Can zebrafish help explain how mutations in this gene contribute to ALS?

Investigating the role of mutations in CHCHD10 using ALS cell and zebrafish genetic models

“The more we understand about all these different genes that we’re discovering in the clinic, we can ask new and better questions in our labs. Major breakthroughs have often come serendipitously, from inquisitive basic science.” – Dr. Gary Armstrong

An interdisciplinary Canadian team headed by Dr. Gary Armstrong of The Neuro (Montreal Neurological Institute-Hospital)  is poised to explore how a recently linked pair of genes contribute to the onset of ALS. In 2014, mutations in a gene called CHCHD10 were newly identified as a genetic cause of ALS; just four years later, Dr. Eric Shoubridge, a geneticist and authority on mitochondrial diseases also based at The Neuro, made a discovery linking the role of CHCHD10 in ALS to another protein, CHCHD2. Yet while it is clear that these two genes are connected to one another, and to ALS, the exact nature of their interrelationship – and the way they contribute to  neurodegeneration  – have yet to be fully understood.

That may soon begin to change. Funded by a Discovery Grant from the ALS Society of Canada and Brain Canada, Dr. Shoubridge will join the team of gene editing specialist Dr. Armstrong, to take the next steps in understanding the connections – and thousands of tiny striped fish, each only two millimetres long when their time in the lab begins, will be their most important helpers.

UNDERSTANDING AN AMBIGUOUS GENETIC TAG-TEAM

Preliminary data suggest that mutations in CHCHD10 and CHCHD2 may lead to impaired functioning of mitochondria, structures within cells that provide the energy the cell needs to survive. “My lab has been working on mitochondrial diseases – basically, diseases of energy metabolism – for twenty-odd years, and CHCHD10 is the first mitochondrial protein that has been linked to ALS,” Shoubridge says.

He explains that in addition to spotlighting CHCHD2, his team found that the two proteins interacted, forming a complex in mitochondria. The two genes are closely related, but neither can fully compensate for the other. “If the energy lack is severe enough, the cells degenerate and die,” Shoubridge explains. “These genes are clearly both involved in neurodegeneration to some degree. Now, we need to understand: what’s their relationship? What are these proteins doing in mitochondria, and how does this cause the disease?”

Much of the groundwork has been done in cell models and patient samples; now, with supportive funding, the Armstrong lab’s zebrafish models will add critical new insights.

WHY ZEBRAFISH?

Zebrafish are well-suited to ALS genetics research. More than 70% of human genes are also found in zebrafish, along with many parallel critical pathways that contribute to development. They are also wonderfully adaptable to genetic modification. For Armstrong, who has engineered zebrafish models for neurodegenerative disease over much of his career, the model is especially useful to investigate defects at the cellular level in the spinal cord of the fish.

Armstrong notes that a revolution in genetic biology has occurred over his dozen years in ALS research: “When I began, there were maybe four genes known to be involved in ALS; now, we have over two dozen, and many more candidate genes are being explored,” he says. “Thanks to gene-editing technologies, we can now make targeted mutations in these fish that are relevant for the disease we’re studying, and genetically more accurate to the mutations carried by patients with ALS.”

This is exactly what Armstrong will do for the funded project: using the CRISPR/Cas9 gene-editing system, his team will create zebrafish animal models with the same mutations in CHCHD10 that have been observed in people with ALS, working with observations at the cellular level made by Shoubridge’s lab. “Then, we can examine the fish to see what’s going wrong – we can look at the muscles, we can look at the brain, we can look the spinal cord.”

Armstrong brings another critical skillset to the table. As a trained electrophysiologist, he can use patch clamp electrophysiology – for instance, attaching electrodes directly to motor neurons, the cells that use electrical signals to stimulate movement in the fish – as a means to examine the functional connectivity between motor neurons and muscle cells. This will be invaluable in unlocking the roles of these two genetic mutations at the critical neuromuscular junction where motor neurons connect to muscle.

A UNIQUE PAIRING OF COMPLEMENTARY STRENGTHS

Dr. David Taylor, Vice President of Research for the ALS Society of Canada, says the project benefits from a distinctive combination, with the Shoubridge and Armstrong labs each focused on unique aspects of the same question; it’s also an important foray into relatively uncharted territory. “There haven’t been many studies done yet that look at understanding the roles of CHCHD10 and CHCHD2 in ALS,” he explains. “It’s exciting to see a mitochondrial expert like Dr. Shoubridge, with his knowledge in molecular genetics and biochemistry, working on the role of these mitochondrial proteins in ALS, and a great pairing given that with zebrafish, the Armstrong lab has an ideal model for them to work with.”

Dr. Viviane Poupon, President and CEO of Brain Canada, agrees that it’s a promising team-up. “The Discovery Grant Program helps bring together unique teams, encouraging researchers like Dr. Armstrong and Dr. Shoubridge to combine their expertise in novel ways,” she says. “We are thrilled to partner with the ALS Society of Canada to fund research projects like theirs, to accelerate breakthroughs and help improve outcomes for Canadians living with ALS.”

TWO “WRONG TURNS” MAKE A RIGHT

As Armstrong tells it, one wrong turn led him into a career in ALS research. “In 2005, I was at the annual conference of the Society for Neuroscience in D.C.,” he relates. “It’s a massive affair, and I Iiterally took a wrong left turn and wound up in a breakout room where Dr. Don Cleveland happened to be talking about ALS, and the gap in knowledge about motor neuron function in existing models.”

“Only a few papers had looked at the functional connectivity between a motor neuron and a muscle cell,” he recalls. “People were dying of this dreadful disease, and there was a clear need, and I had the skill set to help fill it.”

The path was slightly different for Eric Shoubridge. He joined The Neuro in 1985 with the task of setting up their MRI research program, a relatively new technology at the time. As it gained currency, he longed for a fresh challenge. The late Dr. George Karpati, an expert on neuromuscular pathology, urged him to study neuromuscular disease at the DNA level: “And I walked away from my magnetic resonance machines, and I trained myself as a geneticist!” he says, smiling. “Science kind of self-selects, a little bit – you do what you get passionate about.” Today, in addition to his position at The Neuro, he is Chair of the Department of Human Genetics at McGill University.

“So that was my wrong turn,” Shoubridge laughs. “Or should I say, my right turn!”

A FUTURE WITH MORE “EUREKA” MOMENTS

The question of how genes like CHCHD10 and CHCHD2 work fall under the research category of foundational science: painstaking, up-close investigation of the “how” and “why” of very granular biological processes. “The more information about basic biology and how it goes wrong in the disease, the better off we are,” Armstrong says. “ALS Canada and Brain Canada have afforded Eric and me the chance to really pursue this fundamental biology, this fundamental science,” he says. “And that’s critical, because the more we understand about all these different genes that we’re discovering in the clinic, we can ask new and better questions in our labs. Major breakthroughs have often come serendipitously, from inquisitive basic science.”

“That’s what drives basic scientists: the detective work, closely following clues, that can lead to a ‘Eureka!’ moment,” adds Shoubridge. “It’s extraordinarily satisfying work. And I think the Discovery Grant program is really enlightened: it allows you to take a new idea and run with it.”

Funding that makes a difference

The Discovery Grant Program makes these connections possible with a funding model that favours interdisciplinary collaboration, bringing the best minds together to tackle complex problems. Discovery Grants give promising novel ideas the fuel they need to gain traction; in 2021, up to eight projects will benefit from $1M in total funding.

Since 2014, ALS Canada’s partnership with Brain Canada has resulted in more than $23 million being invested in leading-edge ALS research that has helped further understanding of the disease. The Discovery Grant Program is designed to fuel innovation that will accelerate our understanding of ALS, identify pathways for future therapies and optimize care to improve quality of life for people and families affected by this devastating disease.

The Discovery Grant Program has been made possible by Brain Canada, through the Canada Brain Research Fund (with financial support from Health Canada) and the generosity of provincial ALS Societies, ALS Canada donors and community-based efforts, including 40 per cent of net proceeds from the Walk to End ALS.