Source: sitox/Getty Images
Source: sitox/Getty Images

A research team led by scientists at Harvard University said it has found a potential new biomarker and drug target for amyotrophic lateral sclerosis (ALS). Published in Nature Neuroscience, the study (“ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair”) relied on stem cell models of human motor neurons to reveal the gene STMN2 as a potential therapeutic target.

“The findings that ALS patients almost universally display pathological mislocalization of the RNA-binding protein TDP-43 and that mutations in its gene cause familial ALS have nominated altered RNA metabolism as a disease mechanism. However, the RNAs regulated by TDP-43 in motor neurons and their connection to neuropathy remain to be identified. Here we report transcripts whose abundances in human motor neurons are sensitive to TDP-43 depletion. Notably, expression of STMN2, which encodes a microtubule regulator, declined after TDP-43 knockdown and TDP-43 mislocalization as well as in patient-specific motor neurons and postmortem patient spinal cord,” wrote the investigators.

“STMN2 loss upon reduced TDP-43 function was due to altered splicing, which is functionally important, as we show STMN2 is necessary for normal axonal outgrowth and regeneration. Notably, post-translational stabilization of STMN2 rescued neurite outgrowth and axon regeneration deficits induced by TDP-43 depletion. We propose that restoring STMN2 expression warrants examination as a therapeutic strategy for ALS.”

Two ALS drugs have been approved by the FDA, but they act only to slow the disease. In addition to a cure, or even a treatment that is effective for more ALS patients, an effective test for ALS is needed, said the researchers, and that depends on a reliable biomarker.

About 10 years ago, scientists found aggregates of a protein called TDP-43 in post-mortem neurons from ALS patients. This protein should have been in the nucleus of those neurons, but instead it was being flushed out, and building up in the cytoplasm.

Some of the genes at work in the proteasome were interacting with TDP-43 in a way that led to ALS. But which genes are involved, and what they are doing, has not been known.

The gene that encodes for TDP-43 can be mutated to trigger ALS. It is passed on to future generations, who then develop either ALS or, in some cases, frontotemporal dementia (FTD). Since TDP-43 aggregates were discovered in ALS patients, they have been well known as a hallmark of the disease.

TDP-43 is one of many proteins that binds to RNA. The researchers set out to identify, for the first time, all the possible types of RNA that are regulated by the TDP-43 protein in the context of human neurons. Then, they looked at what happened to each gene when they manipulated TDP-43. Until now, studies like this have only been carried out in mice and cancer cell lines.

The researchers reduced the levels of TDP-43 protein in human stem cell-derived motor neurons. Then, using RNA-sequencing, they analyzed how gene expression changed in these cells. Among the thousand or so genes that changed when TDP-43 was manipulated, one stood out: Stathmin2 (STMN2), a gene that is important in neural outgrowth and repair. STMN2 changed consistently in step with TDP-43.

“Once we had a connection between the TDP-43 and the loss of this other critical gene, STMN2, we could see how a motor neuron might begin to fail in ALS,” said Joseph Klim, PhD, postdoctoral fellow in the Harvard department of stem cell and regenerative biology (HSCRB).

“With the discovery that our human stem cell model had predicted exactly what was happening in patients, Joe went on to test in this system whether fixing Stathmin2 could rescue the motor neuron degeneration in our dish caused by disturbing TDP-43. In a beautiful series of experiments that I believe provide great hope for patients, he went on to show this was exactly the case: rescuing expression of Stathmin2 rescued motor neuron growth,” added Kevin Eggan, PhD, professor of stem cell and regenerative biology at Harvard.

The researchers observed that without TDP-43, STMN2’s protein-making instructions turn into nonsense.

“We discovered that when TDP-43 levels are diminished in the nucleus, a cryptic exon is spliced into STMN2messenger RNA. That basically deletes its instructions for making functional protein,” explained Klim. “It becomes impossible for STMN2 to create a vital component for repairing or growing motor neuron axons.”

The next step was to see if their findings reflected the reality of a patient’s biology. They obtained data from RNA sequencing studies that used post-mortem samples from ALS patients. Those datasets, compared with controls, echoed the team’s original findings in human stem cell models. The data from ALS-patient spinal cords mapped to the cryptic exon, but data from the controls did not.

Luis Williams, PhD, director of cell biology at Q-State Biosciences, whose PhD thesis in HSCRB was a key step in this study, added, “Because we had pluripotent stem cells of human origin, we could make cells in a dish that are relevant to ALS and investigate this very specific problem in the right context: with a human genome and all of the genetic factors that regulate motor neurons.”

“These experiments point towards a clear path for testing whether repairing Stathmin2 in patients can slow or stop their disease,” explained Eggan. “The discovery we have made suggests a clear approach for developing a potential therapy for ALS, one that would intervene in all but a very small number of individuals, regardless of the genetic cause of their disease.”

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