An ENU-induced mutation in mouse glycyl-tRNA synthetase (GARS) causes peripheral sensory and motor phenotypes creating a model of Charcot-Marie-Tooth type 2D peripheral neuropathy

Abstract

C201R, with a point mutation that leads to a non-conservative substitution within GARS. Heterozygous mice with a C3H genetic background have loss of grip strength, decreased motor flexibility and disruption of fine motor control; this relatively mild phenotype is more severe on a C57BL/6 background. Homozygous mutants have a highly deleterious set of features, including movement difficulties and death before weaning. Heterozygous animals have a reduction in axon diameter in peripheral nerves, slowing of nerve conduction and an alteration in the recovery cycle of myelinated axons, as well as innervation defects. An assessment of GARS levels showed increased protein in 15-day-old mice compared with controls; however, this increase was not observed in 3-month-old animals, indicating that GARS function may be more crucial in younger animals. We found that enzyme activity was not reduced detectably in heterozygotes at any age, but was diminished greatly in homozygous mice compared with controls; thus, homozygous animals may suffer from a partial loss of function. The Gars C201R mutation described here is a contribution to our understanding of the mechanism by which mutations in tRNA synthetases, which are fundamentally important, ubiquitously expressed enzymes, cause axonopathy in specific sets of neurons.

Trk receptor signaling and sensory neuron fate are perturbed in human neuropathy caused by Gars mutations

“…The greatest sensory deficiency in patients with CMT2D is in the perception of vibration, which is sensed by neurons with large cell bodies and axons (26,27); however, patient sural nerve biopsies show a selective loss of small sensory axons (20,21). This histological finding is also counter to what is observed in CMT2D mice; the milder Gars C201R/+ mice display a general reduction in axon diameter in both the saphenous and sensory femoral nerves (12), whereas the more severe Gars Nmf249/+ allele displays both a reduction in axon diameter and axon number (11); nevertheless, whether specific sensory neuron populations are preferentially atrophied or lost is unknown. We thus set out to interrogate the sensory nervous system of CMT2D mice to better understand how and when Gars mutations cause sensory pathology, its molecular mechanism, and the effect that these mutations have on sensation of the external environment.…”

“…A previous study has shown that expression of TrkC from the TrkA locus caused a developmental fate switch in DRG sensory subtypes (63). Given that GlyRS is expressed during early development (11,12), and that arborization of nociceptive neurons is developmentally impaired in CMT2D mice (Fig. 5F), our work has identified a highly plausible mechanism to account for the sensory neuron identity defects observed in Gars animals; namely, mutant GlyRS binds and spuriously activates multiple Trk receptors, thereby subtly subverting sensory neuron differentiation and/or survival during early stages of development.…”

“…Gars C201R/+ mice do not show loss of motor neuron cell bodies up to at least 4 mo in the lumbar spinal cord (12). α-Motor neurons innervate force-generating extrafusal muscle fibers, whereas the smaller γ-motor nerves innervate intrafusal fibers of muscle spindles (40).…”

“…We began our CMT2D sensory analysis by culturing primary dorsal root ganglion (DRG) neurons from wild-type and Gars C201R/+ mice. This model of CMT2D has a mutagen-induced T456C alteration in the endogenous mouse Gars gene, causing a cysteine-to-arginine switch at residue 201; this produces a range of peripheral nerve defects without affecting survival, reminiscent of CMT2D (12). DRG are heterogeneous collections of neural crest-derived sensory neuron cell bodies found in pairs at each segment of the spinal cord, from where they project to and receive information from target peripheral tissues.…”

“…Several hypotheses have been suggested (9,10), although the exact disease mechanisms remain unknown. Nevertheless, cell-based experiments and studies using two CMT2D mouse models (the mild Gars C201R/+ allele and the more severe Gars Nmf249/+ strain) indicate that CMT2D is likely caused by a toxic gain of function in mutant GlyRS rather than haploinsufficiency due to a loss of aminoacylation activity or a noncanonical function (11)(12)(13)(14)(15). A possible mediator of toxicity was identified when five CMT2D-associated mutations spread along the length of GARS were all shown to induce a similar conformational change in GlyRS, leading to the exposure of surfaces buried in the wild-type protein (16).…”