Axon-seq Lets Researchers Better Analyze mRNAs in Study of ALS

A new method called Axon-seq allows researchers to analyze the content of messenger RNAs (mRNAs) in the motor neuron’s axons, the tips of the nerve cells that communicate with muscle cells, a new study reports.

Using Axon-seq, researchers identified 121 mRNAs that are deregulated in ALS motor neurons, highlighting the new method’s potential to identify new targets for future therapies.

The study, “Axon-seq decodes the motor axon transcriptome and its modulation in response to ALS,” was published in the journal Stem Cell Reports.

ALS is a progressive neurological disease characterized by the death of motor neurons, which control voluntary muscles. Before dying, the nerve cell’s axons — the long, slender projections of a neuron that transmit information (electrical impulses) to the muscle — shrink, making them lose the connection with the muscle cell. The site of communication between motor nerve axons and muscle fibers is called the neuromuscular junction.

This loss of contact between the motor neuron and the target muscle occurs first, long before the death of motor nerve cells and the reason motor neurons in ALS are said to die “backward.”

To better understand this process, researchers at the Karolinska Institutet in Sweden developed a new strategy and examined the content of the messenger RNA — a chemical cousin of DNA and the molecules that contain instructions to make proteins — specifically in the motor neurons’ axons.

While looking at the axons’ mRNA content in both healthy individuals and ALS patients may provide clues to what goes wrong in ALS, it is hampered by the fact that the content of RNA in the axon is very low and it is difficult to isolate and obtain a “pure” collection of axons without any other cell components.

Axon-seq uses microfluidics (a microchip that contains chambers and tunnels through which fluids flow), RNA sequencing (RNA-seq), and bioinformatic analysis.

Eva Hedlund, associate professor at the Department of Neuroscience, Karolinska Institute and the study’s lead author, said in a press release that Axon-seq is “a relatively cheap, simple and highly sensitive method that we’ve described in detail in our study so that it can be used by other researchers interested in studying neuronal processes.”

The researchers used mouse and human stem cells to generate healthy and ALS motor neurons (these carried a mutation in the SOD1 gene, responsible for up to 20% of hereditary cases of ALS), which were cultured in microfluidic devices.

The RNA analysis revealed that the axons’ mRNA content was distinct from that of the nerve cell’s body, a finding that had not previously been reported.

Moreover, while the content in mRNA molecules was low, axons were enriched in mRNAs for energy and protein production.

When comparing the mRNAs from ALS-diseased motor neurons to healthy ones, researchers found that the levels of 121 mRNAs were deregulated in the ALS motor neurons, with several playing key roles in nerve cell function, axon maintenance, and growth. Some of the deregulated mRNAs included those for the Nrp1, Dbn1, and Nek1 genes.

The lower levels of Nrp1 were linked with a loss of function in the neuromuscular junction.

“Many of the genes we found dysregulated in ALS are needed for the normal function of the axon and its contact with the muscle,” said Jik Nijssen, the study’s joint first author. “Many of these genes present possible targets for future therapies.”

Overall, “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,” the study concluded.