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...

Patients with Charcot-Marie-Tooth Type 2D (CMT2D), caused by dominant mutations in Glycl tRNA synthetase (GARS), present with progressive weakness, consistently in the hands, but often in the feet also. Electromyography shows denervation, and patients often report that early symptoms include cramps brought on by cold or exertion. Based on reported clinical observations, and studies of mouse models of CMT2D, we sought to determine whether weakened synaptic transmission at the neuromuscular junction (NMJ) is an aspect of CMT2D. Quantal analysis of NMJs in two different mouse models of CMT2D (Gars P278KY, Gars C201R ), found synaptic deficits that correlated with disease severity and progressed with age. Results of voltage-clamp studies revealed presynaptic defects characterized by: (1) decreased frequency of spontaneous release without any change in quantal amplitude (miniature endplate current), (2) reduced amplitude of evoked release (endplate current) and quantal content, (3) age-dependent changes in the extent of depression in response to repetitive stimulation, and (4) release failures at some NMJs with high-frequency, long-duration stimulation. Drugs that modify synaptic efficacy were tested to see whether neuromuscular performance improved. The presynaptic action of 3,4 diaminopyridine was not beneficial, whereas postsynaptic-acting physostigmine did improve performance. Smaller mutant NMJs with correspondingly fewer vesicles and partial denervation that eliminates some release sites also contribute to the reduction of release at a proportion of mutant NMJs. Together, these voltage-clamp data suggest that a number of release processes, while essentially intact, likely operate suboptimally at most NMJs of CMT2D mice.

Paclitaxel is a microtubule-stabilizing chemotherapeutic agent that is widely used in cancer treatment and in a number of curative and palliative regimens. Despite its beneficial effects on cancer, paclitaxel also damages healthy tissues, most prominently the peripheral sensory nervous system. The mechanisms leading to paclitaxelinduced peripheral neuropathy remain elusive, and therapies that prevent or alleviate this condition are not available. We established a zebrafish in vivo model to study the underlying mechanisms and to identify pharmacological agents that may be developed into therapeutics. Both adult and larval zebrafish displayed signs of paclitaxel neurotoxicity, including sensory axon degeneration and the loss of touch response in the distal caudal fin. Intriguingly, studies in zebrafish larvae showed that paclitaxel rapidly promotes epithelial damage and decreased mechanical stress resistance of the skin before induction of axon degeneration. Moreover, injured paclitaxel-treated zebrafish skin and scratch-wounded human keratinocytes (HEK001) display reduced healing capacity. Epithelial damage correlated with rapid accumulation of fluorescein-conjugated paclitaxel in epidermal basal keratinocytes, but not axons, and upregulation of matrix-metalloproteinase 13 (MMP-13, collagenase 3) in the skin. Pharmacological inhibition of MMP-13, in contrast, largely rescued paclitaxel-induced epithelial damage and neurotoxicity, whereas MMP-13 overexpression in zebrafish embryos rendered the skin vulnerable to injury under mechanical stress conditions. Thus, our studies provide evidence that the epidermis plays a critical role in this condition, and we provide a previously unidentified candidate for therapeutic interventions.aclitaxel is a microtubule-stabilizing chemotherapeutic agent that is widely used in the treatment of common cancers, including breast, ovarian, and lung cancer. Despite its promising anticancerous properties, paclitaxel also damages healthy tissues, most prominently peripheral axons of somatosensory neurons (reviewed in ref. 1). Paclitaxel-induced peripheral neuropathy initiates in the distal extremities and presents as neuropathic pain syndrome, temperature sensitivity, and paresthesia (tingling and numbness). Nerve biopsies from patients suggest that axon degeneration is the primary manifestation of this condition, followed by secondary demyelination and nerve fiber loss in severely affected patients (1, 2). Certain drugs have been shown in vitro and in vivo to protect against paclitaxel-induced nerve damage, including acetyl-L-carnitine, erythropoietin, alpha-lipoic acid, olesoxime, amifostine, nerve growth factor, and glutamate (reviewed in ref.3). However, so far, these agents have either not successfully passed clinical trials or merely alleviate symptoms such as pain without prevention (1). Thus, a better understanding of the underlying causes of paclitaxel-induced peripheral neuropathy is necessary and may help identify new candidate drugs with which to treat this condition.A widely...