A Brief Description of Currently Funded Research Grants 2020 – 2021

Dr. Bénédicte Amilhon
CHU Sainte-Justine Research Center – Université de Montréal
Montreal, QC

INTRODUCTION: Perinatal asphyxia is the most common injury at birth, affecting 1-6/1000 babies, and is associated with increased risk for autism. Motor and cognitive consequences of severe asphyxia are well documented. On the other hand, children exposed to moderate perinatal asphyxia (MPA) have more variable outcomes that may be missed in early childhood. Long-term prognosis after MPA has generally been considered good. Yet, recent studies show that cognitive problems in patients that suffered from MPA do not become detectable before school age, and result in impaired memory, difficulties in social relationships and cognitive deficits that interfere with their daily life.

OBJECTIVES: We use a mouse model of MPA to investigate the vulnerability of selected brain circuits to perinatal asphyxia, and the consequences on cognitive functions. Our model recapitulates clinical features of MPA patients such as normal growth of MPA-exposed pups, absence of detectable brain lesions and absence of motor impairment. At adulthood, MPA exposed mice display cognitive problems such as memory alterations, deficits in social recognition and deficits in cognitive flexibility.
Cognitive flexibility is the ability to rapidly adapt to the ever-changing rules of our environment. This cognitive function is known to rely on the medial prefrontal cortex (mPFC), and to be strongly modulated by the neurotransmitter serotonin. Preliminary analysis of MPA-exposed mouse shows that serotonin is decreased in the mPFC and suggest that this deficit in serotonin might underlie deficits in cognitive flexibility. The objective of our study is to understand the link between alterations of the serotonergic system and mPFC-associated cognitive deficits after MPA.

OUTLINE OF RESEARCH: Our research has three main aims. In Aim 1, we will analyze how MPA affects serotonergic neurons projecting to the mPFC using anatomical studies and electrophysiological recordings in vitro. We will also study how MPA affects the response of mPFC neurons to serotonin. Cognitive flexibility requires plasticity in the mPFC network. In other words, the strength of the connections between neurons in the mPFC needs to dynamically change in response to changes in the environment, to enable learning of new rules. In Aim 2, we will study plasticity of the mPFC network in normal and MPA-exposed mice using in vitro electrophysiology. We will also investigate how changes in serotonin after MPA affects synaptic plasticity in the mPFC. Finally, in Aim 3, we will use optogenetics, a technique allowing to control neuronal activity using light, to i) determine the causal relationship between serotonin in the mPFC and cognitive flexibility and ii) rescue the deficits in MPA-exposed mice by increasing serotonin in the mPFC selectively.

PROJECT BENEFITS AND APPLICATION OF FINDINGS: MPA is one of the leading causes of perinatal injuries and represents major risk factor for autism, yet the precise neural circuits affected by mild asphyxia remain unclear. Treating the deficits induced by MPA and providing rapid interventions at the time of injury requires a better understanding of which neuronal circuits are affected by asphyxia. The results generated by our research will provide novel information on the role of serotonin in brain plasticity in the mPFC and cognitive flexibility deficits after MPA. This research will provide several major advances in our understanding of brain deficits following MPA and paves the way to develop novel pharmacological approaches for rescuing circuit-specific dysfunction following birth asphyxia.

Dr. Wei-Hsiang
Research Institute of McGill University Health Centre
Montreal, QC

INTRODUCTION: Imbalanced excitation (E) and inhibition (I) that favor cortical hyperexcitability is a common feature of autism spectrum disorders (ASDs), which affect 1 in 66 children in Canada. E/I imbalance in ASDs underlies debilitating symptoms including seizures and impaired cognitive functions that negatively affect quality of life for patients and their families. Despite playing a central role in ASD pathogenesis, the synaptic and circuit mechanisms underlying E/I imbalance in ASD remain poorly understood. As a result, we lack disease-modifying treatments.

We will study a monogenic form of ASD associated with epilepsy, Smith-Magenis Syndrome (SMS), to understand the mechanism and develop treatment for brain hyperexcitability in ASDs. SMS is caused by mutations in a chromatin protein, Retinoic Acid Induced 1 (Rai1). My published work showed that in the mouse brain, Rai1 preferentially regulates expression of genes involved in neuronal signaling and excitability, including brain wiring molecules, ion channels, and neurotransmitter receptors. Neural loss of Rai1 in mice results in SMS-like features including motor dysfunction, learning deficits, and hypo-sociability. My work also found that transcriptional deficits and social deficits in SMS mice can be rescued by genetic reactivation of Rai1. These findings suggest that SMS is potentially treatable, and that Rai1 downstream neuronal signaling pathways are promising therapeutic candidates to reverse neuronal hyperexcitability and ASD-like behavioral features.

OBJECTIVES: We will confront two main questions in this grant:
(1) How does Rai1 deletion induce neural hyperexcitability on network and cellular levels?
(2) What are the Rai1-dependent molecular mechanisms responsible neuronal hyperexcitability?

OUTLINE OF RESEARCH: Our preliminary experiments using novel whole brain imaging show that Rai1 loss induces a dramatic increase in neuronal excitability in many brain regions implicated in epilepsy and cognitive functions. Our preliminary study also found that Rai1 mutant mice show misregulation of a signaling pathway strongly associated with epilepsy and autism. In Aim1, we will use calcium imaging (Aim 1.1, year 1) and electrophysiology recordings (Aim 1.2, year 2) to study how Rai1 loss affects E/I imbalance. We expect these experiments to uncover the circuit and synaptic level functions of Rai1. In Aim 2 (year 3), we will use a cutting-edge proteomic approach to identify Rai1 downstream druggable targets to treat ASD-like deficits including social deficits, motor dysfunction, and repetitive motor behaviors induced by Rai1 loss. We will pharmacologically target E/I imbalance to reverse these ASD-like deficits in a mouse model of SMS that we previously generated and characterized.

PROJECTED BENEFITS AND APPLICATION OF FINDINGS: Rai1 is an important gene that regulates cognitive function and E/I balance in the brain. Loss of Rai1 causes SMS, a syndromic ASD associated with learning deficits, epilepsy, social dysfunction, stereotypical motor behaviors, and impaired motor function. These experiments will advance our understanding of the circuit and synaptic mechanisms that link ASD-causing genes to brain E/I imbalance at neuronal and circuit levels. Importantly, we will directly test if rescuing a signaling pathway responsible for E/I imbalance can rescue ASD-like phenotypes associated with Rai1 loss. We expect this project to provide novel insights into how a transcription factor, Rai1, maintains E/I balance in the brain, and explore potential treatments to mitigate cognitive dysfunction in ASDs.

Dr. Jasmine Lalonde
Dr. Jennifer Geddes-McAlister
University of Guelph
Guelph, ON
INTRODUCTION: Schizophrenia (SZ) is a severe brain disorder that affects about 1% of the population and requires treatment throughout the patient’s lifetime. Even though different therapies are available to treat symptoms associated with this mental illness, most options present poor performance against cognitive deficits which have a big impact on the patient’s quality of life. Given the lack of progress in the development of novel drugs to treat cognitive problems in SZ, our research team is therefore determined to identify new candidate targets for therapeutic intervention.

In recent years, genome sequencing of patients led to the discovery of DNA variations that appear to create risk of suffering from SZ. Interestingly, a common finding between these different studies has been that genes coding for synaptic proteins are often affected; an observation that supports the idea that this disorder is consequently connected to problems in the development and/or function of synapses. Despite these findings, however, what is the exact problem with synaptic structures in SZ patients, and how these may create cognitive symptoms, remains a mystery.

OBJECTIVES: To start answering this question, our plan is to: 1) search for changes in the molecular composition of synapses that could be associated with SZ by applying proteomic methods to patient-derived stem cells that are grown into ‘mini-brains’, 2) select the synaptic proteins that we found to be most affected and understand their exact role in the growth of synapses, and 3) use this information to develop a platform that will help us find new targets to change defective signaling pathways and/or protein interactions in ways that would ameliorate synaptic defects associated with the disease.

Until recently, the lack of models for SZ that can take into account human genetic backgrounds has slowed discovery of the affected cellular processes responsible for apparition of the disease. However, the recent ability to reprogram adult cells into stem cells that can then be transformed into ‘mini-brain’ models called cerebral organoids helped overcome this barrier. Most importantly, because cerebral organoids imitate the course of neocortex development, this advance in technology finally allows comparing side-by-side samples derived from SZ patients and healthy control individuals.

Aim 1: Find changes associated with SZ in synaptic protein levels using ‘mini-brain’ models and mass spectrometry (MS). Defects in the molecular organization of synapses responsible for SZ most certainly exist at an early stage of brain development. To test this scenario, we will use stem cells prepared from SZ patients and control individuals to make a collection of ‘mini-brains’. Once these models have reached maturity with many active neurons, we will then extract synapses and measure differences in protein levels with quantitative MS.
Aim 2: Determine the role of affected proteins in SZ to synapse development. As we uncover proteins with abnormal (more or less) abundance in the SZ synapse in comparison to controls, it will be important to validate these findings. Furthermore, in the case of factors for which their role is less well known, it will be necessary to gain insights about their contribution to synapse biology. To achieve these two tasks, we will use single cell and microscopy methods to track the shape, numbers, and activity of synapses when the expression of the protein in question is perturbed.
Aim 3: Identify targets to reverse aberrant synaptic protein in SZ. Here, we will focus on the design of an assay that can be used for discovery of compounds that can change the level of proteins found to be affected in SZ synapses. To test of this assay, we will complete a primary screen with a diverse library of 1280 biologically active compounds. We anticipate that reversing the abundance of proteins we found to be affected in SZ models would improve their synaptic defects.

There exists a critical need to understand the underlying cellular problems causing SZ in order to advance the development of novel treatments, in particular to reduce cognitive dysfunction. This proposal will use unique models in combination with a powerful proteomics method to complete a detailed analysis of SZ synapses. These efforts will finally help create a screening platform to help find new pharmacological options capable of ameliorating synaptic defects associated with this devastating mental illness.

Dr. Melissa Perreault
University of Guelph
Guelph, ON

Autism spectrum disorder (ASD) has a prevalence rate approximately four times higher in males than in females. Sexual dimorphisms also exist in ASD that include differences in memory, cognitive flexibility, verbal fluency, and social communication. A key principle emerging from basic neuroscience research is that brain waves have patterns that are integral to brain communication and are tightly coupled to behavioral states. Our previous work has shown that glycogen synthase kinase-3 (GSK-3), a protein kinase shown to have reduced activity in ASD, can directly regulate these rhythms. As GSK-3 has been shown to be suppressed in both children with ASD and in animal models, this suggests that this protein may play a causal role in the sex-dependent behavioural and cognitive symptoms inherent in the disorder.

OBJECTIVES: Sex differences in GSK-3 activity in ASD, the influence of these differences on brain wave patterns and behaviour, and the potential impact of these processes on ASD susceptibility are unknown. Using the valproic acid (VPA) idiopathic, and the fmr1 KO genetic model systems of ASD, the objectives of the present proposal are to 1) Evaluate sex differences in neurophysiological systems patterns, GSK-3 activity, and behaviour, 2) Identify a role for reduced GSK-3 activity in mediating the disruption of brain wave patterns, and the manifestation of symptoms, and symptom severity, 3) Assess the therapeutic effects of cannflavins A and B in normalizing aberrant GSK-3 activity and neural systems function in vitro.

Aim 1. This Aim will provide novel insights into how males and females differ in brain communication patterns in ASD and the relationship to behavioural output. In this study, we will evaluate sex differences in network activity in male and female adolescent VPA and fmr1 KO rats, and appropriate controls, and we will follow these patterns into early adulthood. Local field potential recordings will be taken from eight brain regions implicated in ASD and correlated to behaviours such as social interactions, repetitive behaviours and cognition. Multitaper spectral power, correlation analysis, and coherence will be analyzed (Matlab). Brains will be extracted and will undergo processing for Western Blot analysis to evaluate sex differences in the expression and activity of GSK-3 in each region (anti-GSK-3, anti-p-GSK-3 Ser9/21). BDNF signaling, enhanced in ASD and also a negative regulator of GSK-3, will also be assessed.
Aim 2. This aim will demonstrate a causal role for GSK-3 in ASD by normalizing oscillatory and behavioural deficits through pharmacogenetic GSK-3 activation. GSK-3 activity will be chronically upregulated in select brain regions using viral-mediated gene transfer with a persistently active form of GSK-3, AAV-hSYN1-GSK-3β(S9A)-HA, as we have done previously, to determine whether the disturbances in brain wave patterns and behaviours can be normalized.
Aim 3. This aim will identify a novel potential therapeutic approach for the treatment of autism via treatment with cannflavins A and B. Increased BDNF signalling and decreased GSK-3 activity have been implicated in ASD. Cannabis administration in ASD appears to be an effective option to relieve symptoms, but the mechanisms remain unknown. We have shown the that the cannabis flavonoids cannflavins A and B are potent inhibitors of the BDNF receptor TrkB. Thus the effects of the cannflavins in normalizing GSK-3 signalling and neural systems function (Axion MEA reader) of primary neuronal cortical, hippocampal, and striatal neurons derived from male and female VPA, fmr1 KO, and control rats, will be compared.

There is an urgent need for basic, preclinical and translational research that combines the understanding of the basic pathophysiology of ASD in males and females with novel strategies that will advance the development of prevention strategies and individualized therapies. This research proposal will provide important insights into how male and female brain communication patterns are distinct in ASD, demonstrate an underlying involvement for GSK-3 in their regulation, and highlight how these sex differences impact behavioural output. Positive findings will not only provide critical information on sex differences in ASD susceptibility but identify potential novel avenues for therapeutic intervention via suppression of GSK-3.