LRRK2 and oxidative stress in Parkinson’s disease

LRRK2基因突变是常染色体显性遗传性帕金森病(PD)的最常见原因，它似乎 所有这些致病突变都与LRRK2激酶活性的异常增强有关。独立的 然而，也有证据表明，LRRK2激酶活性增加有助于 特发性帕金森病的发病机制。目前的项目旨在阐明野生型(非突变)是如何 LRRK2激酶活性被刺激，在细胞中发生这种情况，以及下游的后果是什么。 为了与众所周知的IPD与氧化应激和线粒体功能障碍的联系保持一致，我们关注 与LRRK2相关的这些过程。具体地说，这个项目研究了(I)LRRK2的氧化激活， (Ii)LRRK2在线粒体应激条件下移位到线粒体，和(Iii)LRRK2激酶 活性依赖的氧化应激。这一提议的首要前提是氧化应激和LRRK2 蛋白激酶活性在帕金森病发病机制中密切相关、双向交织。 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LRRK2的氧化激活，我们将(A)评估/比较WT LRRK2的激活 通过生理应激源(H_2O_2和DA)对与PD相关的毒物鱼藤酮、百草枯(PQ)和 三氯乙烯(TCE)；(B)确定莫能菌素和氯喹是否通过氧化激活LRRK2 机制；(C)研究半胱氨酸残基C2024/5在氧化LRRK2激活中的作用。 目的：为了检测LRRRK2易位到线粒体，我们将(A)评估线粒体的定位。 LRRK2对各种线粒体应激源的反应；(B)看看易位是否需要(I)线粒体ROS， (Ii)胞质ROS，(Iii)LRRK2活性或(Iv)Cys2024/5；(C)确定线粒体易位是否 LRRK2在帕金森病大鼠模型体内发生；(D)评估LRRK2与线粒体是否存在关联 在人IPD脑组织中异常增强。 目的3：为了阐明LRRK2激酶在氧化应激中的作用及其下游后果，我们将(A) 使用遗传学和药理学方法来研究致病的LRRK2突变是否会导致氧化应激和 阻断LRRK2激酶活性是否能防止氧化应激；(B)研究Cys2024/5的潜在作用 在驱动LRRK2活性依赖的氧化应激中；(C)确定鱼藤酮是否诱导线粒体 通过抑制/敲除LRRK2减少ROS的产生；(D)确定鱼藤酮是否诱导胞质NOX2 通过抑制/敲除LRRK2来减少激活；(E)评估患者来源的健康人群的ROS产生 对照、IPD和G2019S突变型淋巴母细胞。 总之，这些实验将开始阐明氧化之间的密切和双向关系 应激与LRRK2在帕金森病发病机制中的作用通过这样做，新的治疗策略可能会出现。

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.