Molecular mechanisms underlying mitochondria-lysosome membrane contact sites in neuronal function and neurodegeneration

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Both mitochondria and lysosomes are critical for regulating neuronal metabolism and function, and dysfunction of both organelles has been implicated in multiple neurodegenerative diseases including Parkinson’s and Charcot-Marie-Tooth (CMT) disease. However, the interplay between these two organelles in regulating neuronal homeostasis and driving neurodegeneration are still not well understood. Inter-organelle membrane contacts form between two different organelles and are critical sites for mediating organelle dynamics, metabolite exchange and signaling, but whether mitochondria and lysosomes form similar membrane contact sites to regulate their functional crosstalk was previously unknown. I recently identified the formation and regulation of mitochondria-lysosome membrane contact sites which represent a new pathway for the bidirectional regulation of mitochondria and lysosomes, but the role of these contact sites in neurons has not yet been explored. Importantly, further elucidating the neuronal role of mitochondria-lysosome contacts provides important insight into coupled mitochondrial and lysosomal function in neurons and potential pathways for their coupled dysfunction in multiple neurodegenerative diseases. In this project, I propose to investigate the molecular mechanisms underlying mitochondria-lysosome contact function in healthy and diseased neurons during both the K99 and R00 phases using long-term cultures of human induced pluripotent stem cell (iPSC)-derived neurons grown on micropatterned substrates to facilitate organelle imaging via advanced microscopy techniques including super-resolution imaging, electron microscopy and high spatial and temporal resolution live cell microscopy. In Aim 1, I will investigate how mitochondria-lysosome contacts regulate neuronal health and homeostasis by examining 1) the bidirectional relationship between mitochondrial trafficking and mitochondria-lysosome contacts in axons, and 2) the role of contacts in regulating calcium and lipid dynamics and exchange between mitochondria and lysosomes in neurons. Moreover, as lysosomal Rab7 GTP hydrolysis from GTP-bound state to GDP-bound state driven by a mitochondrial GAP (GTPase activating protein) regulates mitochondria-lysosome contact dynamics, disruption of Rab7 may contribute to neurodegeneration by misregulating contact dynamics and downstream lysosomal and mitochondrial function. In Aim 2, I will investigate the role of Rab7-mediated mitochondria-lysosome contact misregulation in the neurodegeneration of two diseases genetically and functionally linked to both mitochondrial and lysosomal dysfunction: 1) Parkinson’s disease in which various familial genes disrupt Rab7 GTP state, and 2) CMT as autosomal dominant mutations in Rab7 result in CMT Type 2B. Together, the proposed research and career plan offers important new training in experimental techniques and disease modeling, which are essential for my transition to independence and for ultimately achieving my long-term goal of uncovering cellular mechanisms underlying disease pathogenesis at the intersection of inter-organelle contacts andneurodegeneration.