Charcot-Marie-Tooth disease type 2D (CMT2D) is a peripheral nerve disorder caused by dominant, toxic, gain-of-function mutations in the widely expressed, housekeeping gene, GARS. The mechanisms underlying selective nerve pathology in CMT2D remain unresolved, as does the cause of the mild-to-moderate sensory involvement that distinguishes CMT2D from the allelic disorder distal spinal muscular atrophy type V. To elucidate the mechanism responsible for the underlying afferent nerve pathology, we examined the sensory nervous system of CMT2D mice. We show that the equilibrium between functional subtypes of sensory neuron in dorsal root ganglia is distorted by Gars mutations, leading to sensory defects in peripheral tissues and correlating with overall disease severity. CMT2D mice display changes in sensory behavior concordant with the afferent imbalance, which is present at birth and nonprogressive, indicating that sensory neuron identity is prenatally perturbed and that a critical developmental insult is key to the afferent pathology. Through in vitro experiments, mutant, but not wild-type, GlyRS was shown to aberrantly interact with the Trk receptors and cause misactivation of Trk signaling, which is essential for sensory neuron differentiation and development. Together, this work suggests that both neurodevelopmental and neurodegenerative mechanisms contribute to CMT2D pathogenesis, and thus has profound implications for the timing of future therapeutic treatments.aminoacyl-tRNA synthetase | Charcot-Marie-Tooth disease | distal spinal muscular atrophy type V | neuromuscular disease | neurodevelopment C harcot-Marie-Tooth disease (CMT) is a group of genetically diverse peripheral neuropathies that share the main pathological feature of progressive motor and sensory degeneration (1). Although lifespan is usually unaffected, patients display characteristic muscle weakness and wasting predominantly in the extremities, leading to difficulty walking, foot deformities, and reduced dexterity (2). CMT is traditionally divided into type 1/ demyelinating CMTs that display loss of peripheral nerve myelin causing reduced nerve conduction velocity (NCV), type 2/axonal CMTs typified by axon loss with relatively normal NCVs, and intermediate CMTs that share clinical features of CMT1 and -2 (1). Over 80 different genetic loci have been linked to CMT, which is known to affect ∼1/2,500 people, making it the most common group of hereditary neuromuscular disorders (3).Dominant mutations in the glycyl-tRNA synthetase (GlyRS) gene, GARS, are causative of CMT type 2D (CMT2D) [Online Mendelian Inheritance in Man (OMIM) 601472], which normally manifests during adolescence and presents with muscle weakness in the extremities (4). The 2D subtype is one of a number of CMTs associated with mutation of an aminoacyl-tRNA synthetase (ARS) gene (5-8). Humans possess 37 ARS proteins, which covalently link amino acids to their partner transfer RNAs (tRNAs), thereby charging and priming the tRNAs for protein synthesis.This housekeeping function of gl...

The mechanism by which dominantly inherited mutations in the housekeeping gene GARS, which encodes glycyl-tRNA synthetase (GlyRS), mediate selective peripheral nerve toxicity resulting in Charcot-Marie-Tooth disease type 2D (CMT2D) is still largely unresolved. The transmembrane receptor protein neuropilin 1 (Nrp1) was recently identified as an aberrant extracellular binding partner of mutant GlyRS. Formation of the Nrp1/mutant GlyRS complex antagonises Nrp1 interaction with one of its main natural ligands, vascular endothelial growth factor-A (VEGF-A), contributing to neurodegeneration. However, reduced extracellular binding of VEGF-A to Nrp1 is known to disrupt post-natal blood vessel development and growth. We therefore analysed the vascular system at early and late symptomatic time points in CMT2D mouse muscles, retina, and sciatic nerve, as well as in embryonic hindbrain. Mutant tissues show no difference in blood vessel diameter, density/growth, and branching from embryonic development to three months, spanning the duration over which numerous sensory and neuromuscular phenotypes manifest. Our findings indicate that mutant GlyRS-mediated disruption of Nrp1/VEGF-A signalling is permissive to maturation and maintenance of the vasculature in CMT2D mice.

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Charcot-Marie-Tooth disease type 2D (CMT2D) is a peripheral nerve disorder caused by dominant, toxic, gain-of-function mutations in the widely expressed, housekeeping gene, GARS. The mechanisms underlying selective nerve pathology in CMT2D remain unresolved, as does the cause of the mild-to-moderate sensory involvement that distinguishes CMT2D from the allelic disorder distal spinal muscular atrophy type V. To elucidate the mechanism responsible for the underlying afferent nerve pathology, we examined the sensory nervous system in CMT2D mice. We show that the equilibrium between functional subtypes of sensory neuron in dorsal root ganglia is distorted by Gars mutations, leading to sensory defects in peripheral tissues and correlating with overall disease severity. CMT2D mice display changes in sensory behaviour concordant with the afferent imbalance, which is present at birth and nonprogressive, indicating that sensory neuron identity is prenatally perturbed and that a critical developmental insult is key to the afferent pathology. This suggests that both neurodevelopmental and neurodegenerative mechanisms contribute to CMT2D pathogenesis, and thus has profound implications for the timing of future therapeutic treatments. Significance StatementCharcot-Marie-Tooth disease (CMT) is a collection of genetically diverse inherited nerve disorders with the unifying feature of peripheral neuron degeneration. The mechanisms triggering this motor and sensory nerve dysfunction remain unresolved, as does the reason for the lack of sensory pathology observed in distal hereditary motor neuropathies, which can be associated with CMT genes. To unravel the mechanisms leading to afferent deterioration, we have studied the sensory nervous system of CMT Type 2D mice. Our work indicates that the specific cellular identity of sensory nerves is perturbed in mutant mice pre-natally. CMT therefore manifests through the complex interplay between malfunctioning developmental, maturation, and survival programs, which has important ramifications for therapeutic timing.Keywords: aminoacyl-tRNA synthetase (ARS), Charcot-Marie-Tooth disease (CMT), CMT2D, distal spinal muscular atrophy type V (dSMA-V), dorsal root ganglion (DRG), GARS, glycyl-tRNA synthetase (GlyRS), hereditary motor and sensory neuropathy (HMSN), muscle spindle, neurodevelopment, neuromuscular disease, peripheral neuropathy.