A Brief Description of Currently Funded Research Grants 2022 – 2023

Understanding and therapeutically leveraging a rare neocortical neuron type in an ASD mouse model

Dr. Mark Cembrowski
University of British Columbia
Vancouver, BC

INTRODUCTION: Autism spectrum disorder (ASD) imposes considerable challenges on Canadian individuals, families, and communities. Although new drugs for ASD are urgently needed to deal with these challenges, there are currently no drugs that treat the underlying causes of ASD. As such, research aimed at understanding these biological causes of ASD holds immense opportunity for identifying new targets and next-generation therapies that may transform ASD treatment.
Intriguingly, a rare type of excitatory neuron in the brain has been recently implicated in typical neural development, as well as dysfunction in ASD. This neuron type occupies the deepest part of the neocortex, a brain region critical for high-order brain functions. These “deep neocortex” neurons are the earliest born and first active neurons in the neocortex, and thus represent a neuron type that is essential to brain development. Embodying this, a variety of complementary experiments have suggested dysregulation of deep neocortex neurons may drive a variety of neurodevelopmental disorders such as ASD. In collection, deep neocortical neurons represent an excellent opportunity to understand a key cell type in ASD, and to use this understanding to derive new therapies targeted to the deep neocortex.

OBJECTIVE: Here, we will investigate the precise and causal role of deep neocortex neurons in an environmental model of ASD, and use this knowledge to develop and test next-generation drugs for ASD treatment. We will investigate the specific predictions that (1) deep neocortex dysregulation causes cellular and behavioural properties of ASD, and (2) preventing this dysregulation with drugs can prevent these aberrant cellular and behavioural properties. To examine these predictions, our work will integrate molecular, cellular, and behavioural neuroscience using a range of cutting-edge methodologies.

OUTLINE OF RESEARCH: We will first seek to understand ASD-related dysregulation using genomics technology and “Big Data” analysis. To begin, in mice that emulate ASD in a well-controlled experimental setting, we will measure the expression of every gene in the mouse genome for thousands of individual deep neocortex neurons. From these Big Data, we will extract previously unresolved genes that are changed in deep cortex neurons in ASD, and use these results to obtain new drug targets and pharmacological agents. Using state-of-the-art cellular activity recording, we will identify whether these genes may be involved in changing neuronal activity in ASD, and whether these changes can be prevented by manipulating deep neocortical neurons. Finally, we will examine mouse behaviour, seeking to identify whether changing activity in deep neocortex cells with new drugs can prevent ASD-related behaviors. In collection, this work will provide a multidisciplinary and cell-type-specific understanding of ASD, and derive new potential therapeutic targets and agents.

PROJECTED BENEFITS AND APPLICATIONS: This research seeks to identify and regulate causal molecular and cellular components driving neural dysregulation in a model of ASD. This will provide a comprehensive understanding of the basic biology of ASD, generate a framework with which other types of cells involved in ASD may be compared and interpreted, and help to innovate novel targets and approaches to disrupting ASD. In the long term, these results may inform and guide the generation of wholly new therapeutic approaches for preventing ASD in humans.

Developing practical exercise guidelines to improve brain health and cognitive function in adolescents and emerging adults with congenital heart disease

Dr. T. Dylan Olver
University of Saskatchewan
Saskatoon, SK

Introduction: Congenital heart disease (CHD) is characterized by changes in heart structure and function that begin in the womb or during childhood. CHD is the world’s leading birth defect with as many as one in 45 youth currently living with CHD in Canada. A problem facing this population across the life span is brain dysfunction. On average, adolescents and young adults with CHD score lower on standardized cognitive tests. This translates to lower academic achievement, working in lower level jobs and less economic independence. A likely cause of brain dysfunction in CHD is decreased brain blood flow. A single bout of aerobic exercise can increase brain blood flow and improve cognition in the short term. It also causes the release of molecules, called myokines, which help the brain grow (increases blood vessels and neurons in the brain). However, because of the compromised function of their heart, patients with CHD tend to avoid aerobic exercise. Feedback from Lynne Telfer, knowledge user and parent of a young adult with CHD, tells us participation in aerobic exercise, like biking or jogging, is neither
practical nor tolerated well by patients with CHD.

New studies show that isometric handgrip exercise, which consists of repeatedly squeezing a handgrip device for a couple of minutes at a time (similar effort to holding a bag of groceries), may impart similar brain benefits as aerobic exercise. During this type of exercise, neurons in the brain become active and blood flow to the brain increases. Immediately after a single bout, cognitive function is also improved. Most of the studies use a relatively low squeezing effort, but newer studies and pilot work from our lab shows these beneficial effects on brain activation and blood flow might be enhanced with greater levels of squeezing effort. This raises the possibility increasing squeezing effort could also impart greater cognitive benefits. To date, no one has tested this prospect in healthy people or (even studied these phenomenon) in patients with CHD.
Objectives:
Aim1: To determine which isometric handgrip exercise protocol, low- vs. high-squeezing effort, will cause greater brain activation and greater increases in brain blood flow in adolescents and young adults with CHD.
Hypothesis 1: The high-squeezing effort protocol will cause greater brain activation and blood flow responses
than the low-squeezing effort protocol.
Aim 2: To evaluate a more real-world impact of this novel approach, we will determine which exercise protocol,
low- vs. high-squeezing effort, has a greater effect on cognitive function.
Hypothesis 2: Owing to a greater brain activation and blood flow response, the high-effort squeezing protocol
will confer greater cognitive benefits.
Outline of research: A total of 60 patients with uncomplicated CHD will be studied. We will examine the utility of this intervention in 30 adolescents (12-16 years old), divided equally between females and males as well as in 30 young adults (18-24 years old), divided equally between females and males. These cohorts were selected as they represents time periods when one naturally assumes more responsibility for their own health and their brains are highly responsive to exercise. To address our aims 1 and 2, we will conduct non-invasive, safe brain and vascular imaging experiments to examine how low-and high-effort exercise protocols influence brain activation and blood flow. We will also test how low-and high-effort exercise protocols influence cognitive performance, using a number of validated measures (reasoning skills, balance/coordination, reaction time, language skills), and see if the areas of the brain that are engaged during exercise are the same areas that correspond with improvements in cognitive function.

Projected benefits and applications of findings: The proposed work represents the critical first steps in tailoring exercise recommendations for brain health in this vulnerable and underserviced patient group. It holds extraordinary promise in the development of real-world
strategies to improve brain health in a growing number of Canadians living with CHD. IMMEDIATE IMPACT: Our ongoing engagement with parents with lived experience and knowledge users has driven our visible internet presence where our research findings and community-based programming opportunities serve as an invaluable resource for families and patients (mendinglittlehearts.ca).

Symptomatic and Neurotrophic Efficacy of α5-GABAA positive allosteric modulation

Dr. Thomas Prevot
CAMH
Toronto, ON

Introduction: Alzheimer’s disease (AD) is a complex disease with marked symptoms of mood, cognitive decline, increased β amyloid load, tauopathies and progressive loss of brain cell function. On a continuum from stress-related disorders, normal aging to AD, excitation/inhibition imbalance in the brain is reported across disorders, mostly due to decreased inhibition. GABA, the main inhibitory neurotransmitter of the brain is released by various subtypes of cells. We identified a pathway within the GABAergic system, particularly vulnerable and involved in the regulation of cognitive functions. This pathway relies on proper function of the GABAergic receptor containing the α5-subunit (or α5-GABAARs), which represent a target for therapeutic development, away from amyloid and tau pathologies.
The α5-GABAAR contributes to filtering information processing in the brain. In the context of AD, aging or chronic stress, this system is dysfunctional leading to cognitive deficits and neuronal atrophy, worsening the deficit. We developed novel molecules that selectively increase the α5-GABAARs activity and reverse cognitive deficits induced by chronic stress, aging or amyloid load. We recently showed that chronic treatment with one of this new molecule prevents and/or reverses neuronal shrinkage in brain regions involved in cognitive functions across mouse models. The efficacy of this molecule at reversing the cognitive deficits and the neuronal shrinkage provides a novel therapeutic approach to AD symptoms and pathologies.
However, the contribution of α5-GABAARs in the regulation of cognitive functions and their potential at reversing brain cells pathology remains to be clearly demonstrated by direct genetic approaches, addressing the question “Does this molecule truly mediate such efficacy via the potentiation of α5-GABAAR?”
Objectives: We propose to:
1. Characterize the contribution of α5-GABAARs in the regulation of cognitive deficits in an animal model of aging, genetically designed to be insensitive to α5-GABAAR potentiation.
2. Characterize the contribution of α5-GABAARs in the regulation of cognitive deficits in an animal model of amyloid load, genetically designed to be insensitive to α5-GABAAR potentiation.
3. Confirm the requirement for α5-GABAAR potentiation for the neurotrophic effect of the molecule
Outline of Research: In Aim 1, we will study mice bearing a point mutation in the allosteric site of the α5-GABAAR, where the novel molecule binds, but without altering the endogenous function of the receptor. These mice are called α5KI mice. Old α5KI and wildtype littermates will be treated chronically with a novel molecule acting on α5-GABAARs, or with water, for 3 weeks. We will test the mice in cognitive tasks assessing working memory, spatial learning, and cognitive flexibility – three cognitive domains altered in aging and AD. We predict that old mice will show cognitive impairment and old mice bearing the point mutation will not benefit from chronic treatment with this novel molecule.
In Aim 2, we will use a similar design to Aim 1, but using an animal model of amyloid load. We will generate double-transgenic mice by crossing α5KI mice with 5xFAD, known to develop progressive amyloid load. We predict that mice developing amyloid plaques will be cognitively impaired, but chronic treatment will provide beneficial effect on their cognitive functions, only if their α5-GABAARs are still sensitive to our molecule.
In Aim 3, brains of mice from Aim 1-2 will be harvested to investigate the impact of chronic treatment on brain cell morphology (dendritic length and spine density). Since some mice will have their α5-GABAARs insensitive to the novel molecule, we predict that chronic treatment will have no effect on neuronal morphology in these mice. We will also evaluate brain levels of markers altered in AD and linked to cognitive functions, and we will correlate such changes to performances in cognitive tasks, and morphology features to capture the full efficacy of this molecule.
Projected benefits and applications of findings: This project will demonstrate the requirement for α5-GABAAR-mediated potentiation in the regulation of cognitive functions and neuronal morphology. It will further demonstrate the therapeutic potential of using α5-targeting drugs for the treatment of cognitive deficits in neurodegenerative disorders, where the neurotrophic effect will play a critical role in slowing down progression towards disease states.

Contribution of the gut microbiome to symptoms spanning distinct neurodevelopmental disorders in children

Dr. Laura Sycuro
University of Calgary
Calgary, AB

Introduction: Childhood-onset neurodevelopmental disorders (NDDs) – including Autism Spectrum Disorder (ASD), Attention Deficit Hyperactivity Disorder (ADHD), Obsessive Compulsive Disorder (OCD) and Tourette Syndrome (TS) – often co-occur in the same child. Although each NDD is defined by a particular combination of symptoms and developmental deficiencies, many symptoms are common in children with NDDs, such that they cross diagnostic boundaries. Moreover, as neurodiverse children (a term we will use to inclusively refer to children with the one or more of the above-listed disorders) enter adolescence, their symptoms often evolve to include symptoms of anxiety and depression, placing them at greater risk of long-term struggles with these conditions than neurotypical children. These observations suggest there could be shared pathogenic factors, including developmental abnormalities and environmental influences that predispose children to NDDs. One such shared pathogenic factor that is a subject of active research the gut microbiome. Emerging evidence suggests biochemicals produced by gut microbes interact with brain alterations in neurodiverse children to influence symptom development and severity.
Objective: To date, understanding the contribution of the gut microbiome to NDDs has been limited by the extreme heterogeneity of symptoms and co-morbid conditions in neurodiverse children. To unravel the microbial profile of each disorder and assess the influence of dual-diagnoses, we will use a cross-diagnostic approach to examine how rare and co-morbid NDDs, as well as individual symptoms of NDDs, are linked to the gut microbiome. We hypothesize that children diagnosed with ASD, ADHD, OCD and/or TS experience neurological, psychological and gastrointestinal symptoms along a continuum of severity that is influenced by the gut microbiome.
Outline of Research: In the fall of 2020 we began Calgary-based cohort study of NDDs, which included matched co-habitating sibling controls to minimize genetic and environmental factors that are known to confound detection of microbial signatures. To date, we have recruited and collected fecal samples from 70 families (140 participants), including 25 with a diagnosis of ASD, 34 with a diagnosis of ADHD, 20 with a diagnosis of OCD and 38 with a diagnosis of TS. With limitations imposed by the pandemic coming to an end, we anticipate accelerating participant recruitment, allowing us to grow the size of the cohort to 160 families in total by year 2 of the study period. Using this cohort of 360 fecal samples, we will profile microbial signatures using sequence-based approaches (to assess gut microbiome community composition) and biochemical approaches (to detect neuroactive molecules that are produced or influenced by the microbiome). We will employ two powerful forces in Canada’s research arena – the International Microbiome Centre (IMC) at the University of Calgary and The Metabolomics Innovation Centre (TMIC) at the University of Alberta – to better resolve bacteria, including novel species, and more accurately quantify specific neuroactive metabolites than preceding studies. Integrative statistical analyses of bacteria and their biochemical products will provide a powerful framework for discovering novel intersections between the microbiome, neuroactive biochemicals, and clinical phenotypes.
Projected Benefits and Applications: By defining pathogenic mechanisms through which the microbiome contributes to symptoms, this work promises to illuminate new avenues of therapeutic development. We will also create a valuable biobank of cultivatable microbiota that will enable future mechanistic studies in mouse models. Critically, this seminal work could justify the inclusion of more broadly defined neurodiverse patient populations in clinical trials for precision microbiome therapies already on the horizon.