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.

Public Health Relevance

Parkinson's disease (PD), one of the most common neurodegenerative diseases, afflicts millions of people worldwide with tremendous global economic and societal burdens. An effective treatment is desperately needed but the underlying molecular and cellular mechanisms of PD's destructive path remain poorly understood. This proposal aims to dissect the cellular functions of a gene implicated in PD, LRRK2, and is therefore relevant to public health and the mission of the NIH, as it will illuminate the pathological causes of PD and provide novel targets for therapeutic intervention.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
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Neural Oxidative Metabolism and Death Study Section (NOMD)
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Sieber, Beth-Anne
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Stanford University
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Shaltouki, Atossa; Hsieh, Chung-Han; Kim, Min Joo et al. (2018) Alpha-synuclein delays mitophagy and targeting Miro rescues neuron loss in Parkinson's models. Acta Neuropathol 136:607-620
Course, Meredith M; Hsieh, Chung-Han; Tsai, Pei-I et al. (2017) Live Imaging Mitochondrial Transport in Neurons. Neuromethods 123:49-66
Wang, Xinnan (2017) Destructive cellular paths underlying familial and sporadic Parkinson disease converge on mitophagy. Autophagy 13:1998-1999
Hsieh, Chung-Han; Shaltouki, Atossa; Gonzalez, Ashley E et al. (2016) Functional Impairment in Miro Degradation and Mitophagy Is a Shared Feature in Familial and Sporadic Parkinson's Disease. Cell Stem Cell 19:709-724
van der Bliek, Alexander; Wang, Xinnan (2016) Organelles and spatial organization of the cell: organelle homeostasis and turnover. Mol Biol Cell 27:873-4