RNAseq of fluidically isolated human and mouse motor neuron somas and axons in vitro

Spinal motor axons traverse large distances to innervate target muscle, and thus require local control of cellular events for proper functioning of the distal axon. To interrogate axon-specific processes we developed Axon-seq, a refined method incorporating microfluidic devices and stringent bioinformatic quality controls. Axon-seq demonstrates improved sensitivity and accuracy in whole-transcriptome sequencing of axons compared to previously published studies. Importantly, we show that axon transcriptomes are distinct from those of somas, displaying fewer detected genes and no contaminating astrocytic markers. We identified >5,000 transcripts in stem cell-derived spinal motor axons required for local oxidative energy production and ribosome generation. Axons contained unique transcription factor mRNAs, e.g. Ybx1, with implications for local axonal functions. Cross-comparison with existing mouse motor axon datasets, as well as our own human motor axon data identified a common axon transcriptome. As motor axons degenerate in amyotrophic lateral sclerosis (ALS), we investigated their response to the disease-causing SOD1G93A mutation, identifying 121 ALS-dysregulated transcripts. Several of these are implicated in axonal and dendritic outgrowth, including Nrp1, Dbn1, and Nek1, a known ALS-causing gene. In conclusion, Axon-seq proves a robust and improved method for RNA-seq of axons, furthers our understanding of peripheral axon biology, and identifies novel therapeutic targets to maintain neural connectivity in disease. Overall design: A total of 118 samples were analyzed here. Of these, 32 are microfluidic cell culture samples derived from mouse embryonic stem cells differentiated into motor neurons, where axons and somas were separated for RNA isolation. This was performed for 2 cell lines (control and SOD1G93A), resulting in the following 4 groups: control somas (n=13), control axons (n=5), SOD1G93A somas (n=10) and SOD1G93A axons (n=4). An additional 34 single mouse motor neurons were sequenced (n=12 for control, n=22 for SOD1G93A). Human control motor neurons derived from induced pluripotent stem cells were also cultured in microfluidic devices, and separate axon and soma fractions were harvested (n=6 axon, n=7 soma). An additional n=39 human single motor neurons were sequenced.