A Brief Description of Currently Funded  Research Grants 2019-2020

Dr. Simon Chen
University of Ottawa
Ottawa, ON
INTRODUCTION: Autism Spectrum Disorders (ASDs) comprise a group of severe neurodevelopmental disabilities that affect approximately 1 in every 88 children. Many identified genetic causes associated with ASDs have been shown to affect the formation, functional efficacy, or plasticity of excitatory and inhibitory synapses in the brain. Aberrant perturbations in synaptic functions could lead to an elevation of `noise’ or `excitability’ among neural networks; hence, resulting in the behavioral deficits observed in ASDs. While many studies on ASD are focused on disabilities such as social impairments, repetitive stereotyped behaviors, and communication deficits, clinical studies have also reported that ASD patients exhibit deficits in basic motor coordination and motor learning. These motor deficits could result in frustration for patients with ASD and interfere heavily with their everyday routines and social interactions.
Recent identifications of gene mutations or copy number variations (CNV) associated with ASDs have contributed to a significant breakthrough toward a better understanding of these disorders. Our lab studies the 16p11.2 deletion (16p11.2+/-) mouse model of ASDs. We have obtained preliminary data that indicates 16p11.2+/- mice exhibit delayed motor learning without deficits in motor execution and coordination. In addition, we identified a decrease in axonal projection of a major neuromodulator, noradrenaline, in the motor cortex of 16p11.2+/- mice. Hence, the overall aim of this proposal is to examine whether the delay in motor learning observed in the 16p11.2+/- mice is caused by perturbed noradrenaline activity in the motor cortex. A careful dissection of the neural circuits within the M1 will not only provide a better understanding of motor learning, but will also aid to pinpoint network dysfunctions in ASD.
OBJECTIVES: we propose the following aims to test the hypothesis
I. Do neurons in the motor cortex of 16p11.2+/- mice show abnormal network activity during motor learning?
II. Is there also reduced activity in noradrenaline neurons in the 16p11.2+/- mice?
III. Does activating noradrenaline neurons rescue delayed motor learning and network abnormalities in 16p11.2+/- mice?
OUTLINE OF RESEARCH: We are currently studying the 16p11.2+/- mouse model of ASDs. The 16p11.2 chromosomal copy number deletion, which encompasses a 593 kb region encoding 29 genes, is a strong genetic risk factor in developing ASD, which contributes to 1% of case prevalence. A homologous chromosome region, 7qF3, and the same 29 genes were identified in mice, and deletion of this chromosomal region has shown behavioral resemblances to the human. Preliminary data from our lab shows that 16p11.2+/- mice do not exhibit movement-related deficits, but require a prolonged training to acquire new motor movements. This is accompanied by a significant reduction in noradrenalin axonal innervations in the motor cortex.
We have designed a series of experiments employing chronic in vivo two-photon imaging, which permit us to follow the dynamics of abnormal networks, with hundreds of neurons at single-neuron resolution, in the motor cortex of 16p11.2+/- mice over weeks of training. We will combine with different cutting-edge techniques to manipulate noradrenaline activity in the 16p11.2+/- mice and examine whether this will be sufficient to rescue the delayed motor learning.
PROJECT BENEFITS AND APPLICATIONS: ASD can be a debilitating disorder, affecting not only cognition and communication but also the ability to acquire common everyday movements. Together, the experiments proposed here are designed to significantly advance our understanding of the circuit plasticity underlying motor learning, and how disrupted noradrenaline signaling may contribute to neural network dysfunction in the brains of ASD patients, using in vivo two-photon imaging in awake and behaving mice. We expect this body of work will have a significant impact on the development of therapeutic strategies for counteracting neural circuit dysfunctions associated with motor-related deficits in ASDs.

Dr. David Gosselin
Université Laval
Quebec City, QC
Introduction: Autism spectrum disorders (ASD) are a group of disabilities that include significant social, communication, and behavioral impairments. In Canada, ASD are among the most common forms of neurodevelopmental disorders, affecting 1 in 66 children. There are currently no cures for ASD. Therefore, there is a high incentive to conduct research on ASD to develop efficient disorder-modifying treatments that will improve the lives of individuals living with ASD.
The brain possesses a specialized immune cell called microglia. Microglia are important to protect the brain from infection and also play an important role in ensuring normal brain development. Accumulating evidence suggests that there is a persistent inflammatory response and altered microglial activity in the brain of individuals with ASD. However, the molecular mechanisms that underlie this chronic microglial inflammation remains largely unknown.
Genetic evidence indicates that mutations affecting proteins involved in the regulation of expression of genes are significant genetic risk factors predisposing for the development of ASD. Notably, our data shows that human and mouse microglia express most of these regulatory proteins. Together, these observations support our hypothesis that defective functions of these regulatory proteins may be a major factor contributing to elevated microglial expression of inflammatory genes in the brain of individuals living with ASD.
Objectives: The ultimate goal of our research project is to gain a better understanding of the mechanisms that contribute to deregulation of gene expression in microglia in ASD. To this end, two objectives are pursued in this research project proposal. In Objective 1, we will assess whether absence of a regulatory protein called Capicua (Cic) transcription repressor leads to higher expression of inflammatory genes in microglia. In Objective 2, we will investigate the consequence of loss of Cic functions in microglia on brain development and behavior.
Outline of research: We will perform our studies using mice as a model of brain development. Note that our previous research showed that mouse and human microglia share extensive similarities in common with respect to the mechanisms of regulation of gene expression. In a first series of experiments, we will perform comprehensive analyses using next-generation DNA sequencing to reveal the subsets of genes regulated by Cic. Notably, we will also examine microglial DNA architecture to gain insights into the signaling pathways and epigenetic mechanisms regulated by Cic. Following these analyses, we will conduct imaging analyses using state-of-the-art electron microscopy to assess how absence of Cic disrupts the interactions of microglial cells with neuronal structures. Finally, we will perform a series of behavioral analyses to assess the consequence of impaired microglial gene regulation and cell functions in absence of Cic on brain functions.
Projected benefits and application of findings: Collectively, the studies proposed here are highly innovative because they will explore with unparalleled resolution the molecular mechanisms underlying defective gene regulation in microglia associated with ASD. Of note, validation of our hypothesis will provide the first mechanistic evidence that microglia can display higher inflammatory activity in absence of infections in the brain. This would represent a novel conceptualization of the development of ASD. Furthermore, our research approach will allow us to identify signaling pathways regulated by Cic. This is significant because the molecular components of these pathways represent accessible targets for pharmacological interventions. Thus, the identification of signaling pathways implicated in ASD is key for the design of treatments aimed at mitigating brain dysfunctions in ASD. Overall, this research project is extremely well-positioned to make game-changing discoveries that will make major advancements in our understanding of the development of ASD. This knowledge will in turn provide new insights that will guide future biomedical research on ASD and, importantly, possibly lead to new innovating forms of treatment.

Dr. Jeehye Park
Hospital for Sick Children
Toronto, ON
Introduction: Frontotemporal dementia (FTD) is a neurodegenerative disease leading to behavioral changes and social deficits. ALS is a neurodegenerative disease resulting in muscle weakness, atrophy and paralysis followed by respiratory failure and death. As stated, FTD and ALS were first described as clinically distinct neurodegenerative diseases. Over several decades of clinical studies reported the existence of patients with both FTD and ALS. Recent pathological and genetic studies corroborated that FTD and ALS are closely linked. However, not all ALS patients develop FTD and not all FTD patients develop ALS. Plus, mutations within the same disease gene could give rise to three different clinical outcomes, only FTD, only ALS and both FTD and ALS. This intriguing link between FTD and ALS and the underlying complex molecular mechanisms leading to FTD and/or ALS are not clearly understood. If FTD and ALS are closely linked, do these two disorders share a common molecular mechanism? What are the distinct molecular pathways leading to only FTD or only ALS?
Objective: In this proposal, we exploit one such gene MATR3 whose mutations lead to different clinical outcomes; S85C mutation identified in familial ALS with an age of onset between 30s and 40s, whereas F115C identified in both ALS and dementia between the ages of 50s and 70s. We generated MATR3 mutant mice harboring a single pathogenic mutation in the mouse Matr3 gene using Crispr/Cas9 system (MATR3 S85C or F115C knock-in mice). Our MATR3 S85C knock-in mice show motor deficits reminiscent of ALS, whereas MATR3 F115C knock-in mice do not show any overt behavioral phenotype at one-year of age. To investigate the molecular mechanisms by which S85C and F115C mutations cause shared (ALS) and distinct (with or without FTD) clinical consequences, we will first investigate whether MATR3 F115C knock-in mice develop ALS and FTD at later ages (Objective 1a). In addition, we will compare the S85C and F115C knock-in mice for FTD-like neuropathological features (Objective 1b). Furthermore, we will delineate the cellular and molecular mechanisms by which S85C and F115C mutations lead to ALS and FTD/ALS, respectively (Objective 2).
Outline of Research: OBJECTIVE 1: To determine whether MATR3 F115C knock-in mice exhibit FTD-like behavioral and pathological features. 1a) To characterize the cognitive behavioral phenotypes of MATR3 knock-in mice. FTD is characterized by behavioral impairments such as apathy, lack of empathy, social withdrawal and repetitive compulsive behavior. Therefore, we will test whether MATR3 F115C knock-in mice show social impairment and repetitive-like behavior. We plan to perform several behavioral analyses including three-chamber test (social cognition test) and marble burying test (repetitive compulsive-like behavior).
1b) To investigate the brain pathology in MATR3 knock-in mice. FTD patients show pyramidal neuron degeneration, gliosis and ubiquitinated cytoplasmic inclusions in the neocortex and hippocampus. We will conduct immunohistochemistry on both MATR3 S85C and F115C knock-in mouse brain to compare and contrast the pathology. We expect that MATR3 F115C knock-in mice show FTD-related brain pathology whereas MATR3 S85C knock-in mice do not. OBJECTIVE 2: To investigate the cellular and molecular mechanisms by which different MATR3 mutations lead to diverse phenotypic outcomes within the FTD/ALS disease continuum. Our preliminary data strongly suggest that differential protein solubility of S85C and F115C may underlie the different phenotypic outcomes. We will test our hypothesis using primary cortical and motor neurons isolated from our S85C and F115C knock-in mice. We expect that S85C and F115C mutants show different solubility characteristics in primary cortical and motor neurons.
Projected benefits and applications of findings: Altogether, these unprecedented studies will reveal the role of MATR3 in brain function and dementia and elucidate the detailed molecular mechanisms by which different MATR3 mutations lead to diverse phenotypic consequences within the FTD/ALS disease spectrum. Our MATR3 mouse models will be useful for further mechanistic studies as well as preclinical studies for FTD/ALS. This knowledge will provide fundamental insights into our understanding of how FTD/ALS-linked mutations in other genes contribute to varying clinical features within the disease continuum.

Dr. Anne Wheeler
Hospital For Sick Children
Toronto, ON
Introduction: Concussions are very common in children who often experience injuries while engaged in physical activities. Most children experience symptoms for only a short period of time, however, approximately 1 in 4 children with concussion suffer from long-term disability after a concussion, including problems with thinking and behaviour. It is not known why some children recover and some children experience long-lasting symptoms and there are no treatments available for these symptoms. The barrier to addressing these issues is that we don’t know what brain processes are responsible for symptoms and recovery from symptoms after concussion in children.
The forces that act on the brain during a traumatic injury damage white matter, the cables that connect different brain regions. White matter is made of long axons that are wrapped in myelin, a fatty substance that supports and insulates axons. Once myelin is damaged or lost the brain has the inherent ability to add new myelin and repair this type of injury which may be a process that helps children recover from symptoms after injury. This may explain why the youngest children who have the highest rates of myelination are more likely to recover than older children. Similarly, known differences in myelin between boys and girls may account for the higher rates of long-lasting symptoms in girls.
Objective: The objective of this project is to establish whether myelin loss and remyelination are responsible for behavioural impairment and recovery following concussion in development.
Outline of research: Using a closed-skull impact model of concussion in mice we will tackle 3 aims to achieve our objective. Aim 1. Establish the time course of white matter and behavioural impairment after a concussion at 3 stages of development in male and female mice. Different groups of male and female mice will receive a concussion-like impact at 3 stages of development: juvenile, early adolescence and late adolescence. We will characterize behaviour and white matter structure at each of 3 post-injury timepoints that correspond to acute, subacute and chronic phases of recovery from concussion in children. The results of this aim will provide a description of normal age- and sex- related differences in the vulnerability of white matter and repair capacity of myelin in this model of concussion as well as a description of the relationship of these brain changes to changes in behaviour after a concussion. Aim 2. Block the production of new myelin after concussion in young mice and determine whether this prolongs impairment. The first aim will provide evidence for a relationship between myelin and behaviour changes after concussion in young mice. However, a causal link between myelin and symptoms will still be missing. Our pilot data suggest that blocking the production of new myelin after concussion prolongs behavioural impairment in adult male mice. In the following experiments, we will examine this effect in young male and female mice. This experiment is designed to test the hypothesis that new myelin is an important factor in recovery after concussion such that blocking new myelin will lead to worse symptoms in young mice. This experiment will also inform whether new myelin is equally important for recovery in young male and female mice. Aim 3. Enhance the production of new myelin after concussion in young mice and determine whether this accelerates recovery. If normal remyelination contributes to recovery from a concussion then enhancing the production of new myelin may accelerate recovery after concussion. Here we will test if promoting remyelination leads to decreased symptoms in young male and female mice. Myelination will be enhanced in two different ways, with an environmental and pharmacological intervention.
Projected benefits and application of findings: Successful demonstration of a role for new myelin in recovery from a concussion would suggest that children with long-lasting symptoms have problems with their natural ability to remyelinate after a concussion and that these children may benefit from interventions that promote the production of new myelin. This would present the exciting opportunity to test and/or develop new therapeutic strategies to facilitate recovery after concussion by enhancing myelin production in children.