Probing mRNA Populations from Axons of ALS Mutant iPSC-derived Spinal Motor Neurons


Translational Research

Poster Number: 296


Madison James, MS, Johns Hopkins University School of Medicine, Arun Venkatesan, MD, PhD, Johns Hopkins University School of Medicine, Mohamed Farah, PhD, Johns Hopkins University School of Medicine

It has been established that axons contain mRNA and locally synthesize proteins, including those involved in axonal regeneration, health, and maintenance, and increasing evidence points to dysregulation of axonal RNA as a major contributor to axon degeneration in Amyotrophic Lateral Sclerosis (ALS). Reprogramming adult cells has made it possible to derive patient-specific neurons from induced pluripotent stem cells (iPSCs) to both investigate molecular mechanisms of neurodegeneration, and to identify potential therapeutic targets. ALS patient-derived spinal motor neurons have revealed insights into mutation-specific pathogenesis, and RNASeq studies have been done looking for differences in gene expression profiles between iPSC-derived motor neurons from patients with ALS disease-causing mutations and controls, as these may give key insights into disease mechanisms. The initial pathology of ALS, however, begins at distal axons and neuromuscular junctions, so axonal gene expression specifically may contribute to disease. As such, we’ve focused this work on isolating axonal RNA. We have cultured induced pluripotent stem cell (iPSC)-derived motor neurons in microfluidic devices that allow us to isolate the distal axons from the cell bodies. Importantly, single cell RNAseq analyses have shown that a single neuron has more RNA than pooled axons, so a few neuronal or non-neuronal cell bodies that escape through the microchannels to the axonal compartments can contaminate the extracted RNA. To overcome this potential contamination, we designed modified microfluidic devices with two sets of channels in a row to completely sequester neuronal cell bodies away from axons. We have also been able to extract high quality RNA from control and ALS-mutant cells for sequencing from the spatially separated compartments, allowing us to analyze axonal RNA expression profiles independent of neuronal cell body expression. Presently, we are conducting bioinformatic analysis to interrogate axonal mRNA profiles in the context of ALS-disease causing mutations.