Projects Funded 2018

Table of Contents

ALS Canada Project Grants

ALS Canada Trainee Awards

ALS Canada Project Grants

Can a revolutionary gene-editing tool create better animal models for studying ALS?

Peripheral and central neuronal circuit defects in ALS genetic models

$125,000 awarded to Dr. Gary Armstrong at the Montréal Neurological Institute

The loss of connection between muscles and motor neurons at neuromuscular junctions occurs early and throughout the progression of ALS. Defects arising in synapses, the spaces between neurons where electrical and chemical signals pass from one neuron to another, are not well understood.

In previous work, Dr. Armstrong has successfully created zebrafish models of ALS and conducted lab experiments to study the nature of defects in neuromuscular junctions and synapses in the spinal cord. Zebrafish are uniquely suited to these studies because they are transparent, allowing researchers to see biological responses easily. However, with older technology, the zebrafish ALS models may not represent the human disease in the right way.

With this grant, Dr. Armstrong will use a powerful new gene editing tool called CRISPR-Cas9 to genetically modify zebrafish to express the TDP-43 and FUS forms of ALS. He hopes that by more accurately representing the genetic situation in human ALS he will have a more in-depth opportunity to examine the cellular defects that arise in synapses within the spinal cord and in neuromuscular junctions that connect the brain and spinal cord to muscles. New insights about the circuitry between muscles and motor neurons may provide a more complete understanding of how ALS arises and one day, could lead to new treatment approaches. Further, if CRISPR-Cas9 proves to be a successful gene-editing tool to create better zebrafish models of ALS, the learning will pave the way for using it to create better models of ALS in other animals in future research.

Can overactivated immune cells in the brain be reprogrammed to prevent or slow ALS?

Targeting SRSF3 to reprogram immune response in ALS

$125,000 awarded to Dr. Jasna Kriz at l’Université Laval

Microglia are the primary immune cells of the brain, providing the first and main form of defense against toxic substances. Microglia are initially helpful, but in many neurodegenerative disorders, including ALS, chronic overactivation during progression causes them to change and become toxic. Scientists have tested therapeutics in human clinical trials that target microglia in a generalized manner to reduce inflammatory and immune responses in a non-specific way, however this approach has not been successful to date. In this project, Dr. Kriz will attempt to target specific activation within the microglia of people living with ALS.

Dr. Kriz’s recent research indicates that the activation of a protein called serine/arginine-rich splicing factor 3 (SRSF3) can cause microglia to transform from their healthy, helpful state into abnormal cells that promote inflammation. In her work, she will investigate the role of SRSF3 in mice microglia that have been genetically reprogrammed to have human ALS and are chronically activated. She also hopes that inhibiting SRSF3 will not only reduce the toxic activation of microglia in ALS, but also restore their normal supportive function.

If successful, Dr. Kriz suggests that this SRSF3 activation mechanism will be an exciting new target for therapies that may be able to prevent ALS or slow its progression by reverting over-activated microglia back to normal and restoring their healthy immune response.

A new tool to measure ALS-specific health-related quality of life in clinical trials

Co-production of a preference-based measure of health-related quality of life for ALS

$125,000 awarded to Dr. Ayse Kuspinar and Dr. Vanina Dal Bello-Haas at McMaster University

Currently, no available assessment tool has been developed in collaboration with Canadians living with ALS that sufficiently accounts for or values their needs and preferences in the context of the Canadian healthcare system. Conventional measurement tools tend to focus on the physical factors of the disease and ignore the value of a potential treatment on quality of life.

With this grant, Dr. Kuspinar and Dr. Dal Bello-Haas will develop a new tool called the Preference-Based ALS Index (PB-ALS) with essential input from Canadians living with ALS and ALS expert clinicians in the Canadian ALS Research Network (CALS). Participating volunteers will identify specific aspects of their life that has been affected by ALS, such as their social life, relationship with family and friends and ability to work. Each participant will rate how they are doing for each domain and prioritize the domains where they see the most need for improvement. The tool will attach an economic value to each factor, allowing the researchers to calculate an overall score that incorporates gains in one health area with losses in another.

Dr. Kuspinar and Dr. Dal Bello-Haas anticipate that gathering this cost-effectiveness information during clinical trials will help measure the value of therapies on the quality of life of someone living with ALS. The goal is to enable this information to be used during the drug access pathway in order to expedite Health Canada approval and provincial funding decisions for new ALS drugs in the future, resulting in Canadians gaining faster access to new treatments.

Can new understandings about stress granules explain subtypes of ALS?

Defining the RBP repertoires of ALS-associated membrane-less granules

$125,000 awarded to Dr. Eric Lécuyer at the Institut de recherches cliniques de Montréal

DNA holds the master instructions for making proteins in the body. Other molecules called RNA are made from the master instructions and have several critical roles in cells, including being responsible for protein production. When cells are internally or externally exposed to potentially harmful factors, RNA is protected by specific proteins that bind it and accumulate in little structures called stress granules. Over the past several years, ALS researchers have learned that the composition, distribution and formation of stress granules within motor neurons are significant factors in the development of ALS and frontotemporal dementia.

With an ALS Canada-Brain Canada Discovery Grant awarded in 2016, Dr. Lécuyer used antibodies to explore and describe stress granules in greater detail. For this project, he will advance his previous work. Using the antibodies and advanced imaging techniques, he will investigate the relationship between stress granules and RNA binding proteins in different types of human ALS cells, with an emphasis on toxicity caused by the most prominent genetic form of ALS, C9ORF72. Using these results, he will also examine how stress granules are associated with neurodegeneration in ALS by conducting experiments with fruit flies that have been genetically modified to have human ALS. An important aspect of this project is that Dr. Lécuyer will create an open ALS RNA Binding Protein Imaging Database to share his imaging data with the global ALS research community.

Can new technologies discover whether ALS pathology is unique in different parts of the brain and spinal cord?

Linking brain cell subtype-specific gene expression and epigenomic mosaicism in C9orf72-ALS

$125,000 awarded to Dr. Janice Robertson at the University of Toronto

ALS disease onset and progression vary significantly from person to person and scientists need to understand why this variation happens in order to effectively diagnosis and treat the disease.

One of the landmark discoveries in understanding ALS was that the most common genetic cause of ALS and frontotemporal dementia is a mutation where the C9orf72 gene has abnormal repetitive pieces of DNA called a repeat expansion. After several years of studying C9orf72 in people with ALS, scientists have learned that abnormalities appear to be different in one area of the brain and spinal cord versus another, but this has been difficult to confirm.

In this pilot project, Dr. Robertson and her Postdoctoral Fellow, Dr. Paul McKeever, will be among the first researchers to apply two powerful new techniques; single-nucleus RNA-sequencing (sNuc-Seq) and assay for transposable-accessible chromatin sequences (ATAC-Seq), towards ALS research. Using archived samples of brain and spine tissue from people who had ALS with a mutated C9orf72 gene, people who had sporadic ALS, and people without ALS, she will perform sNuc-Seq and ATAC-Seq on the tissue in order to better understand the differences in ALS pathology at a single cell level.

Drs. Robertson and McKeever’s work will not only determine if there are differences in single cell level pathology from one brain region to another, but also whether differences can occur within a single region and in different cell types. Their research suggests this deep-dive analysis will confirm that the variations in ALS might be explained by pathological differences from cell-to cell. If correct, Drs. Robertson and McKeever hope to scale up this research to test a much larger number of people in order to revolutionize our understanding of the disease, and our ability to treat it.

Can a new way of measuring biological age explain why ALS affects people differently?

Investigation of epigenetic modifiers in ALS

$125,000 awarded to Dr. Ekaterina Rogaeva at the University of Toronto

We usually count age as the number of years since birth, but emerging evidence shows that the biological age of our cells may differ from chronological age. DNA can be altered by gene mutations or by environmental factors that leave marks on DNA without changing its underlying structure. in a process called DNA methylation. By measuring the accumulation of these marks over time in DNA from blood samples scientists can calculate DNA methylation levels, or the individual’s DNA methylation age, which may be the most accurate way to estimate their biological age.

Dr. Rogaeva’s work will determine whether DNA methylation age can help to explain why the onset of sporadic ALS, the most common form of the disease, can occur so differently in different people. Using clinical data and blood samples collected at diagnosis from 250 people with sporadic ALS, Dr. Rogaeva will analyze the clinical characteristics of ALS and DNA methylation levels in order to determine whether accelerated biological age is correlated with the disease. If an accelerated biological age is found in these samples, she will then validate her findings by conducting the same analysis using the thousands of whole genome sequence DNA profiles collected for Project MinE.

If successful, this research could someday lead to the development of a blood test that measures biological age and detects genetic variations associated with ALS. If DNA methylation age does influence the timing of disease onset, the test may prove to be a valuable tool for understanding susceptibility to ALS and enable earlier diagnosis, resulting in an earlier administration of treatments.

Do “hidden proteins” play a role in ALS?

Cracking the FUS code in ALS: From the dual-coding gene FUS, alt-FUS engages too in the pathology

$125,000 awarded to Dr. Xavier Roucou, at l’Université de Sherbrooke

Conventional science assumes that the known genes in our DNA contain coding information, with a single start and end, which produces a single protein with specific function that is critical for a cell to survive. However, Dr. Xavier Roucou has discovered that some genes may also contain a hidden set of instructions that make what are called alternate proteins (altProts). AltProts are not just variations of the expected proteins; they are entirely different.

Abnormal FUS protein has been identified as one of the causes of familial ALS, the inherited form of the disease. Dr. Roucou and colleagues recently discovered a new altProt created from a piece of DNA within the FUS gene, called altFUS, and believe that it may be a unique driver of the disease. They found that in ALS, alt-FUS cooperates with FUS in three processes within neurons: (1) by altering the energy-producing structures called mitochondria; (2) by impairing the ability to clear out waste products in a process known as autophagy; and (3) by encouraging FUS proteins to accumulate. Notably, they found that some of the problems related to ALS only occurred when both FUS and altFUS were present.

With this grant, Dr. Roucou will look for differences in altFUS protein in human ALS by comparing post-mortem brain tissue with and without the disease. Next, he will investigate if altFUS contributes to ALS on its own, by examining if abnormal forms of altFUS can cause disease in cells and fruit flies in the absence of full length FUS protein. Throughout his research, Dr. Roucou will add the altProt data generated in this project to his recently created open source database called OpenProt, which is freely available to other researchers online, in order to share his research and make it available to other investigators.

If altFUS is observed to be a key contributor to developing ALS, this project could revolutionize the biological understandings of the disease. The insights gained may highlight the importance of exploring altProts in other ALS-related genes, especially since alternative protein-coding instructions have been found on 75 per cent of the top 50 ALS-related genes.

Does a previously unstudied protein play an important role in ALS?

Defining the biological and disease relevance of hnRNP A1B, a TDP-43 dependent splice variant of hnRNP A1

$125,000 awarded to Dr. Christine Vande Velde at the University of Montréal

The TDP-43 protein is usually found inside the cell nucleus and is responsible for regulating many cellular processes. In 97 per cent of ALS cases and nearly half of frontotemporal dementia, scientists have discovered that the TDP-43 protein is misplaced to an area outside the cell nucleus called the cytoplasm.

Dr. Vande Velde recently discovered that reducing the amount of TDP-43 protein in the nucleus caused an ALS gene called hnRNPA1 to be abnormally read, thus creating a new protein that she labeled hnRNP A1B. Dr. Vande Velde suspects this new protein could be a previously undiscovered toxic form. In her work, Dr. Vande Velde will examine how hnRNP A1B functions and whether it plays a role in known mechanisms of ALS pathology. She will conduct cell and mice experiments first and then validate her findings using ALS tissue samples generously donated to the Douglas-Bell Canada Brain Bank in Montréal and through other ALS labs.

Dr. Vande Velde hopes that a better understanding of hnRNP A1B’s function in ALS will reveal it as a potential target for new therapies and biomarkers in the future.

ALS Canada Trainee Awards

Doctoral Scholarships

Can advanced imaging techniques effectively track disease progression?

MRI biomarkers of cerebral degeneration in ALS and their pathological validation

$75,000 awarded to Abdullah Ishaque, an MD/PhD student, in Dr. Sanjay Kalra’s lab at the University of Alberta

A biological marker, or “biomarker” for short, is a measurable indicator associated with a disease state that helps determine risk, severity and response to therapy. For example, the level of cholesterol in the blood is a biomarker for the risk of heart disease and is used as an indicator of response to cholesterol-lowering drugs.

Today, biomarkers are being incorporated into an increasing number of clinical trials. They can help advance drug development by providing researchers a way to monitor disease progression and by helping to identify and enroll people who are most likely to respond to a new treatment. In comparison to clinical trials that include people with many disease variations in one group, enrolling only those likely to respond can help researchers discover whether a therapy works in a faster and more effective way.

Validated biomarkers for ALS are urgently needed to help researchers develop a path to unique treatments for each person living with ALS. Magnetic resonance imaging (MRI) has emerged as a promising source of non-invasive biomarkers for ALS. Abdullah Ishaque, working with his PhD supervisor Dr. Sanjay Kalra, has recently developed two imaging biomarkers called texture analysis (TA) and quantitative T2 (qT2) mapping. TA measures subtle patterns and relationships in brain images and qT2 allows researchers to evaluate degeneration in the brain by analyzing water content, iron content, demyelination (damage to the outer protective covering of motor neurons) and inflammation.

In this project, Ishaque will investigate whether these two imaging biomarkers can monitor degeneration in the brain, upper motor neuron dysfunction and disease progression associated with ALS in people living with the disease. To validate his findings, he will also perform MRI scans on post-mortem tissue samples donated generously from people who had ALS and compare results with other measures of disease progression in the samples including neuron loss, changes to glial cells and demyelination.
For his work, Ishaque will be using brain MRI images obtained from the Canadian ALS Neuroimaging Consortium (CALSNIC), a project that was funded through the largest grant given through the ALS Canada-Brain Canada Foundation partnership with money raised through the Ice Bucket Challenge.

Is a loss of C9orf72 responsible for the most common protein abnormality in ALS?

Investigating the effects of c9orf72 haploinsufficiency on TDP-43 pathology in ALS

$75,000 awarded to Lilian Lin, a PhD student in Dr. Janice Robertson’s lab at the University of Toronto

Normal cells have a “self-cleaning” process called autophagy that breaks down and clears out cellular waste, so the cell can function properly. In the majority of ALS cases, a protein known as TDP-43 becomes misfolded and accumulates in motor neurons, causing toxicity. The accumulation of misfolded TDP-43 also occurs in people living with ALS who have a mutation in the C9orf72 gene, the most common genetic cause of ALS. The C9orf72 protein is known to play a role in autophagy and that role is diminished when the gene is mutated.

Recent animal experiments suggest that boosting autophagy with an experimental drug treatment can clear excess TDP-43 accumulation. Using human cells grown in culture and genetically-modified mice with depleted C9orf72, Lilian Lin will investigate whether the C9orf72 gene mutation and the associated loss of normal autophagy function increase TDP-43 protein misfolding and accumulation. If her hypothesis is correct, it will show an important connection between the two most prominently studied proteins in ALS and suggest that targeting this loss of autophagy might be a key avenue for the development of new ALS therapies.

Does a viral infection play a role in ALS initiation and progression?

Characterizing enterovirus disruption of autophagy as a disease mechanism for ALS

$75,000 awarded to Yasir Mohamud in Dr. Honglin Luo’s lab at the University of British Columbia

A group of viruses called enteroviruses can cause a variety of infectious illnesses that are usually mild with symptoms that may include fever, respiratory distress, flu-like muscle aches and rashes. However, some enteroviruses are more serious, like enterovirus D68 that can cause severe respiratory illnesses or the poliovirus that causes polio. Some researchers have long suspected that enteroviruses may be linked to ALS due to their ability to harm motor neuron function, but the available evidence of a causal link has so far been inconclusive.

Yasir Mohamud and colleagues at the University of British Columbia recently discovered that infecting motor neurons with enteroviruses can cause changes that are remarkably similar to those seen in ALS. The similarities include TDP-43 protein abnormalities and a reduced ability for motor neurons to clear and recycle cellular waste in a process called autophagy. Based on these observations, Mohamud believes that enterovirus infection may play an important role in how ALS begins and progresses.

In this project, Mohamud will use mouse models to investigate whether enterovirus infection causes TDP-43 toxicity and disrupts the autophagy process. He will also see if he can restore autophagy using compounds designed to inhibit the virus and examine whether enteroviral infection encourages the spread of misfolded TDP-43 and SOD1 from cell to cell, possibly explaining progression of ALS throughout the body.

As the majority of ALS cases are sporadic, meaning that they occur without any apparent family history of disease, Mohamud hopes that this project will provide a deeper understanding of the underlying triggers of sporadic ALS and reveal a promising new target for treatment development.

Post-Doctoral Fellowships

Can restoring motor neuron inhibition prevent or stop ALS progression?

Examining the role of KCC2 in maintaining the balance between excitation and inhibition in ALS

$165,000, in partnership with La Fondation Vincent Bourque, awarded to Dr. Sahara Khademullah, a postdoctoral candidate from Dr. Yves De Koninck’s lab at Université Laval.

Each muscle in the body receives electrical signals through motor neurons that will stimulate it to contract. Other specialized neurons inhibit these signals, allowing muscles to relax. When this inhibitory process does not occur, motor neurons become overexcited, a hallmark feature of ALS before symptoms appear, particularly in the upper motor neurons of the motor cortex.

In this project, Dr. Sahara Khademullah will establish whether a protein called KCC2 (potassium chloride cotransporter 2), which is found to be lower in people with sporadic ALS than in people without the disease, is a viable target to prevent or stop ALS progression. This research builds on two projects previously funded by ALS Canada (one in partnership with Brain Canada Foundation) that explored how enhancing the inhibitory response in the motor cortex may be protective in ALS. Dr. Khademullah will determine the extent of KCC2 deficits and when they occur in mice that have been genetically altered to mimic aspects of human ALS. Using newly-developed and non-invasive technology, she will also measure a readout of KCC2 loss in real time as the disease progresses in mice.

Dr. Khademullah will also determine whether KCC2 in cerebrospinal fluid (CSF) could be a reliable biomarker for ALS. She will collect CSF from ALS mice before they have developed symptoms and throughout disease onset and progression and compare the results to CSF from normal mice. Learning when KCC2 deficits occur may provide essential clues about optimal timing for treatment. Finally, Dr. Khademullah will test whether an experimental drug developed by Dr. De Koninck called CLP290 can retain or recover the ability to inhibit motor neurons by restoring KCC2 levels, which may slow or even stop disease progression. Overall, this research may identify KCC2 as a viable target for future drug development and a reliable biomarker to speed diagnosis and measure responses to new treatment options in clinical trials.

Can using worm and stem cell models of ALS to screen for new ALS drugs identify a treatment that slows disease progression?

Pre-clinical drug discovery against hexanucleotide repeat-containing C9orf72 toxicity using C. elegans and patient-derived induced motor neurons

$165,000 awarded to Dr. Prateep Pakavathkumar in Dr. Alex Parker’s lab at Université de Montréal

A few years ago, Dr. Parker and researchers in his lab genetically engineered worms that mimicked human ALS through motor neuron degeneration and paralysis. He then tested them with a battery of potential treatments to see if they could find one that would have a positive effect on motor neuron health and function. Through this work, they identified the drug pimozide as having a potential impact on functionality in worm models and investigators are now studying the effectiveness of pimozide in a Phase 2 clinical trial in eight locations across Canada.

In this project, Dr. Pakavathkumar will use a similar approach to screen potential therapies using new worms that have been modified in C9ORF72, which is the most common genetic cause of ALS in humans. If he finds promising candidates, he will validate the therapies in further lab experiments using motor neurons derived from people with the C9orf72 form of ALS. Any therapies that continue to show potential will be shared with other researchers to test them in zebrafish and mouse models with the hope of eventually moving them into human clinical trials.

Can antibodies detect a misfolded protein associated with ALS in cerebral spinal fluid?

Detection of misfolded TDP-43 in cerebral spinal fluid of ALS cases: Diagnostic potential of confirmation-specific antibodies

$165,000 awarded to Dr. Yulong Sun in Dr. Avi Chakrabartty’s lab at the University of Toronto

A more streamlined way to diagnose ALS earlier is desperately needed as current methods can take up to two years and rely heavily on ruling out other conditions that share similar symptoms. One of the hallmarks of ALS for 97 per cent of cases is the accumulation of misfolded TDP-43 protein in motor neurons. One theory about how the disease spreads from cell-to-cell is that misfolded TDP-43 is expelled from motor neurons in ALS and picked up by other cells. Some of this expelled TDP-43 may end up in cerebral spinal fluid (CSF).

Dr. Sun has developed two antibodies which bind exclusively to misfolded TDP-43 with the aim of making them more detectable in body fluids, something current lab tests cannot accomplish. He and his colleagues have previously shown pilot data that these antibodies are capable of detecting misfolded TDP-43 in post-mortem patient brain tissue.

In this project, he seeks to develop a new lab test that can detect misfolded TDP-43 in CSF. First, using advanced imaging technologies, he will test the two antibodies to see if they can detect misfolded TDP-43 in 40 samples of CSF already collected from people living with ALS at the Sunnybrook ALS Clinic. If successful, he will then develop a simple lab test that may help doctors diagnose ALS earlier and monitor disease progression.