Investigating axonal length-dependent pathology in a human stem cell-based model of Charcot-Marie-Tooth disease (type 2D) - PROJECT SUMMARY AND ABSTRACT This F32 research proposal will evaluate the axonal length-dependent pathology of Charcot-Marie-Tooth disease, type 2 (CMT2) using a human stem cell-based model. CMT is the most common inherited peripheral neuropathy in humans and is characterized by distal muscle weakness caused by motor and sensory neuron dysfunction. CMT2 is a genetically dominant axonal form, with mutations in eight aminoacyl-tRNA synthetases (ARS) identified as causative. Interestingly, while ARS mutants share similar pathological features, studies have yet to identify a common gain- or loss-of-function underpinning disease etiology. Furthermore, it remains to be determined why these mutations impact neurons in an axonal length-dependent manner. The only known shared function of ARS is protein translation, in which ARS conjugate amino acids with their cognate tRNAs. However, previous research has established CMT2 mutations do not consistently impact enzymatic function. Interestingly, a recent study discovered that a CMT2 GARS mutation resulted in aberrant interaction of GARS with stress granule protein G3BP1. Further, G3BP1 and other granule proteins have been recently identified as components of naturally occurring RNA transport granules. Thus, I hypothesize that GARS is regularly transported along axons in RNA granules, and CMT2 mutations impact transport of granules, reducing the amount of ARS and other species reaching axon terminals in an axonal length-dependent manner, altering axonal transcriptomes and proteomes. To test this hypothesis, I will first examine the interaction of ARS proteins with various granule protein molecules in disease-relevant human induced pluripotent stem cell-derived motor neurons. I will employ co-immunoprecipitation and imaging analyses to confirm ARS proteins, in particular GARS, associate with RNA granules (Aim 1). I will next evaluate the impact of a GARS mutation on length-dependent transport and local translation in motor neurons. Utilizing microfluidic chambers, I will culture GARS mutants and isogenic control motor neurons in chambers with increasing lengths, thus controlling the length of the axonal projections. I will then evaluate axonal transport and local protein synthesis deficits in the mutant neurons (Aim 2). The data generated from these studies will include axonal-specific transcriptomic and proteomic profiles for both normal and mutant motor neurons. This will provide the field with an in-depth analysis of the local changes occurring in motor neurons, giving new insight into CMT2 pathology, identification of novel targets for therapies, and a greater understanding of the mechanisms underpinning axonal protein synthesis. Further, these studies, and future work building off this project, will expand our understanding of neuronal development and evolution. The PI of the project will train with well-established, highly regarded professors at the University of Washington in the fields of neuroscience, stem cell biology, and bioengineering, and the training plan will build strong professional skills – publications, communication, mentoring – to foster growth into a well-rounded independent research scientist.
