Axon-Seq Decodes the Motor Axon Transcriptome and Its Modulation in Response to ALS.

Jik Nijssen,Julio Aguila,Eva Hedlund,Rein Hoogstraaten,Nigel Kee

doi:10.1016/j.stemcr.2018.11.005

Jik Nijssen, Julio Aguila + Show 3 more

Open Access

https://doi.org/10.1016/j.stemcr.2018.11.005

Copy DOI

Abstract

SummarySpinal motor axons traverse large distances to innervate target muscles, thus requiring local control of cellular events for proper functioning. To interrogate axon-specific processes we developed Axon-seq, a refined method incorporating microfluidics, RNA sequencing (RNA-seq), and bioinformatic quality control. We show that the axonal transcriptome is distinct from that of somas and contains fewer genes. We identified 3,500–5,000 transcripts in mouse and human stem cell-derived spinal motor axons, most of which are required for oxidative energy production and ribogenesis. Axons contained transcription factor mRNAs, e.g., Ybx1, with implications for local functions. As motor axons degenerate in amyotrophic lateral sclerosis (ALS), we investigated their response to the SOD1G93A mutation, identifying 121 ALS-dysregulated transcripts. Several of these are implicated in axonal function, including Nrp1, Dbn1, and Nek1, a known ALS-causing gene. In conclusion, Axon-seq provides an improved method for RNA-seq of axons, increasing our understanding of peripheral axon biology and identifying therapeutic targets in motor neuron disease.

Highlights

Spinal motor neurons (MNs) are highly polarized cells
After being plated in microfluidic devices, motor axons were recruited to the vacant chamber by a gradient of glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) (Figures 1A and 1F)
An initial concentration of 50 ng/mL of GDNF/BDNF was followed by a lower concentration of 5 ng/mL, once axons had crossed the microchannels, to avoid growth cone collapse (Figure 1A)

Summary

Introduction

Spinal motor neurons (MNs) are highly polarized cells. Their somas and dendrites are located in the spinal cord, while their axons traverse the body and connect to muscle fibers. The large distance between the MN soma and its synapse implies that the distal axon must contain a microenvironment able to independently respond to internal and external triggers. Vesicles containing proteins and RNAs travel slowly, at 0.1–10 mm per day (Lasek et al, 1984). Protein transport alone does not suffice to sustain the dynamics of the axon and synapse. Local synaptic translation is important for temporal control of protein synthesis and synaptic plasticity (Holt and Schuman, 2013)

Results

Discussion

Conclusion