Investigating the Contribution of Motor Neuronal Mitochondria to Charcot Marie Tooth Type 2D (CMT2D)

PROJECT SUMMARY Charcot-Marie Tooth disease (CMT) is a rare, heritable, and heterogeneous peripheral neuropathy that can affect both motor and sensory neurons. There is currently no cure or pharmacological treatments, and the many CMT subtypes make finding common therapeutic routes difficult. Healthy mitochondria are critical for proper neuronal function and dysfunctional mitochondria have been shown to be associated with neurodegenerative diseases. Many subtypes of CMT have causative mutations in nuclear-encoded mitochondrial proteins, such as MFN2, GDAP1, and COX6A1. The goal of this project is to characterize mitochondrial morphology, function, and proteomes in CMT2D (caused by mutations in GARS), which is a genetically bifunctional tRNA synethetase, catalyzing the addition of glycine to tRNAGly in both mitochondrial and cytosolic translation. Mouse models of CMT2D have a cell-type specific reduction in mitochondria levels in motor neurons, and transcription signatures indicating mitochondrial dysfunction. The long-term goal of this project is to elucidate how perturbations in motor neuronal mitochondria are impacting the functionality of motor neurons in CMT2D and extend these findings to related peripheral neuropathies. This proposal is novel because it is the first to investigate neuronal compartment- and cell-type specific mitochondrial protein expression in CMT and how changes lead to length-dependent peripheral neurodegeneration. I hypothesize that mutated glycyl tRNA synthetase (GARS) will impact mitochondrial translation leading to altered expression and reduced functionality of mitochondrial proteins (largely electron transport chain (ETC) complexes Aim 1 will investigate Gars human iPSC-derived motor neurons, cultured in microfluidic devices to distinguish neuronal compartments. Aim 2 will measure mitochondrial protein levels through cell-type specific isolation of GFP- tagged mitochondria in Gars mice, with biochemical and immunofluorescence validation. Mitochondrial proteomics will be conducted in a neuronal compartmental specific fashion (distal axon vs. soma), alongside mitochondrial morphological, functional, and validation assays. Together, these experiments will not only establish how motoneuronal mitochondria are perturbed, but also compare cell-type and compartment-specific similarities and differences. My goals for training include (i) acquiring an experimental skillset that will enable me to probe cellular and molecular neurobiological mechanisms, (ii) gain proficiency at implementing a robust cell culture experimental model, and (iii) improve scientific communication and mentoring skills. These will assist me in becoming an independent research scientist in investigating the cellular mechanisms behind physiological phenomena in disease contexts. My graduate program, institution, and thesis mentor have successful training records, providing me with a resource-rich environment to execute the research strategy and achieve my training goals.