Neuropathy‐associated histidyl‐tRNA synthetase variants attenuate protein synthesis in vitro and disrupt axon outgrowth in developing zebrafish

Abstract

Charcot‐Marie‐Tooth disease (CMT) encompasses a set of genetically and clinically heterogeneous neuropathies characterized by length‐dependent dysfunction of the peripheral nervous system. Mutations in over 80 diverse genes are associated with CMT, and aminoacyl‐tRNA synthetases (ARS) constitute a large gene family implicated in the disease. Despite considerable efforts to elucidate the mechanistic link between ARS mutations and the CMT phenotype, the molecular basis of the pathology is unknown. In this work, we investigated the impact of three CMT‐associated substitutions (V155G, Y330C, and R137Q) in the cytoplasmic histidyl‐tRNA synthetase (HARS1) on neurite outgrowth and peripheral nervous system development. The model systems for this work included a nerve growth factor‐stimulated neurite outgrowth model in rat pheochromocytoma cells (PC12), and a zebrafish line with GFP/red fluorescent protein reporters of sensory and motor neuron development. The expression of CMT‐HARS1 mutations led to attenuation of protein synthesis and increased phosphorylation of eIF2α in PC12 cells and was accompanied by impaired neurite and axon outgrowth in both models. Notably, these effects were phenocopied by histidinol, a HARS1 inhibitor, and cycloheximide, a protein synthesis inhibitor. The mutant proteins also formed heterodimers with wild‐type HARS1, raising the possibility that CMT‐HARS1 mutations cause disease through a dominant‐negative mechanism. Overall, these findings support the hypothesis that CMT‐HARS1 alleles exert their toxic effect in a neuronal context, and lead to dysregulated protein synthesis. These studies demonstrate the value of zebrafish as a model for studying mutant alleles associated with CMT, and for characterizing the processes that lead to peripheral nervous system dysfunction.

Drosophila Models for Charcot–Marie–Tooth Neuropathy Related to Aminoacyl-tRNA Synthetases

“…On one hand, it will be of great interest to test if CMT causing mutations in other aaRS cause comparable phenotypes and reduction in protein synthesis. Interestingly, similar observations regarding protein synthesis have been made for hYARS1 CMT and hHARS1 CMT in vertebrate cells [45,96], suggesting that an overarching theme might indeed be in place. On the other hand, it will be important to dissect the exact molecular mechanisms by which aaRS CMT affect global protein synthesis and to establish if and how this effect is related to other findings regarding aaRS and CMT.…”

“…Both in vitro and in vivo approaches have been employed to provide insights into this appealing biological question. Because aaRSs are evolutionary conserved [26], several groups have attempted to study these enzymes and their mutations in lower organisms, including yeast, zebrafish, worm, mice or fruit flies [21,33,35,[41][42][43][44][45]. Among those model systems, D. melanogaster stands out as the organism where the highest number of CMT-causing aaRS mutations have been modeled and where important breakthroughs have been made.…”

A humanized yeast model reveals dominant-negative properties of neuropathy-associated alanyl-tRNA synthetase mutations

“…We hypothesize that dominant pathogenic variants in these other neuropathy-associated ARS genes will show dominantnegative effects, because these genes also encode homo-dimeric ARS enzymes (53)(54)(55)(56). Classifying pathogenic variants in these additional ARS loci as dominant-negative alleles will also contribute to defining a common mechanism of ARS-mediated dominant neuropathy, and will provide critical insight into how missense ARS variants lead to depleted pools of charged tRNA, which ultimately impairs protein translation (36,(38)(39)(40)(41).…”

Roles of Aminoacyl-tRNA Synthetases in Cancer

“…Generally, ARSs and AIMPs are considered as housekeeping molecules. However, increasing evidence indicates that these molecules are involved in various physiological and pathological processes, including cysteine polysulfidation, angiogenesis, posttranslational modification, immune response and nervous system development ( Akaike et al, 2017 ; Fu et al, 2017 ; He et al, 2018 ; Mullen et al, 2020 ; Zhu et al, 2020 ). Strikingly, ARSs and AIMPs are closely related to tumorigenesis ( Yin et al, 2016 ; Gao et al, 2019 ; Zhou Z. et al, 2020 ).…”