PROJECT SUMMARY/ABSTRACT
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.
