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Principal Investigator: Robert W. Burgess
Organization: JACKSON LABORATORY
Fiscal Year: 2024
Award: $265,500
Funding agency: National Institute of Neurological Disorders and Stroke
PROJECT SUMMARY
Charcot-Marie-Tooth disease (CMT) is a collection of inherited peripheral neuropathies with a cumulative
incidence of ~1:2500 people. There is no approved treatment for any of the 100 genetic subtypes of CMT,
presenting a large unmet clinical need. At least five forms of CMT are caused by dominant mutations in tRNA
synthetase genes, the housekeeping enzymes that charge amino acids onto their tRNAs for translation. We
recently proposed a mechanism for this disease wherein the mutant enzyme binds its tRNA, but does not release
it to the ribosome, effectively sequestering the substrate. This leads to ribosome stalling and activation of the
integrated stress response (ISR) through the sensor kinase GCN2. The activation of the ISR contributes to the
disease severity, and when ISR activation is blocked by genetically deleting or pharmacologically inhibiting
GCN2, the neuropathy is much milder in mouse models of CMT type 2D, caused by dominant mutations in Glycyl
tRNA synthetase (Gars1). The activation of the ISR has two primary effects: 1) eukaryotic initiation factor 2-
alpha (eIF2) is phosphorylated, suppressing cap-dependent translation, and 2) the transcription factor ATF4 is
selectively translated, promoting expression of cellular stress response genes. The goal of this project is to
determine which of these two outcomes of ISR activation are exacerbating the neuropathy in Gars1/CMT2D
mouse models. Towards this, we propose two aims. In Aim 1, we will examine the levels of translation in
Gars1/CMT2D mice with and without Gcn2 deletion. We have previously shown that motor neurons have
reduced translation in the Gars1 mutant mice, but whether translation remains low when the ISR is not activated,
or whether it recovers, paralleling the improvement in the neuropathy phenotype, is unknown. We will use
fluorescent non-canonical amino acid tagging to assay translation in motor neurons and other spinal cord cell
types in these mice. In Aim 2, we will address the possible role of ATF4. In one experiment, we will overexpress
a conditional ATF4 transgene in motor neurons in an otherwise wild-type background. Our preliminary data
suggest this recapitulates some phenotypes seen in Gars1 mutant mice, implicating ATF4 target gene
expression as a way in which ISR worsens the Gars1 phenotype. In the second experiment, we will use a
conditional knockout of Atf4 to delete the gene from motor neurons in a Gars1/CMT2D genetic background to
see if eliminating ATF4 target gene expression alleviates the neuropathy phenotype, as predicted by our ATF4
overexpression preliminary data. In Aim 2, the mice will be evaluated using behavioral, neurophysiological,
histopathological, and gene expression assays that we have established as clinically relevant and central to the
disease process in the Gars1 mice. Upon completion, these experiments will indicate whether it is decreased
translation or expression of ATF4 target genes (or a combination) that is contributing to the ISR-mediated
neuropathy in the CMT2D mice. These results may reveal novel, more focused points of therapeutic intervention
in this disease.
Terms: <Affect><Amino Acids><Amino Acyl T RNA Synthetases><Amino Acyl-tRNA Ligases><Amino Acyl-tRNA Synthetases><Aminoacetic Acid><Aminoacyl Transfer RNA Synthetase><Aminoacyl-tRNA Synthetase><Axon><Basal Transcription Factor><Basal transcription factor genes><Behavioral><Binding><Body Weight><Breeding><Cell Body><Cells><Cellular Stress><Cellular Stress Response><Charcot Marie Disorder><Charcot Marie Muscular Atrophy><Charcot Marie Tooth Disorder><Charcot Marie Tooth muscular atrophy><Charcot-Marie Disease><Charcot-Marie-Tooth><Charcot-Marie-Tooth Disease><Charcot-Marie-Tooth neuropathy><Charge><Clinical><Codon><Codon Nucleotides><Collection><Data><Defect><Disease><Disorder><Dominant Genes><Double-Stranded RNA><EIF-2><EIF2><Enzyme Gene><Enzymes><Eukaryotic Initiation Factor-2><Eukaryotic Initiation Factors><Eukaryotic Peptide Initiation Factor-2><Eukaryotic Peptide Initiation Factors><Eukaryotic Translation Initiation Factors><Frequencies><GARS gene><Gene Deletion><Gene Expression><Gene Expression Monitoring><Gene Expression Pattern Analysis><Gene Expression Profiling><Gene Family><General Transcription Factor Gene><General Transcription Factors><Genes><Genetic><Genetic Alteration><Genetic Change><Genetic defect><Glycine><Glycine-Specific tRNA><Glycyl-tRNA Synthetase><Goals><Hereditary><Housekeeping><Housekeeping Gene><Housework><Human><Incidence><Inherited><Kinases><Mediating><Medulla Spinalis><Mice><Mice Mammals><Modern Man><Molecular Interaction><Motor><Motor Cell><Motor Neurons><Murine><Mus><Mutant Strains Mice><Mutation><Nerve Cells><Nerve Conduction><Nerve Unit><Neural Cell><Neural Conduction><Neurocyte><Neurons><Neuropathy><Outcome><PNS Diseases><Paper><Peptide Initiation Factor EIF-2><Peripheral><Peripheral Nerve Diseases><Peripheral Nervous System><Peripheral Nervous System Diseases><Peripheral Nervous System Disorders><Peripheral Neuropathy><Peripheral Sensory Neuropathy><Peroneal Muscular Atrophy><Persons><Phenotype><Phosphorylation><Phosphotransferase Gene><Phosphotransferases><Process><Protein Phosphorylation><Proteins><Reproducibility><Research><Ribosomes><Role><Severities><Severity of illness><Spinal><Spinal Cord><Supportive Therapy><Supportive care><Therapeutic Intervention><Transcript Expression Analyses><Transcript Expression Analysis><Transcription Factor Proto-Oncogene><Transcription factor genes><Transfer RNA><Transfer RNA Acylation><Transfer RNA Amino Acylation><Transfer RNA Aminoacylation><Transfer RNA Charging><Transfer RNA Synthetase><Transgenes><Transgenic Organisms><Translating><Translations><Transphosphorylases><Triplet Codon-Amino Acid Adaptor><Viral Diseases><Virus Diseases><aberrant folded protein><aberrant folded proteins><abnormal folded protein><abnormal folded proteins><aminoacid><aminoacid tRNA ligase><analyze gene expression><axonal degeneration><biological adaptation to stress><cell stress><cell type><clinical relevance><clinically relevant><conditional knock-out><conditional knockout><decreased muscle mass><degenerative axon><disease severity><dominant genetic mutation><dominant mutation><dsRNA><experiment><experimental research><experimental study><experiments><gene deletion mutation><gene expression analysis><gene expression assay><genome mutation><improved><in vivo><intervention therapy><low muscle mass><misfolded protein><misfolded proteins><motoneuron><mouse model><mouse mutant><murine model><mutant><neuronal><neuropathic><neurophysiological><neurophysiology><neuroprotection><neuroprotective><novel><overexpress><overexpression><pharmacologic><primary outcome><proteotoxic protein><proteotoxin><reaction; crisis><reduced muscle mass><sensor><sensory neuropathy><social role><stress response><stress; reaction><stressor><tRNA><tRNA Acylation><tRNA Amino Acylation><tRNA Aminoacylation><tRNA Charging><tRNA Synthetase><tRNAGly><therapeutic target><transcription factor><transcriptional profiling><transfer Ribonucleic acids><transgene><transgenic><translation><translation assay><viral infection><virus infection><virus-induced disease>