Animal models enable scientists to study human diseases in lab settings. They help scientists learn about the biological changes that occur during disease onset and progression, and they can also speed the identification of promising therapies for testing in future clinical trials with human volunteers.
In 2005, when he was still a graduate student, Dr. Gary Armstrong attended a Society for Neuroscience conference in Washington, D.C. He decided to hear a talk on ALS, since it was an area he was not yet familiar with. Dr. Don Cleveland, a leading ALS researcher was presenting. “Dr. Cleveland said that it was great that scientists had created new mouse models of a human form of genetic ALS, but what the field needed next were experts to study the electrical signalling processes between motor neurons and muscles,” said Dr. Armstrong. “I had special training in electrophysiology, the study of electrical signals between cells. His talk really sparked my decision to focus my research on studying neuromuscular junctions in ALS.”
Zebrafish models of ALS
Today, Dr. Armstrong is a neuroscientist and assistant professor at the Montréal Neurological Institute at McGill University. He has published several papers about the loss of communication between motor neurons and muscles at the neuromuscular junctions, the tiny places where motor neurons connect to individual muscle fibres.
Through his work, Dr. Armstrong has successfully created zebrafish models of human ALS with TARDP and FUS gene mutations, two of the mutations associated with familial ALS. The very small zebrafish are only about six centimetres long, but they are an excellent animal for studying ALS for several reasons. Like humans, they have the same genes that provide the master instructions for making TDP-43 and FUS proteins that are associated with some of the known genetic causes of ALS in humans. Zebrafish grow quickly into adults in a few days, so lab experiments can be conducted fairly quickly. They are also transparent, making it easy for researchers to examine their anatomy in fine detail. “Under a microscope, I can look at the spinal cord, then zoom in closer to see the neuromuscular junctions,” said Dr. Armstrong. “They are beautiful to see at the biological level; they look like little pretzels.”
A powerful new gene editing tool
Previously, Dr. Armstrong successfully created genetically-engineered zebrafish to express human TDP-43 and FUS gene mutations using standard gene-editing technology, which involves inserting a human ALS gene into a zebrafish embryo so that it will hopefully mimic the signs and symptoms of the disease. Some of the key drawbacks with this method, however, are the difficulty in controlling precisely where the altered gene is inserted, and the composition of the abnormal human protein, which is typically produced in much greater quantities than in humans with ALS.
In 2012, scientists discovered a powerful new gene-editing tool called CRISPR-Cas9 (“CRISPR”) that has revolutionized lab research worldwide. It works like a pair of molecular scissors, cutting DNA in a specific place to remove a defective gene so that a new gene can be inserted. CRISPR is a much faster, less expensive technique for making genetically modified animal models of disease compared to older methods. Furthermore, by inserting a mutation directly into an existing zebrafish gene, it creates an animal model that has the correct amount of abnormal protein, rather than unnaturally high levels.
Dr. Armstrong realized right away that CRISPR could allow him to make improved zebrafish models of ALS. But first, he had to find a way to modify the CRISPR technique to cut and paste just one of the building blocks that make up DNA, a single nucleotide, not a whole gene consisting of a sequence of nucleotides, so that he could model the genetic mutations seen in human ALS as closely as possible. “Imagine we are using scissors to cut apart words in a ‘sentence’ of the DNA code. With regular CRISPR, we can cut out the word ‘the.’ But we needed to take out just the letter ‘t,’ to reduce ‘the’ to ‘he,’ more accurately modeling the single nucleotide mutations that cause ALS,” said Dr. Armstrong. “My colleagues and I tried several times to make this work. When we were finally successful, it was one of the happiest moments of my research career so far.”
Advancing ALS research
With a $125,000 project grant from the 2018 ALS Canada Research Program, which builds on two previous grants — an ALS Canada-Brain Canada Career Transition Award in 2015 and an ALS Canada-Brain Canada Discovery Grant in 2016 — Dr. Armstrong will use his innovative CRISPR technique to create zebrafish models with TDP-43 and FUS genetic mutations of ALS. He will determine if the CRISPR models are better than those made with traditional gene-editing by measuring their activity in Petri dishes over time with an automated data capture system he created previously. He will examine the fish to see if the gene editing worked as planned and if they developed mutated TDP-43 and FUS proteins and defects in the neuromuscular junctions of their spinal cords.
If he is successful, this project will advance future ALS research in two ways. First, it will validate this modified CRISPR technique, paving the way for using it to create more accurate models of ALS in other animals, like mice, in future research.
Second, Dr. Armstrong could scale up the production of CRISPR zebrafish models of ALS and use them to screen for drugs. Compared to other animal models, screening drugs with zebrafish is much faster because they grow so quickly, and experimental drugs can be added directly to the water, treating many fish at once rather than one animal at a time.
“Our innovation may help other researchers develop better animal models of ALS, helping to advance promising therapies into clinical trials much faster than current timelines,” Dr. Armstrong said.