; Human; Hydrogen Peroxide; Idiopathic Parkinson Disease; Ionophores; Knock-out; LRRK2 gene; Link; Microglia; Mitochondria; Mitochondrial Proteins; Modeling; Monovalent Cations; Morphology; Mutate; Mutation; Nerve Degeneration; Neurons; Oxidation-Reduction; Oxidative Stress; Oxidative Stress Induction; Paraquat; Parkinson Disease; Pathogenesis; Pathogenicity; Patients; Phosphotransferases; Play; Prevention; Process; Production; Protein Import; Proteins; Rattus; Regulation; Reporting; Research Personnel; Risk Factors; Rodent Model; Role; Rotenone; Signal Transduction; Specificity; Stress; Substantia nigra structure; Toxic effect; Trichloroethylene; Variant; autosome; brain tissue; design; dihydroethidium; dopaminergic neuron; enzyme activity; experimental study; in vivo; kinase inhibitor; mitochondrial dysfunction; mutant; neurotoxicity; non-genetic; novel; novel therapeutic intervention; oxidation; pharmacologic; physiologic stressor; prevent; receptor; response; risk variant; stressor; tool; toxicant; trafficking

Mutations in LRRK2 are the most common cause of autosomal dominant Parkinson's disease (PD) and it appears that all such pathogenic mutations are associated with aberrantly enhanced LRRK2 kinase activity. Independent of mutations, however, there is also evidence that increased LRRK2 kinase activity contributes to the pathogenesis of idiopathic PD (iPD). The current project is designed to elucidate how wildtype (non-mutated) LRRK2 kinase activity is stimulated, where in the cell this occurs, and what the downstream consequences are. In keeping with the well-known association of iPD with oxidative stress and mitochondrial dysfunction, we focus on these processes in relation to LRRK2. Specifically, this project examines (i) the oxidative activation of LRRK2, (ii) the translocation of LRRK2 to mitochondria under conditions of mitochondrial stress, and (iii) LRRK2 kinase activity-dependent oxidative stress. The overarching premise of this proposal is that oxidative stress and LRRK2 kinase activity are intimately and bidirectionally intertwined in PD pathogenesis. The project has 3 broad Specific Aims to address these issues: Aim 1: To elucidate the oxidative activation of LRRK2 kinase, we will (a) assess/compare WT LRRK2 activation by physiological stressors (H2O2 and DA) to the PD-associated toxicants: rotenone, paraquat (PQ) and trichloroethylene (TCE); (b) Determine whether monensin & chloroquine activate LRRK2 via oxidative mechanisms; (c) Examine the role of cysteine residues C2024/5 in oxidative LRRK2 activation. Aim 2: To examine translocation of LRRRK2 to mitochondria, we will (a) Assess mitochondrial localization of LRRK2 in response to various mitochondrial stressors; (b) See if translocation requires (i) mitochondrial ROS, (ii) cytosolic ROS, (iii) LRRK2 activity or (iv) Cys2024/5; (c) Determine whether mitochondrial translocation of LRRK2 occurs in vivo in rat models of PD; (d) Evaluate whether LRRK2 association with mitochondria is aberrantly enhanced in human iPD brain tissue. Aim 3: To elucidate the role of LRRK2 kinase in oxidative stress and its downstream consequences, we will (a) Use genetic and pharmacological approaches to see if pathogenic LRRK2 mutations cause oxidative stress and whether blocking LRRK2 kinase activity prevents oxidative stress; (b) Examine the potential role of Cys2024/5 in driving LRRK2 kinase activity-dependent oxidative stress; (c) Determine if rotenone-induced mitochondrial ROS production is reduced by LRRK2 inhibition/knockout; (d) Determine if rotenone-induced cytosolic NOX2 activation is reduced by LRRK2 inhibition/knockout; (e) Assess ROS production in patient-derived healthy control, iPD and G2019S mutant lymphoblastoid cells. Together, these experiments will begin to elucidate the intimate and bidirectional relationship between oxidative stress and LRRK2 in PD pathogenesis. By doing so, new therapeutic strategies are likely to emerge.

LRRK 2突变是常染色体显性帕金森病（PD）的最常见原因， 所有这些致病突变都与异常增强的LRRK 2激酶活性有关。独立 然而，也有证据表明，LRRK 2激酶活性的增加有助于基因突变。 特发性PD（iPD）的发病机制。目前的项目旨在阐明野生型（非突变） LRRK 2激酶活性被刺激，在细胞中发生，以及下游后果是什么。 与众所周知的iPD与氧化应激和线粒体功能障碍的相关性一致，我们关注 与LRRK 2有关的这些过程。具体而言，该项目检查（i）LRRK 2的氧化活化， (ii)在线粒体应激条件下LRRK 2向线粒体的易位，和（iii）LRRK 2激酶 活性依赖性氧化应激。这项提议的首要前提是，氧化应激和LRRK 2 激酶活性在PD发病机制中紧密且双向地交织。 该项目有3个广泛的具体目标来解决这些问题： 目的1：为了阐明LRRK 2激酶的氧化活化，我们将（a）评估/比较WT LRRK 2活化 生理应激源（H2 O2和DA）对PD相关毒物：鱼藤酮、百草枯（PQ）和 （B）确定莫能菌素和氯喹是否通过氧化活化LRRK 2 （c）检查半胱氨酸残基C2024/5在氧化LRRK 2活化中的作用。 目的2：为了检测LRRRK 2向线粒体的易位，我们将（a）评估LRRRK 2的线粒体定位， LRRK 2响应于各种线粒体应激物;（B）观察易位是否需要（i）线粒体ROS， (ii)细胞溶质ROS，（iii）LRRK 2活性或（iv）Cys 2024/5;（c）确定是否存在线粒体易位， LRRK 2在PD的大鼠模型中体内发生;（d）评估LRRK 2与线粒体的缔合是否是在PD的大鼠模型中发生的。 在人类iPD脑组织中异常增强。 目的3：为了阐明LRRK 2激酶在氧化应激及其下游后果中的作用，我们将（a） 使用遗传和药理学方法来观察致病性LRRK 2突变是否会导致氧化应激， 阻断LRRK 2激酶活性是否防止氧化应激;（B）检查Cys 2024/5的潜在作用 在驱动LRRK 2激酶活性依赖性氧化应激中的作用;（c）确定鱼藤酮诱导的线粒体氧化应激 通过LRRK 2抑制/敲除减少ROS产生;（d）确定鱼藤酮诱导的细胞溶质N 0X 2 通过LRRK 2抑制/敲除降低活化;（e）评估患者来源的健康人中的ROS产生。 对照、iPD和G2019 S突变型淋巴母细胞样细胞。 总之，这些实验将开始阐明氧化和细胞凋亡之间的密切和双向关系。 应激和LRRK 2在PD发病机制中作用通过这样做，可能会出现新的治疗策略。