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Mullen, Patrick

Neuroscience Graduate Program

2022

Ph. D.

The aminoacyl-tRNA synthetases (ARS) are a large family of enzymes that catalyze the aminoacylation reaction, in which an amino acid is attached to cognate tRNAs. This reaction is essential for the process of protein synthesis, as it allows tRNAs to carry amino acids to the ribosome during translation. In recent years, genome sequencing has revealed an association between ARS and inherited diseases of the nervous system. Dominant ARS mutations are linked to the inherited peripheral neuropathy Charcot-Marie-Tooth disease (CMT), while recessive ARS mutations cause multisystem disorders that often include severe neurodevelopmental defects. Additionally, bi-allelic mutations in 2 of the 3 aminoacyl-tRNA synthetase multifunctional interacting proteins (AIMPs) are associated with developmental encephalopathies that overlap phenotypically with recessive ARS disorders. A key outstanding question regarding inherited ARS disorders is whether loss of enzyme function and subsequent protein synthesis defects underlie disease pathogenesis. Additionally, the effects of ARS mutations on cellular and organismal physiology remain unclear. A better understanding of the processes linking ARS mutations to neurological diseases may ultimately lead to novel therapeutic strategies for these currently untreatable disorders. In this work, I investigated histidyl-tRNA synthetase (HARS) mutations that are associated with CMT. Previous work in the lab determined that CMT-HARS variants compromise enzyme activity, but the effects on neuronal function had not been explored. In studies using rat pheochromocytoma (PC12) cells, I found that expression of CMT-HARS impaired neurite outgrowth in response to nerve growth factor. Consistent with these in vitro findings, transgenic zebrafish injected with mRNAs encoding V155G and Y330C mutant HARS displayed axonal abnormalities and concomitant disruption of touch responsiveness and swimming behavior. Effects on neurite outgrowth were recapitulated by inhibiting HARS or global protein translation, suggesting that dysregulation of protein synthesis is a key component of the observed phenotypes. In support of this hypothesis, I found that PC12 cells expressing mutant HARS proteins displayed attenuated protein synthesis and increased phosphorylation of eIF2[alpha], which is a signaling event that reduces protein translation in response to cellular stress. Owing to the fact that CMT-HARS variants confer loss-of-function effects, it is possible that mutant proteins decrease protein translation through a dominant negative inhibition of aminoacylation. In support of this hypothesis, I found that mutant proteins dimerize with wild-type subunits. In sum, these data demonstrate that inhibition of protein synthesis is a common effect of ARS-CMT variants and provide support for the involvement of a dominant negative mechanism. I also investigated the effects of novel bi-allelic mutations in the aminoacyl-tRNA synthetase multifunctional interacting protein 2 (AIMP2). These mutations were identified in patients with developmental encephalopathy, and lead to a profound reduction in AIMP2 protein expression. At the cellular level, the consequences of loss of AIMP2 included decreased protein synthesis and delayed progression through the G1-S checkpoint of the cell cycle. Loss of AIMP2 caused a reduction in the levels of an interacting protein, methionyl-tRNA synthetase (MARS), which is known to regulate protein synthesis initiation and cell cycle progression. These results indicated that dysregulation of protein synthesis and impaired progression through the cell cycle are key pathogenic processes in AIMP2-linked developmental encephalopathies. Notably, decreased AIMP2 expression caused by mutations encoding premature stop codons was rescued by anticodon-engineered tRNAs, which may represent a viable therapeutic strategy for a select group of patients. Together, these data suggest that dominant HARS mutations and bi-allelic AIMP2 mutations cause neurological disease through dysregulation of proteostasis, with differential downstream consequences on neurite outgrowth and cell-cycle progression, respectively.