How dominant mutations in glycyl tRNA synthetase (GARS) cause Charcot-Marie-Tooth disease Type 2D (CMT2D) peripheral neuropathy is still unclear and controversial. The technical challenge of studying the mammalian peripheral axon in vivo has contributed to the lack of a disease mechanism. The long-term goal of this project is to understand how mutations in ubiquitously expressed GARS lead to the specific and progressive degeneration of peripheral axons. The immediate objective is to use an in vivo, cell type- and compartment-specific approach to test the hypothesis that mutations in Gars cause impaired translation in two mouse models of CMT2D. Evidence points to a toxic gain-of-function of mutant GARS as the cause of neuropathy, but a dominant negative mechanism has not been ruled out. In Drosophila, overexpression of human mutant GARS in peripheral neurons causes neurodegeneration and reduced translation without altering aminoacylation. Because at least four other tRNA synthetases are linked to Charcot-Marie-Tooth, impaired translation is an attractive potential disease mechanism, and its detailed testing through the following experiments will further elucidate the gain-of function vs. dominant negative question: 1) We will test the hypothesis that mutant GARS impairs translation in motor neurons using two in vivo, cell type-specific techniques; non-canonical amino acid-tagging (NCAT) will provide the location, identity, and quantity of newly translated proteins, and ribosome-tagging will catalog ribosome-associated RNA. Motor neuron cell bodies are gathered from the spinal cord and axons from the sciatic nerve, providing cell compartment-specificity. Although local translation in adult, mammalian axons has not been established, it is required for normal regeneration after injury and preliminary data show that ribosomes are present and associated with mRNA in motor axons of the sciatic nerve. Wild-type sciatic nerve controls will be used to establish axonal translation. 2) We will test the hypothesis that translational impairments are independent of transcriptional changes. Thiouracil (4-TU)-tagging, a third in vivo, cell type-specific technique, will be used to catalog newly transcribed RNA in motor neuron cell bodies and newly transported RNA in axons. 3) Finally, we will test the hypothesis that mutant Gars axons attempt regeneration, but fail because of impairments in translation. NCAT, ribosome-, and 4-TU-tagging will be performed using wild-type regenerating motor neurons and the data compared to Gars mutant samples. The proposed experiments will test a hypothesized disease mechanism and uncover new motor neuron biology. Their completion will result in a comprehensive profile of translation and transcription in CMT2D, wild-type, and regenerating wild-type motor neuron cell bodies and axons. Identifying local translation in peripheral axons will represent a departure from the current model of axonal homeostasis, revealing the axon as a cell compartment with unique translational needs and its own ways of meeting them. Dependence of axons on local translation could explain their sensitivity to mutations in tRNA synthetases.
Charcot-Marie-Tooth disease is the most common inherited peripheral neuropathy, but the technical limitations of studying the peripheral neuron contribute to the current lack of treatment. The goal of this project is to understand the mechanisms of peripheral neuron degeneration using recently developed in vivo techniques. A better understanding of neurodegeneration may reveal new therapeutic avenues for Charcot-Marie-Tooth and other related neuropathies.
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