The overall goal of this study is to use linked population health databases to characterize the epidemiology of mitochondrial disease and the co-occurrence with mental health conditions in Ontario. Our specific objectives are to examine: (1) the health care burden and costs associated with mitochondrial disease in a population- based cohort in Ontario: (2) the association between mood disorders and other mental health conditions in patients with mitochondrial disease: and(3) the joint impact of mental health conditions on the health care use. costs and mortality.

This study represents the first to establish a methodological approach to measure mitochondrial disease from population data across Canada. For the first time in Canada. we will contribute key epidemiological evidence to inform health and health care for those living with mitochondrial conditions. and further support hypotheses and research on the relationship between mitochondrial disease and mental health This project will build the fundamental methodology that can be replicated in other Canadian provinces, and enable future international research. including comparisons of mitochondrial disease populations and further study of health care utilization and costs

Nominated PI: Laura Rosella. Associate Professor. Dalla Lana School of Public Health. University of Toronto

Co-PtTrevor Young. Professor. Department of Psychiatry and Pharmacology. University of Toronto

“Mitochondrial Metabolism Vulnerabilities in Breast Cancer”

$80,000 (over 2 years)

Breast cancer (BC) is the number one cancer killer of women Improving survival and quality of life for patients with BC is limited by our ability to effectively personalize systemic therapies. reducing the risk of treatment resistance and disease recurrence. while minimizing the risks of toxicity associated with ineffective chemotherapeutic agents. Selection of an appropriate therapy for patients with locally advanced breast cancer (LABC). which typically involves treatment with chemotherapy prior to surgery. involves significant trial-and- 5 ; error. Most patients receive a standard cocktail of drugs. but only about one-third have a positive and complete response. We need to gather more information about how each patient's tumour cells can be killed: in effect. Pa personalizing treatment

Here we propose to identify genes in mitochondria of cancer cells that may contribute to their growth. survival and ability to develop resistance to chemotherapy. We now know that cancer cells can use nutrients differently from normal cells, which raises the possibility of new strategies to kill them We call these 4 ‘mitochondrial metabolism vulnerabilities’ Our novel approach will be to silence mitochondrial genes in LABC cells taken directly from patients using state-of-the-art genetic and imaging tools to identify tumour-specific vulnerabilities. These vulnerabilities can then be targeted alone or in conjunction with lower doses of established therapies to kill tumours more effectively reducing side effects. An exciting outcome of this work is the development of patient-specific therapies not based on trial-and-error. but a personalized approach based on the patient's own tumour's demonstrated drug sensitivities.

Perrault syndrome is an autosomal recessive rare mitochondrial disorder characterized by hearing loss in both males and females with females also experiencing ovarian failure. However. the disease is clinically heterogenous and affected patients exhibit additional neurological symptoms including spasticity. ataxia. and neuropathy. Mutations in six different genes. five of which encode mitochondrially-targeted proteins. have so far been linked to Perrault syndrome. One of these genes is CLPP. which encodes the mitochondrial ClpP protease. Human ClpP is a nuclear-encoded serine protease that is translocated to the mitochondria viaan N- terminal targeting sequence. In the mitochondria. ClpP assembles into a tetradecameric cylinder hat associates with an ATPase cap The resulting ATPase-protease complex is involved in the degradation of several critical mitochondrial matrix and inner membrane proteins.

Currently. it is not known how CLPP mutations result in Perrault syndrome and there are no disease-modifying curative therapies Current management is symptomatic only. egcochlear implantation and ovarian hormone replacement. Effective therapies are urgently needed. especially to address the neurodegenerative aspects of this disease. In this work. we propose to investigate and characterize model and patient-derived cell lines carrying CLPP mutations to determine the molecular pathogenic basis of this mitochondrial disease.Furthermore. the effect of compounds targeting ClpP on cell biology and physiology will also be studied The project is a close collaboration between two groups with excellent expertise in ClpP biochemistry. biology. and treating patients with Perrault syndrome. The project has clear translational potential and is expected to advance the field of mitochondrial medicine.

Involvement of the brain in mitochondrial disease is common and as a consequence. neurological manifestations. including also neuropsychiatric symptoms such as mood alterations. are very frequent. There 4 has been no systematic study of these clinical aspects which are often missed and greatly disabling. therefore the neuropsychiatric aspects of mitochondrial disease remains very poorly defined. Information on how to do accurate and precise assessment of neuropsychiatric symptoms in mitochondrial disease is largely missing. which limits the ability of developing treatment trials targeting these symptoms. Furthermore. there is a critical need of defining biomarkers for brain involvement in mitochondrial disease, to learn how to best measure the effects of new therapeutics, in order to promote discovery of novel effective medications.

Our study will integrate precise neuropsychiatric assessment. based on use of validated diagnostic instruments, cognitive testing. and tracking of mood fluctuations, with state-of-the-art neuroimaging techniques to assess brain metabolism and micro-structure, in adult patients with mitochondrial disease. The findings from this research will improve the evaluation of therapeutic interventions and signposting to appropriate services. and will enable precise assessment in future trials of novel therapeutics improving mitochondrial function in the brain. Our project will therefore directly benefit patients with mitochondrial disease by contributing to better management of these disabling symptoms. and to the discovery of new effective treatments. The inter-disciplinary nature of the proposed research, integrating aspects of physical. brain, and mental illness. will contribute to tackle the stigma of mental health that patients with pathologies of the brain still regularly experience.

Nominated PI: Alessandro Colasanti. Senior Clinical Lecturer in Psychiatry: Brighton and Sussex Medical School

Co-PtIris Asllani, MR Physicist and Research Associate Professor. Brighton and Sussex Medical School

Collaborators:Robert Pitceathly. MRC Clinical Scientist. UCL Institute of Neurology and National Hospital or Neurology and Neurosurgery

There are millions of Canadians that suffer from diseases stemming from Mitochondrial dysfunction. including Alzheimer's disease. The underlying molecular mechanism and its role in Alzheimer's disease pathogenesis remains poorly understood Alzheimer's disease patients and mouse models exhibit an imbalance within mitochondrial fission and fusion and this change within mitochondrial dynamics has significant consequences on synaptic and neuronal function.

Re-establishing an equilibrium within mitochondrial dynamics and morphology may serve as a potential therapeutic target for recovering mitochondrial function and neuronal homeostasis. Sigma-1-receptors. situated at the mitochondrion-associated ER membrane, are reduced in early Alzheimer's disease patients and their agonist. Pentazocine. was previously shown to elicit potent neuroprotective effects. For these reasons. we are investigating the pharmacological significance of Sigma-1-receptors and their respective role on mitochondrial function. Our future research endeavours seek to identify whether the FDA approved drug Pentazocine can be repurposed as a therapeutic strategy for the treatment of Alzheimer's disease and various other mitochondrial specific diseases.

Awardee Royea, Jessika

PIKhacho. Mireille. Biochemistry. Microbiology. and Immunology. University of Ottawa

Co-Pi(s)Bergeron, Richard. Cellular and Molecular Medicine, Ottawa Hospital Research Institute

“Regulation of PINK1-Parkin Mitophagy by Molecular Chaperones”

$30,000 (1 year)

Parkinson's disease (PD) is a common disabling neurodegenerative disorder. affecting an increasing number of Canadians. for which we have no cure This deficiency is due to the lack of treatments that target the key molecular pathways involved in the underlying pathogenesis of PD. We have recently identified a chaperone protein that modulates protein quality control and cell death by disrupting the removal of damaged mitochondria. These organelles are known as the powerhouses of the cell since they supply energy and regulate pathways important for cell survival known to be affected in PD. In this project we will use an approach that combines gene therapy and specialized targeting sequences that can engage this chaperone protein to test if this improves outcomes in preclinical models of PD. This work will provide the foundation to develop therapeutics that regulate chaperone proteins in the brain as a novel approach to treat PD.

Awardee: Kapadia, Minesh

PI: Kalia. Suneil. UofT -Dept of Surgery. UofT

Division of Neurosurgery Dept of Genetics and Development. UHN

The ability to preserve lungs prior to the time of transplantation has made lung transplantation a clinical reality for those with end-stage lung disease. Currently. lung preservation is performed by flushing the organ with an organ-specific solution. and subsequently storing the organ on ice where it rests at approximately 4'C. Using this approach. preservation times are limited to clinical times of approximately 6-8 hours. Longer preservation times will allow for the overcoming of geographical hurdles faced in organ donation. allow for more optimized donor and recipient matching. and progress lung transplantation towards a semi-elective procedure. Previous reports have shown increased mitochondrial degeneration during prolonged cold storage periods. with an association of worsening graft function. In this project. we aim to explore the specific role of organ temperature during lung preservation on mitochondrial health during the preservation period. with hopes of optimizing the current temperature being used to store lungs. With these findings. we further hope to explore new therapeutic approaches of mitochondrial protection during the cold preservation period such as mitochondrial transplantation and anti-oxidative metabolite preservation solution supplementation

Awardee: Ali. Aadil

Pi:Cypel. Marcelo. Institute of Medical Sciences UofT. Thoracic Surgery. UHN

Currently. treatments for mitochondrial dysfunction/disease are limited to antioxidants and compounds targeting specific mitochondrial proteins. however. an emerging approach is the transplant of freshly isolated mitochondria to injury sites. The use of mitochondrial transplants presents several challenges. including the need to identify autologous mitochondria with low levels of mitochondrial DNA (mtDNA) mutations. Therefore,the overall objective of this study is to identify and optimize novel protocols for autologous mitochondrial transplantation in patient-derived induced- pluripotent stem cells (iPSCs). Specifically. we will: (2) Define the best method to reduce or eliminate mutant mtDNA in patient-derived iPSCs: and (2) Establish an efficient and stable mitochondrial delivery method. To date. we have finalized the mitochondrial isolation protocol and are in the process of optimizing the reintroduction of isolated mitochondria into po cells lacking mtDNA.

Awardee: Bodenstein, David

Pi: Andreazza. Ana. Department of Pharmacology & Toxicology. UofT

Co-PI: Hurd Thomas, Department of Molecular Genetics. UofT

Mitochondrial DNA (mtDNA) is indispensable as it encodes components essential for ATP production. Despite its importance. limited recombination and repair mechanisms result in high rates of mutations in mtDNA which contributes to disease. Unfortunately, most tissues are incapable of selectively recognizing and degrading mutant mtDNA. The female germline. the tissue which gives rise to eggs. is unique. in that it is capable of selectively eliminating mutant mtDNA We aim to uncover the mechanism through which the germline does this in order to manipulate these processes to eliminate mutant mtDNA from diseased tissues. We have identified a number of proteins that play a critical role in eliminating mutant mtDNA from the germline and are currently determining mechanistically how they work together to selectively eliminate mutant mtDNA.

Awardee:Jeedigunta. Swathi

Pi:Hurd Thomas, Department of Molecular Genetics. UofT

Co-PI:Andreazza. Ana. Department of Pharmacology & Toxicology

Genetic interactions (GI) occur when mutations in multiple genes combine to generate an unexpected phenotype. and aid in our understanding of genotype-to-phenotype relationships in both health and disease. By investigating Gl's, we can map functional relationships between genes that can be used for identifying genetic elements involved in biological processes and disease. elucidate gene function. and discover therapeutic targets. The disease that has become a driver of my interest in how GI's involving two or more genes can contribute to an individual's disease phenotype is Barth Syndrome. rooted in mutations in the gene TAZ There is extreme variability observed in Barth Syndrome patients even though one gene is responsible for this disease. suggesting that there are modifier genes or additional variants of TAZ that could be involved. My project aims to generate the first genetic interaction profile of the TAZ gene using a highly sensitive genome- wide CRISPR/Casg screening platform. In parallel this project aims to generate the first comprehensive reference genetic interaction map of the mitochondria in human cells. exploring relevant disease alleles and GI's involved in both physiological and pathological conditions.

Awardee:Masud. Sanna

Pi:Moffat. Jason. Department. Molecular Genetics. UofT

Co-PI:Boone. Charlie, Department Molecular Genetics. UofT

The project is designed to establish a link between psychiatric illnesses risk gene FXR1 and mitochondrial function that could explain mitochondrial malfunction in the patients with disorders such as schizophrenia and bipolar disorder. Firstly. we are planning to establish a molecular phenotype of mitochondria using cell culture models and CRISPR-Casg approach to target the gene of interest. Secondly. we will pursue a similar approach to knockout the gene of interest in cortical neurons of adult mice using viral delivery to check for mitochondrial phenotype. And at last. we plan to test if the protein of interest exhibits protective function against oxidative stress in mitochondria.

Student: Marakhovskaia, Aleksandra

PI:Beaulieu. Martin. Department of Pharmacology & Toxicology. UofT

Co-PI:McPherson. Peter. Department of Pharmacology & Toxicology. UofT

Strong evidence suggests a key role for adult neurogenesis in tissue regeneration and cognitive function.Understanding the regulation and persistence of neural stem cells (NSC) in the adult brain is critical for real-life application in neurodegenerative diseases. Our group has shown that mitochondrial function plays a pivotal role in regulating NSC and cognitive function. Our goal is to identify how mitochondrial dynamics and metabolism regulate neurogenesis and learning and memory. Unbalanced mitochondrial dynamics results in mitochondrial fragmentation. which is seen during aging and at an accelerated rate in neurodegenerative diseases Optic atrophy 1 (OPA1. a gene implicated in Parkinson's Disease and Dominant Optic Atrophy) facilitates mitochondrial fusion and regulates energetics We use Opat inducible knockout in mouse NSCs to model accelerated aging and neurodegeneration. We show that Opai loss leads to mitochondrial dysfunction which induces a cascade of cellular stress response that regulates cell survival at the expense of NSC proliferation.

This study reveals how mitochondrial dysfunction. typical of neurodegenerative diseases alters NSC fate decisions and identifies new regulatory targets by which to enhance mitochondrial function in the context of neurodegeneration. Modulating such stress conditions posed here opens new avenues in neurodegenerative disease therapeutic strategies

Awardee: Iqbal. Mohamed Ariff

Pr:Slack, Ruth, Cellular and Molecular Medicine, University of Ottawa