The goal of this proposal is to understand how dominant mutations in tRNA synthetase genes cause axonal degeneration in the peripheral nervous system. Mutations in glycyl tRNA synthetase (GARS) and tyrosyl tRNA synthetase (YARS) cause Charcot-Marie-Tooth type D and Dominant Intermediate Charcot-Marie- Tooth type C (CMT2D and DI-CMTC) respectively. These enzymes are well characterized for their function in charging amino acids onto their cognate tRNAs for translation. However, there are few correlations between the effects of these mutations on this activity and the disease phenotypes. We have demonstrated this using a mouse model of CMT2D, in which an amino acid substitution in GARS causes a dominant peripheral neuropathy despite normal enzymatic activity of the mutant protein, whereas a loss-of-function allele that eliminates Gars expression does not cause a dominant phenotype despite lower overall levels of enzyme activity. These results lead us to hypothesize that mutant forms of GARS cause disease either through the loss of an unknown, noncanonical function, or through a pathological gain-of-function. We therefore propose three aims to better understand the disease mechanisms of CMT2D and DI-CMTC.
In Aim 1, we will create a mouse model of DI-CMTC using a "Conditional knockin" strategy to introduce human disease alleles into the mouse Yars gene. Preliminary studies indicate that decreasing Yars expression to 35% of wild type levels does not cause a peripheral neuropathy, suggesting that like Gars, a mutant form of the YARS protein is required to cause disease. The Yars conditional knockin mice will allow us to further explore whether GARS and YARS mutations do indeed share a common pathogenic mechanism.
In Aim 2, we will test a proposed disease mechanism based on in vitro and biochemical data from our collaborators, Drs. Schimmel and Yang. This model proposes that altered interaction between GARS and DAXX, a protein implicated in other motor neuron disease pathways, may underlie CMT2D. We will test this in vivo by again using a conditional knockin strategy to target residues in GARS that are essential for the DAXX interaction. If the proposed mechanism is correct, this should cause a severe neuropathy phenotype.
In Aim 3, we will combine mouse and human genetics to understand the genetic basis for the variable severity of CMT2D. CMT2D patients may present with purely motor deficits (diagnosed as distal Spinal Muscular Atrophy type V, dSMAV), whereas others carrying the same mutation present with both motor and sensory deficits. We are able to reproduce this variability in mice and have mapped a locus on Chr. 1 that contributes to the preservation of sensory axons. We will use mouse genetics to identify the modifying gene on Chr. 1. We will also determine if the same region of the human genome contributes to variable severity in CMT2D patients in collaboration with Dr. Albena Jordanova. The identification of modifier loci will improve our understanding of CMT2D mechanisms, suggest therapeutic strategies, and enhance diagnosis/prognosis of CMT2D patients.
This proposal seeks to combine research in mouse models of hereditary diseases of the peripheral nervous system (Charcot-Marie-Tooth Disease) with research in human genetics to better understand disease mechanisms and to suggest new therapeutic strategies. This work will lead to improved genetic diagnoses in humans, create directly relevant animal models for preclinical testing of therapies, and identify new therapeutic targets for these diseases, and possibly other motor neuron diseases such as Amyotrophic Lateral Sclerosis (ALS).
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|Bais, Preeti; Beebe, Kirk; Morelli, Kathryn H et al. (2016) Metabolite profile of a mouse model of Charcot-Marie-Tooth type 2D neuropathy: implications for disease mechanisms and interventions. Biol Open 5:908-20|
|Grice, Stuart J; Sleigh, James N; Motley, William W et al. (2015) Dominant, toxic gain-of-function mutations in gars lead to non-cell autonomous neuropathology. Hum Mol Genet 24:4397-406|
|Niehues, Sven; Bussmann, Julia; Steffes, Georg et al. (2015) Impaired protein translation in Drosophila models for Charcot-Marie-Tooth neuropathy caused by mutant tRNA synthetases. Nat Commun 6:7520|
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|He, Weiwei; Bai, Ge; Zhou, Huihao et al. (2015) CMT2D neuropathy is linked to the neomorphic binding activity of glycyl-tRNA synthetase. Nature 526:710-4|
|Amin, Neal D; Bai, Ge; Klug, Jason R et al. (2015) Loss of motoneuron-specific microRNA-218 causes systemic neuromuscular failure. Science 350:1525-9|
|Tezuka, Tohru; Inoue, Akane; Hoshi, Taisuke et al. (2014) The MuSK activator agrin has a separate role essential for postnatal maintenance of neuromuscular synapses. Proc Natl Acad Sci U S A 111:16556-61|
|Sleigh, James N; Grice, Stuart J; Burgess, Robert W et al. (2014) Neuromuscular junction maturation defects precede impaired lower motor neuron connectivity in Charcot-Marie-Tooth type 2D mice. Hum Mol Genet 23:2639-50|
|Sleigh, James N; Burgess, Robert W; Gillingwater, Thomas H et al. (2014) Morphological analysis of neuromuscular junction development and degeneration in rodent lumbrical muscles. J Neurosci Methods 227:159-65|
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