Xinnan Wang - US grants

Project Summary Misregulation of Mitochondrial Motility in Parkinsonian Pathogenesis I set out to understand the regulatory mechanisms underlying mitochondrial transport in cells as my long-term career goal. Mitochondria move and undergo fission and fusion in all eukaryotic cells, but the need to supply mitochondria to the far-flung extremities of neurons creates a particular urgency for mitochondrial transport in neurons. Misregulation of the transport and distribution of mitochondria in axons can be a critical component of neurodegeneration. I propose that the transport of mitochondria is particularly vital for maintaining neuronal function and that even subtle perturbation of their traffic may contribute to neurodegenerative disorders. Starting with a motor/adaptor complex including kinesin-1 heavy chain (KHC), milton and Miro that transports axonal mitochondria anterograde and having elucidated the mechanism how Ca++ regulates mitochondrial motility via this complex (Wang and Schwarz, 2009a), I now would like to investigate the involvement of this complex in neurodegeneration as my immediate goal. Specifically, I propose to focus on PINK1 and Parkin, mutations of which cause Parkinson's disease in humans. Because both proteins can localize to mitochondria and genetically interact, and because PINK1 resides in the outer mitochondrial membrane and interacts with KHC/milton/Miro complex (Zhou et al., 2008; Weihofen et al., 2009), I hypothesize that PINK1 and Parkin also participate in the regulation of mitochondrial transport by regulating KHC/milton/Miro activity, misregulation of which may explain the Parkinsonian neurodegeneration. I therefore propose to look at animal models of Parkinsonism that might involve impaired mitochondria, to determine if mitochondrial transport is abnormal, and to examine the underlying mechanisms. I plan to establish a link between misregulation of mitochondrial motility and Parkinsonian neurodegeneration in my mentored phase here in Children's Hospital Boston and Harvard Medical School, and continue to investigate the underlying mechanisms and the involvement of mitochondrial motility in other neurodegenerative diseases as an independent principal investigator.

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.

We seek to understand the regulatory mechanisms that control the movements of mitochondria in cells. Mitochondria move and undergo fission and fusion in all eukaryotic cells, but the need to supply mitochondria to the far-flung extremities of neurons creates a particular urgency for mitochondrial transport in neurons. Defects in mitochondrial movements can give rise to peripheral neuropathies and degeneration 1. We and our colleagues have identified a protein complex that includes milton, Miro, and the kinesin-1 heavy chain (KHC) and have shown that this complex is essential for the anterograde ((+)-end directed) transport of mitochondria in neurons. We have determined that Miro is degraded by the proteasome via PINK1/Parkin- dependent phosphorylation and ubiquitination. Additionally, and most relevantly to this proposal, we have obtained exciting preliminary results demonstrating a novel machinery spanning both the mitochondrial outer and inner membranes that stabilizes Miro. We now propose to further our understanding of this machinery by examining in greater detail how it is regulated by intracellular signals and neuronal activity. These signaling pathways are crucial to the ability of mitochondria to properly distribute themselves within a cell and to permit cells to respond to stresses and changes in activity. Aim 1: To determine the nature of the ternary complex spanning the mitochondrial membranes. Aim 2: To determine the regulatory signals of this ternary complex. Aim 3: To determine the functional relevance of this ternary complex.

We seek to understand the regulatory mechanisms that control the movements of mitochondria in cells. A protein complex that includes milton, Miro, and the kinesin-1 heavy chain (KHC) is essential for mitochondrial transport in neurons. Miro resides at the central hub to allow multiple cellular signals to control mitochondrial motility. These signals include mitophagy, calcium, hypoxia, and nutrient availability etc. Additionally, and most relevant to this proposal, we have obtained exciting preliminary results demonstrating that a novel machinery spanning both the mitochondrial outer and inner membranes?the mitochondrial intermembrane space bridging (MIB) complex, stabilizes Miro and regulates mitochondrial motility. This new discovery and our past work raise a burning question at the intersection of fundamental cell biology and neurobiology: How these discrete Miro-pathways functionally coordinate or converge on mitochondrial integrity in response to various stimuli and stresses? In this proposal, we will probe these questions using classical cell biological and neurobiological approaches combined with a powerful genetic tool (fruit flies) for functional validation. Aim 1: To dissect the functional significance of the MIB-Miro complex. Aim 2: To determine the nature of Miro interaction with its binding partners. Aim 3: To explore the interplay of the regulatory signals and machineries of Miro.