Regulation of mitochondrial motility and mitophagy by LRRK2.

描述（由申请人提供）：神经元轴突中的顺行线粒体运输由初级马达/衔接子复合物介导，所述初级马达/衔接子复合物包括马达蛋白KHC（驱动蛋白重链）和两个线粒体衔接子米尔顿和Miro（Glater等人，2006年）。在目前的模型中，Miro，一种线粒体外膜（OMM）蛋白，与米尔顿结合，milton又与KHC结合，以将线粒体募集到马达和微管（Guo等人，2005年，Fransson等人，2006年，Glater等人，2006年）。在我的博士后培训期间，我发现两个 PD蛋白PINK 1和Parkin靶向Miro降解以促进损伤诱导的线粒体自噬（Wang et al.，2011年）。这与我们在自己的实验室中进行的初步观察一致，即来自一名PD患者的突变型帕金成纤维细胞在线粒体损伤后未能降解Miro，并且线粒体自噬受损。最常见的遗传性PD是由LRRK 2基因中的G2019 S突变引起的。LRRK 2编码一个多结构域的Ser/Thr激酶，其功能未知，底物尚未确定。令人惊讶的是，我们发现Miro也保留在来自一名患有LRRK 2G 2019 S的PD患者的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LRRK 2G 2019 S iPSC（诱导型多能干细胞）衍生的神经元中，受损的线粒体不能停止并经历线粒体自噬，这让人想起在突变体PINK 1或Parkin啮齿动物模型中发现的那些（Wang et al.，2011年）。因此，LRRK 2G 2019 S就像PINK 1或Parkin突变一样，阻止Miro蛋白在受损线粒体上降解，破坏线粒体运动和线粒体自噬。然而，没有证据直接将LRRK 2与Miro和线粒体转运联系起来。不同PD突变导致相同表型的机制是什么？在本提案中，我们的目标是解开这个谜团。我们假设LRRK 2和PINK 1/Parkin以平行途径运作，但最终会聚集在共同的底物Miro上。在另一种模型中，LRRK 2可能不会直接磷酸化Miro，而是可以与PINK 1或Parkin发生遗传或物理相互作用并调节PINK 1或Parkin，从而通过影响Miro的翻转来控制受损的线粒体转运和清除。我们将在本研究提案中定义的分子机制将提供对LRRK 2相关PD发病机制的深入了解，特别是对于LRRK 2G 2019 S患者，其约占总病例的5-6%（博尼法蒂，2006）。除了与PD的相关性外，我们还希望我们的结果阐明神经元线粒体转运和清除的机制，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LRRK 2影响Miro周转的机制。目的2：确定LRRK 2和Miro之间的物理关系。目的3：探讨LRRK 2与PINK 1/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.