Research Projects

Dominant mutations in 7+ tRNA synthetase (aaRS) genes cause forms of Charcot-Marie-Tooth disease (CMT), a rare neuromuscular disorder characterized by progressive degeneration of motor and sensory nerves. In 2021, our lab reported that CMT-associated mutations in glycyl tRNA synthetase (GARS, CMT type 2D) and tyrosyl tRNA synthetase (YARS1, dominant intermediate CMT type C) sequester tRNAs, leading to ribosome stalling, and activation of the integrated stress response (ISR) via the sensor kinase, GCN2. Genetic KO or pharmacological inhibition of GCN2 in mice with pathogenic Gars/CMT2D mutations reverses ISR activation and the neuropathy phenotype. REF1, REF2, REF3

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While preventing ISR activation through knockout or inhibition of GCN2 provides therapeutic benefit, it is still unclear how the downstream effects of decreased translation, and selective expression of the transcription factor ATF4 and stress response genes (SRGs), each contribute to the disease pathogenesis. Preliminary results from Tim's K99-funded project show that motor neuron-specific overexpression of ATF4 (ATF4-OE) is sufficient to produce a CMT-like phenotype in mice, while motor neuron-specific ATF4 knockout (ATF4-KO) in Gars/CMT2D mice ameliorates relevant neuromuscular deficits, such as grip strength and nerve conduction velocity (NCV), and almost completely reverses gene expression changes in the Gars/CMT2D mice.

Although we have identified an aaRS-CMT disease mechanism (tRNA sequestration, ISR activation) and successfully tested a potential drug targeting this mechanism (GCN2iB) in mice, it is not known if the ISR is activated in the human disease. Because we are studying an uncommon subtype of a rare disease, obtaining quality postmortem patient tissue samples to validate our proposed disease mechanism is not feasible. Therefore, we are using cultured motor neurons derived from human induced pluripotent stem cells (hiPSCs) with CMT-associated aaRS mutations genetically engineered into them and performing molecular and biochemical assays to test for tRNA sequestration, ribosome stalling, ISR activation, and ATF4 expression. To enhance the translatability of our research, we are collaborating with Drs. Alec Smith and Mark Bothwell at University of Washington to generate cutting-edge hiPSC-derived neuromuscular cultures in which we can measure electrophysiological properties including conduction velocity, neuromuscular transmission, and muscle force. This platform can be adapted for other neuromuscular diseases, like ALS, and used for high throughput testing of mechanistic and therapeutic strategies.

In a project recently funded by the Merkin Peripheral Neuropathy and Nerve Regeneration Center, we are making hiPSCs with a HaloTagged ATF4 gene knocked into the endogenous ATF4 locus and pathogenic aaRS-CMT mutations. The HaloTag system consists of the self-labeling HaloTag fused to the protein of interest (ATF4) and a diverse set of Halo ligands that allow for live-cell fluorescent imaging, molecular and biochemical assays, and even targeted protein degradation. This will enable us to fully characterize how ATF4 is regulated with experiments such as fluorescent labeling in live or fixed cells to visualize ATF4 expression and localization, immunoprecipitations to identify posttranslational modifications and dimerization partners that could affect downstream gene expression, and ChIPseq or CUT&RUN to determine DNA targets. We hope this work will lead to the identification of novel therapeutic targets for aaRS-CMT.

A mouse line with ataxia and muscle wasting was derived from a mutagenesis screen for neuromuscular phenotypes. Genetic mapping and whole genome sequencing revealed segregating mutations in two genes: Stk36Y1003N and Tuba4aQ176P. CRISPR-engineering these variants into C57BL/6J mice confirmed Tuba4aQ176P as the causative mutation, while Stk36 mice were overtly normal. The phenotype of Tuba4aQ176P mice includes Purkinje neuron degeneration and skeletal muscle wasting. The manuscript describing these mice is in preparation to submit for publication.