Scientific discoveries are like puzzles. At first, two puzzle pieces may not appear to fit together, but then a new way of comparing them makes it possible to see how they connect, helping to fill in the picture.
Mutations in the C9ORF72 gene are the most common genetic cause of ALS. Another abnormality that occurs in a majority of all ALS cases is the misplacement of TDP-43 protein from the cell nuclei of motor neurons into the cytoplasm, the jellylike substance found inside cells. In the cytoplasm, the mislocalized TDP-43 protein aggregates clump up, becoming potentially toxic to the motor neurons.
Lilian Lin, a PhD student in Dr. Janice Robertson’s Lab at the University of Toronto believes that the loss of the ability of the C9ORF72 gene to function properly and the aggregation of TDP-43 may be related events. She hypothesizes that when the C9ORF72 gene is mutated, it loses the ability to perform its normal functions, causing an accumulation of misplaced TDP-43 in the cytoplasm. With a Trainee Award of $75,000 over three years from the ALS Canada Research Program, Lin will perform experiments to find out if her hypothesis is true.
“TDP-43 mislocalization is seen in all C9ORF72-ALS cases so far, so it is very important to see if there is a relationship,” said Lin. “If we can discover the underlying mechanisms to explain how and why these events occur, we can test whether it is possible to modify them to halt or slow disease progression.”
Exploring the relationship
The C9ORF72 protein is presumed to normally perform at least two roles to keep cells healthy: shuttling proteins in and out of the cell nucleus in a process called nucleocytoplasmic transport, and cleaning out damaged, accumulated proteins in a process known as autophagy.
For her project, Lin will create genetically-modified mice with depleted C9ORF72 function—some with no function and some with partial function. Then she will introduce TDP-35 protein into the brains of the mice. TDP-35 is a novel version of TDP-43 developed in Dr. Robertson’s lab that is an excellent model for studying the behaviour of TDP-43.
Over the course of about a year, Lin expects the C9ORF72-deficient mice will develop the signs and symptoms of ALS. She will assess their progress with behavioural and cognitive tests and examine changes in brain and spinal cord tissue at three, six and twelve months to evaluate disease progression compared to healthy mice.
The project is underway and Lin has already noticed some changes. “So far, it’s hard to pinpoint exactly what’s different with the mice, but they don’t explore as much as healthy mice when placed in a new environment,” Lin said. She suspects the mice may also be showing signs of dementia. ALS and frontotemporal dementia (FTD) are related diseases that likely exist on one clinical spectrum, with levels of cognitive and behavioural impairment ranging from no FTD to significant FTD symptoms.
Lin will also study cell cultures to see how reduced function of the C9ORF72 gene affects TDP-43 aggregation. Finally, she will perform lab experiments to see if she can reverse TDP-43 aggregation, by moving TDP-43 protein back into the cell nuclei or by boosting autophagy.
Advancing the field of ALS research
Lin started her academic career studying protein behaviour in a chemical engineering lab at the National Taiwan University. After coming to Canada, she focused more on protein aggregation as it relates to neurodegenerative diseases. Her work on ALS began in Dr. Martin Duennwald’s lab at Western University, where she studied protein aggregation in yeast and human cell models of ALS. Now, in Dr. Robertson’s lab at the University of Toronto, Lin is looking forward to finding out if there is a relationship between C9ORF72 deficiency and the mislocalization of TDP-43 by working with mouse models.
“Before we can find a way to halt or slow ALS disease progression, or prevent it from occurring, we need to understand where we may be able to intervene in the process,” said Lin. “I’m excited to be working on a project that may provide important insights about the mechanisms of ALS that may, one day, lead to the discovery of a new therapy.”