Molecular Hallmarks Of Motor Neuron Death In ALS Identified

A protein known as Splicing Factor, proline- and glutamine-rich (SFPQ), which normally resides inside the cell nucleus, exits the nucleus in diseased motor neurons, a new study from The Francis Crick Institute shows.

The finding is the result of a collaborative group of clinical neurologists, molecular biologists and computer scientists working together to solve the mystery of why motor neurons die in patients with amyotrophic lateral sclerosis (ALS).

Clinical neurologist, Rickie Patani, who sees first-hand the impact that ALS has on his patients, said:

“It’s a really devastating disease. Patients progressively lose the ability to move, eat, speak and ultimately breathe. We set out to uncover the molecular events that lead to ALS, in the hope that one day we can develop new treatments for patients.”

That was three years ago.

RNA Deregulation

Previous studies had implicated deregulation of RNA – a molecule closely related to DNA that has a vital role in coding, decoding, regulating and expressing genes – in ALS. For instance, patients with a hereditary form of ALS often have genetic mutations that prevent their RNA from functioning properly.

But even with RNA expert Jernej Ule on board, comparing RNA sequencing in healthy and diseased motor neurons couldn’t provide the full picture.

So, using cutting-edge stem cell technology, scientists in Rickie’s lab took skin cells from healthy volunteers and patients with ALS and turned them into stem cells capable of becoming many other cell types.

Intron retention is the predominant splicing change during early motor neurogenesis and occurs prematurely in VCPmu cultures.

Intron retention is the predominant splicing change during early motor neurogenesis and occurs prematurely in VCPmu cultures.
Credit: Raphaelle Luisier, Giulia E. Tyzack, et al. CC-BY

Then, using specific chemical signals, they ‘guided’ the stem cells into becoming motor neurons that they could study in the lab.

“By turning back the clock, we could watch what happened to the motor neurons over time to lead to the disease.It was really amazing,”

Giulia Tyzack, a researcher in Rickie’s lab, said.

Bioinformatic Treasure Hunt

Armed with a whole load of RNA sequencing data from healthy and diseased motor neurons at different stages of disease progression, Jernej and Rickie turned to Nick Luscombe and Raphaelle Luisier to drill down into the data and work out exactly what was going wrong. Nick and Raphaelle are bioinformaticians; highly skilled scientists who develop advanced computational techniques to study biological data.

“Initially, using conventional analysis, we didn’t detect any differences in RNA sequencing between healthy and diseased motor neurons,” said Raphaelle. “But we knew something must have been going wrong to make the ALS motor neurons die, so we wrote a new program to dig deeper into the genetic code – and when the results came back, we knew we were on to something.”

The analysis unearthed what was going wrong in ALS motor neurons.

Parts of the RNA sequence that don’t code for proteins are usually cut out before the RNA is translated into protein, but in the ALS motor neurons this wasn’t happening as effectively. This guided the team to collectively discover that the protein SFPQ, which normally resides inside the cell nucleus, was in fact leaving the nucleus in diseased motor neurons.

“It was like one big treasure hunt. We had the map, and knew where we were looking, and with enough digging we found the gold,”

said Nick.

Motor Neuron Death Link

The team had uncovered these molecular hallmarks inside human stem cell models of hereditary ALS. They next confirmed that animal models of hereditary ALS also shared the same features.

But to see if the same events could explain non-hereditary forms of the disease, they looked at post-mortem spinal cord tissue from patients.

SFPQ nuclear clearance is a molecular hallmark of genetic and sporadic ALS.

SFPQ nuclear clearance is a molecular hallmark of genetic and sporadic ALS.
(A) Analysis of the subcellular localisation of SFPQ in MNs in the ventral spinal cord of wild-type, VCPA232E and SOD1G93A mice.
(B) Analysis of the subcellular localisation of SFPQ in MNs in the ventral spinal cord of healthy controls and patients with sporadic ALS (sALS).
Credit: Raphaelle Luisier, Giulia E. Tyzack, et al. CC-BY

They found that the loss of SFPQ protein was consistent across the board, whether they looked at cells, mouse models or post-mortem tissue confirming that they had discovered an important molecular hallmark of ALS.

“Now that we know these key events are linked to motor neuron death in people with ALS, we can start to think about how we could develop new ways to detect and treat the disease,”

said Rickie.

The research was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK, the UK Medical Research Council, and the Wellcome Trust. Additional support came from an MRC eMedLab Medical Bioinformatics Infrastructure Award, the UCL Grand Challenges Award, a Marie Curie Post-doctoral Research Fellowship and an Advanced Postdoc Mobility Fellowship from the Swiss National Science Foundation.

Raphaelle Luisier, Giulia E. Tyzack, Claire E. Hall, Jamie S. Mitchell, Helen Devine, Doaa M. Taha, Bilal Malik, Ione Meyer, Linda Greensmith, Jia Newcombe, Jernej Ule, Nicholas M. Luscombe & Rickie Patani
Intron retention and nuclear loss of SFPQ are molecular hallmarks of ALS
Nature Communications volume 9, Article number: 2010 (2018) doi:10.1038/s41467-018-04373-8

Top Image: Joanna Wardyn, Wellcome Images