Charcot-Marie-Tooth–Linked Mutant GARS Is Toxic to Peripheral Neurons Independent of Wild-Type GARS Levels

Abstract

Charcot-Marie-Tooth disease type 2D (CMT2D) is a dominantly inherited peripheral neuropathy caused by missense mutations in the glycyl-tRNA synthetase gene (GARS). In addition to GARS, mutations in three other tRNA synthetase genes cause similar neuropathies, although the underlying mechanisms are not fully understood. To address this, we generated transgenic mice that ubiquitously over-express wild-type GARS and crossed them to two dominant mouse models of CMT2D to distinguish loss-of-function and gain-of-function mechanisms. Over-expression of wild-type GARS does not improve the neuropathy phenotype in heterozygous Gars mutant mice, as determined by histological, functional, and behavioral tests. Transgenic GARS is able to rescue a pathological point mutation as a homozygote or in complementation tests with a Gars null allele, demonstrating the functionality of the transgene and revealing a recessive loss-of-function component of the point mutation. Missense mutations as transgene-rescued homozygotes or compound heterozygotes have a more severe neuropathy than heterozygotes, indicating that increased dosage of the disease-causing alleles results in a more severe neurological phenotype, even in the presence of a wild-type transgene. We conclude that, although missense mutations of Gars may cause some loss of function, the dominant neuropathy phenotype observed in mice is caused by a dose-dependent gain of function that is not mitigated by over-expression of functional wild-type protein.

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

“…S3D). L5 was chosen due to its size and because the resident sensory neurons target distal tissues of the hind limbs, where neuromuscular pathology occurs in Gars mice (11,15,28). Counting βIII-tubulin + (red) cell profiles to estimate the number of neurons per DRG, we found no difference between wild-type and mutant ganglia (SI Appendix, Fig.…”

“…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).…”

Regulation of mRNA Translation in Neurons—A Matter of Life and Death

“…However, the underlying molecular mechanism of these disorders has remained elusive. Many CMT-linked mutations seem to have no significant effect on the aminoacylation activity of the synthetase, and recent studies support a toxic gain of function mechanism, possibly involving the exposure of a neomorphic binding surface and resulting in aberrant protein interactions (He et al, 2015; Motley et al, 2011; Seburn et al, 2006; Sleigh et al, 2017). In contrast, other CMT-linked mutations have been reported to cause a loss of charging activity, suggesting that haploinsufficiency of the enzyme may be a possible contributing factor (Griffin et al, 2014; McLaughlin et al, 2012).…”

A novel AARS mutation in a family with dominant myeloneuropathy

“…2 Two Gars mutations cause peripheral neuropathy in mice, 38,39 and overexpression of wild-type human GARS failed to mitigate severity of the disease in these models, suggesting a toxic gain-of-function mechanism. 40 Despite this, the mechanism of toxicity has not been identified and loss-of-function assays in yeast continue to be among the most specific tests for disease pathogenesis.…”