Regulation of mitochondrial motility and mitophagy by LRRK2.

描述(由申请人提供)：神经元轴突中的线粒体顺行运输是由一个初级运动/适配器复合体介导的，该复合体包括运动蛋白KHC(运动蛋白重链)和两个线粒体适配器Milton和Miro(Glate等人，2006年)。在目前的模型中，Miro是一种外线粒体膜(OMM)蛋白，与Milton结合，Milton又与KHC结合，将线粒体招募到发动机和微管(Guo等人，2005年，Fransson等人，2006年，Glate等人，2006年)。在我的博士后培训期间，我发现有两个 PD蛋白PINK1和Parkin针对Miro进行降解，以促进损伤诱导的有丝分裂(Wang等人，2011年)。这与我们在自己的实验室进行的初步观察一致，即来自一名PD患者的突变Parkin成纤维细胞在线粒体损伤后未能降解MIRO，并损害了有丝分裂。遗传性帕金森病最常见的遗传形式是由LRRK2基因G2019S突变引起的。LRRK2编码一个功能未知、底物未知的多结构域丝氨酸/苏氨酸蛋白激酶。令人惊讶的是，我们发现Miro也保留在一名患有LRRK2G2019S的PD患者成纤维细胞中受损的线粒体上。此外，我们有初步证据表明，在来自两名PD患者的LRRK2G2019S IPSC(可诱导多能干细胞)来源的神经元中，受损的线粒体无法停止并进行有丝分裂，这让人想起在突变的PINK1或Parkin啮齿动物模型中发现的那些线粒体(Wang等人，2011年)。因此，LRRK2G2019S就像PINK1或Parkin突变一样，阻止Miro蛋白对受损线粒体的降解，扰乱线粒体的运动性和有丝分裂。然而，目前还没有证据表明LRRK2与MIRO和线粒体运输直接相关。由不同的PD突变引起的相同表型背后的机制是什么？在这项提案中，我们的目标是解开这个谜团。我们假设LRRK2和PINK1/Parkin平行运行，但最终在共同的底物MIRO上收敛。在另一种模型中，LRRK2可能不会直接磷酸化MIRO，而是可以在基因或物理上与PINK1或PARKIN相互作用并调节，通过影响MIRO的翻转来控制受损的线粒体运输和清除。我们将在这项研究中定义的分子机制将有助于深入了解LRRK2相关的PD发病机制，特别是LRRK2G2019S患者，约占总病例的5-6%(Bonifati，2006)。除了与帕金森病的相关性外，我们还希望我们的结果阐明神经元线粒体运输和清除的机制，以阐明线粒体生物学和神经生物学的基本原理。因此，我们建议使用果蝇遗传学、培养细胞，甚至PD患者成纤维细胞和衍生神经元的组合来追求以下特定目标。目的1：确定LRRK2影响Miro周转的机制。目的2：确定LRRK2和MIRO之间的生理联系。目的：分析LRRK2与PINK1/Parkin通路的关系。

DESCRIPTION (provided by applicant): Anterograde mitochondrial transport in neuronal axons is mediated by a primary motor/adaptor complex which includes motor protein KHC (kinesin heavy chain), and two mitochondrial adaptors milton and Miro (Glater et al., 2006). In the current model, Miro, an outer mitochondrial membrane (OMM) protein, binds to milton which in turn binds to KHC, to recruit mitochondria to motors and microtubules (Guo et al., 2005, Fransson et al., 2006, Glater et al., 2006). During my postdoctoral training, I discovered that two PD proteins PINK1 and Parkin target Miro for degradation to promote damage-induced mitophagy (Wang et al., 2011). This is consistent with our preliminary observation conducted in my own laboratory that mutant Parkin fibroblasts from one PD patient are failed to degrade Miro after mitochondrial damage and are impaired in mitophagy. The most common genetic form of hereditary PD is caused by a G2019S mutation in the LRRK2 gene. LRRK2 encodes a multi-domain Ser/Thr kinase with unknown functions and unconfirmed substrates. Surprisingly, we found that Miro is also retained on damaged mitochondria in fibroblasts from one PD patient with LRRK2G2019S. In addition, we have preliminary evidence that in LRRK2G2019S iPSC (inducible pluripotent stem cells)-derived neurons from two PD patients, damaged mitochondria fail to stop and to undergo mitophagy, reminiscent of those found in mutant PINK1 or Parkin rodent models (Wang et al., 2011). Therefore, LRRK2G2019S, just like PINK1 or Parkin mutations, prevents Miro protein from degradation on damaged mitochondria, disrupting mitochondrial motility and mitophagy. However, there has been no evidence directly linking LRRK2 to Miro and mitochondrial transport. What is the mechanism underlying the same phenotype caused by distinct PD mutations? In this proposal, we aim to unravel this puzzle. We hypothesize that LRRK2 and PINK1/Parkin operate in parallel pathways but eventually converge on the common substrate Miro. In an alternative model, LRRK2 may not directly phosphorylate Miro, but rather it could genetically or physically interact with and regulate PINK1 or Parkin to control damaged mitochondrial transport and clearance by influencing turn-over of Miro. The molecular mechanism we will define in this research proposal will provide insight into LRRK2-related PD pathogenesis, especially for patients with LRRK2G2019S, which represents about 5-6% of the total cases (Bonifati, 2006). In addition to its relevance to PD, we also expect our results elucidating the mechanisms underlying neuronal mitochondrial transport and clearance to illuminate basic principles of mitochondrial biology and neurobiology. Thus, we propose to use a combination of Drosophila genetics, cultured cells, and even PD patient fibroblasts and derived neurons to pursue the following Specific Aims. Aim 1: To determine the mechanism by which LRRK2 influences the turnover of Miro. Aim 2: To determine the physical relationship between LRRK2 and Miro. Aim 3: To dissect the relationship between LRRK2 and the PINK1/Parkin pathway.