Axonal pathogenesis of human iPSC-derived motor neurons

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Project Summary: Reprogramming adult cells has made it possible to differentiate patient-specific neurons from induced pluripotent stem cells (iPSCs). These patient-derived neurons have become invaluable in the investigation of molecular mechanisms of neurodegeneration and identification of potential therapeutic targets. Amyotrophic Lateral Sclerosis (ALS) patient-derived spinal motor neurons have revealed insights into mutation-specific pathogenesis, but previous studies have almost exclusively addressed deficits within motor neurons such as stress granule formation, hyperexcitability, and reduced autophagy. However, the initial pathology of ALS, and many other motor neuron diseases, begins at the distal axon and neuromuscular junction. Normal adult axons contain thousands of diverse sets of mRNAs whose protein products are locally translated to maintain axonal homeostasis and health. However, the expression of axonal mRNA in human motor neurons derived from iPSCs harboring ALS-linked mutations are poorly understood. Additionally, iPSC-derived neuromuscular synapses are rarely utilized as a system to interrogate the pathogenesis of axon degeneration, in part, because of a lack of robust and systematic evaluations of neuromuscular synapses formed by different iPSC- derived cells. The main questions that this application addresses are: 1) Are there differences in the expression of axonal mRNA between patient and healthy control iPSC- derived human spinal motor neurons? 2) What are the effects of distinct disease-causing mutations on the formation and maintenance of human muscle fiber innervations? We hypothesize that ALS-causing mutations may reduce the abundance of locally translated axonal mRNA whose protein products are involved in axonal health and function. We will subject axonal RNA extracted from control and mutant samples to RNAseq, and we are collaborating with bioinformatic group at our institution to probe for effected axonal pathways. We plan to take advantage of a microfluidic device platform that separate neuronal cell bodies from axons/muscle fibers, permitting us to obtain pure axonal mRNA and create mature human-derived neuromuscular synapses. We will test human cell lines containing distinct mutations: SOD1 and C9orf72 repeat expansions, and we will first examine SOD1A4V lines and C9orf72 lines for which isogenic controls have been created. In addition, we have access to cell lines in which the genetic cause of disease has not been determined (sporadic ALS) through the Johns Hopkins ALS center and Answer ALS that we can use for future studies.