The genotype-to-phenotype relationship in health and disease is complex, influenced by environmental and genetic factors. Unmasking the non-trivial pairwise genetic interactions on a genome-wide scale remains an active area of investigation in many prominent disease models. The diverse spectrum of phenotypes observed in Barth Syndrome (BTHS), caused by mutations in the gene TAZ is consistent with such complex genotype-to-phenotype interplay, however, systematic efforts to understand these interactions remain lacking. Furthermore, numerous mutations in the TAZ gene have been documented in BTHS patients, including frameshift and point mutations, as well as mutations that disrupt alternative splicing of TAZ.
This diverse pattern of TAZ mutations suggests that several independent mechanisms contribute to a loss or altered function of Tafazzin, the protein product of TAZ, which may account for the broad phenotypic variance observed in patients. Despite the documented mutations, little is known about the genetic and protein interactions of TAZ/Tafazzin, or the genetic architecture of TAZ and which mutations are functionally consequential. Moreover, it remains unclear whether Tafazzin moonlights in alternative processes unrelated to its highly conserved acyltransferase that may contribute to the pathology of BTHS. Using deep mutational scanning approaches, we propose to further characterize the TAZ gene to understand other functional roles of Tafazzin in yeast and human cells.

Supervisor : Jason Moffat, Department of Molecular Genetics University of Toronto

Co-Supervisor : Charlie Boone, Department of Molecular Genetics, University of Toronto

Some of the challenges faced by the field of lung transplantation with the highest potential impact on outcomes are improving organ preservation in a way that allows viability to be protracted, providing time for additional innovative therapies to be applied, and recovering injured lungs that would otherwise not be acceptable for transplantation, which is both a consequence of improved preservation and an additional hurdle in itself. Current standard practice of cold static preservation (CSP) of donor lungs with ice cooling at approximately 4ºC limits storage of the organs for approximately 6-8 hours. The Toronto’s Ex-Vivo Lung Perfusion (EVLP) technique, developed in 2008 by our lab, is an innovative platform that simulates an in vivo environment prior to transplantation. In our most recent publication, we were able to demonstrate for the first time the feasibility of 3-day lung preservation by adding two four-hour cycles of EVLP to a 10ºC CSP, achieving exceptional functional results. In common to our previous findings in which extended CSP at 10 degrees showed benefits over the current standard of 4ºC, this successful approach seems to be correlated with improved mitochondrial health.
Albeit remarkable, these encouraging results observed in healthy lungs preserved for extended periods may not always reflect the reality of many of the organs clinically available for transplantation, which frequently present some degree of injury and inflammation. In a paper from our lab recently submitted to the Journal of Heart and Lung Transplantation, we show for the first time that 10°C can also be a superior storage temperature in the context of aspiration-induced lung injury. Taken together, the data notably suggested that protecting the mitochondria could be an effective therapeutic target for future investigations. Since 2009, the concept of mitochondrial transplantation (MT) emerged as a potentially effective means for reducing ischemia-reperfusion injury (IRI) and extending organ preservation in heart, brain, kidney, and lung. Furthermore, these findings have been already translated to a clinical trial

Supervisor : Marcelo Cypel, Department of Surgery, Toronto General Hospital

Co-Supervisor : Ana Andreazza, Department of Pharmacology & Toxicology, U of T

Exportin 1 (XPO1), a nuclear export protein, is overexpressed in almost all patients with solid and hematological cancers . In normal cells, XPO1 mediates the export of RNA, ribosomal subunits, and various proteins from the nucleus to the cytoplasm. However, cancer cells hijack the activity of XPO1 to mislocalize proteins for their benefit. For example, XPO1 can mediate the cytoplasmic mislocalization of DNA damage response proteins such as p53, resulting in cancer cell growth and chemotherapy resistance. Selinexor is a novel anti-cancer agent that inhibits the nuclear export of these proteins via the obstruction of XPO1. Although selinexor has been approved for use in relapsed/refractory diffuse large B cell lymphoma (DLBCL) [3], knowledge gaps in understanding precisely what XPO1 exports in DLBCL and how selinexor impacts this prevents the drug from being used optimally in the clinic. Of increasing importance is the role of mitochondrial metabolism, as evidence in recent years has indicated that cancer cells commandeer oxidative phosphorylation to increase the availability of substrates for growth and invasion.
However, the mechanisms by which nuclear export influences mitochondrial function are lacking. Our preliminary data indicates that selinexor is able to decrease oxidative metabolism and ATP production in DLBCL while leaving glycolysis unaffected. This indicates that XPO1 mediated nuclear export plays a role in mitochondrial function. By identifying what proteins are exported by XPO1 in DLBCL and how they influence the mitochondria, a more in-depth understanding of the XPO1-mitochondrial axis will be delineated. This will allow us to better understand the role of nuclear export in regulating mitochondrial function, which may be pertinent not only to cancer, but other human diseases as well. In addition, this will enable us to further understand selinexor’s mecha.

Supervisors :

John Kuruvilla: Department of Medical Oncology and Hematology, Princess Margaret Cancer Centre

Armand Keating, IBBME, University of Toronto

Severe undernutrition, referred to as malnutrition in this proposal, is associated with a high risk of morbidity and mortality in children under five years of age, markedly in resource-poor countries. Poor host responses and the occurrence of repeated bacterial infections, allows these infections to progress more easily into severe clinical septic phenotypes that underlie these adverse outcomes. The underlying immune and metabolic dysregulations are not corrected by the standardized WHO guidelines1 . Therefore, relapse is common, and hospital fatality rate remains high1 . While malnutrition is known to influence innate immunity, the first line of defense that initially responds to pathogenic microbes, a greater mechanistic understanding is required of these processes to address this global issue. Neutrophils, found to be increased in the circulation of children with malnutrition, are first responder cells of the innate immune system that influence and regulate the inflammatory response for pathogen clearance through their large repertoire of effector functions (chemotaxis, phagocytosis, ROS formation, degranulation, neutrophil extracellular traps (NETs) formation, and production of cytokines and other inflammatory mediators)
As result, I aim to (1) Define, in mice, effects of severe malnutrition on differentiation and immune functions of neutrophils actively responding to infection in the bone marrow and tissues, respectively. (2) Delineate, in mice, the malnutrition-induced metabolic disturbances that impair neutrophil development and functions.

Supervisor : Robert Bandsma, Department of Pediatrics, Hospital for Sick Children

Co-Supervisor : Michael Glogauer, Professor, Faculty of Dentistry, University of Toronto

Parkinson’s disease (PD) is a neurodegenerative disease affecting approximately 3% of the population over the age of sixty-five and is characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pas compacta (SNpc). There is no cure for PD and the best treatment options for patients only provide symptomatic relief. Thus, understanding the underlying pathways of PD is crucial for determining the etiology and generating a cure for this disease. While PD is largely a sporadic disorder, several genes have been linked to heritable forms, revealing underlying molecular pathways that may be implicated in this disease. For example, mutations in the alpha synuclein (αsyn) encoding SNCA gene, such as A53T, A30P, or E46K missense mutations are linked to autosomal dominant forms of familial PD3. Interestingly, mitochondria dysfunction has been highly implicated in the pathogenesis of PD5 , and αsyn has been demonstrated to have detrimental effects on mitochondria function. Using the dual fluorophore reporter FIS1-mcherry-EGP (‘mito-QC’)9 I have demonstrated increased mitochondria autophagy (mitophagy) in primary cortical neurons overexpressing A53T, human induced pluripotent stem cell-derived dopaminergic neurons (hiPSC-DA) harboring the A53T mutation, and in an in vivo rat model of A53T. Evidently, mutant αsyn has downstream consequences on mitochondria health and thus determining its upstream regulator could prove vital in decreasing a syn damage to mitochondria. The co-chaperone carboxyl terminus of heat shock protein 70- interacting protein (CHIP) is a promising candidate for regulating levels of αsyn in the cell. CHIP is an E3 ubiquitin ligase and molecular cochaperone and previous work by our lab has shown that CHIP ubiquitinates toxic αsyn oligomers, targeting them for degradation.
This suggests that CHIP plays an important role in regulating αsyn. However, despite the importance of CHIP in the pathogenesis of PD, its regulation remains poorly understood. Using mass spectrometry, our lab, in collaboration with the Schmitt-Ulms lab, has demonstrated increase in serine 19-phosphorylated CHIP (pCHIP-S19) upon treatment of the mitochondria toxin carbonyl cyanide m-chlorophenyl hydrazine. I have identified a kinase that phosphorylates CHIP at S19, and my preliminary results suggest that pCHIP-S19 may enhance the ability of CHIP to degrade αsyn (Data not shown). Thus, I am interested in exploring the potential functional implications of pCHIP-S19 on αsyn regulation and associated mitochondria dysfunctions.

Supervisor : Suniel Kalia, Department of Laboratory Medicine and Pathobiology, UHN

Co-Supervisor : Joel Watts, Department o Biochemistry, University of Toronto

Approximately 1% of Canadians develop epilepsy in their lifetime with ~1/3rd being treatment resistant. This leads to chronic morbidity, greatly burdening the health care system, particularly in pediatric patients suffering from a severe epileptic encephalopathy. Epilepsy Canada reports that each year in Canada, an average of 15,500 people learn they have epilepsy; 75-85% before age 18. About one-quarter of patients with mitochondrial disease have epilepsy, most commonly present in pediatric patients with classical mitochondrial disease syndromes such as MELAS, MERFF, Leigh disease or POLG-related disorders. Overall, studies have shown that 40-60% of patients with mitochondrial disease develop seizures. Most cases of epilepsy with mitochondrial disorders are drug-resistant, associated with a very poor prognosis, often with a fatal outcome. Seizures, irrespective of their origin, represent an excessive acute energy demand in the brain. Accordingly, secondary mitochondrial dysfunction has been described in various epileptic disorders, including disorders that are mainly of non-mitochondrial origin making the role of mitochondrial dysfunction in epilepsy much more widespread than just originating from primary mitochondrial disorders.
The underlying biological hypotheses are: 1. Mitochondrial dysfunction is a frequent underlying cause of epilepsy, particularly in pediatric epileptic encephalopathies, and conversely, that recurrent seizures can cause mitochondrial dysfunction. 2. hCOs provide a model of severe epileptic encephalopathies useful for diagnosis, uncovering pathophysiology, and assessing various pharmacological and other treatment options. In the proposed study, as an engineer, I will focus on developing a robust and pathophysiological discovery platform using organoids to understand the impact of mitochondrial dysfunction on seizures and epilepsy.

Supervisors :

Peter Carlan, Department of Neurology, Krembil Research Institute

Taufik Valiante, Department of Neurology, Krembil Research Institute

Heart disease is the leading cause of morbidity and mortality worldwide, imparting significant medical and socioeconomic costs. Owing to its continuous mechanical work, the heart requires large amounts of ATP to function optimally (between 20-70 times its own weight daily)1,2. Healthy and efficient mitochondria are therefore required for maintaining homeostatic energy demands. Adverse changes in mitochondrial structure and function are prominent features of cardiomyocyte dysfunction in the failing heart3 . Preventing or mitigating these adverse changes has the potential to improve the efficiency of cardiomyocyte metabolism and consequently improve organ function, representing a novel therapy for heart failure. The pediatric human heart has a higher inherent capacity for self-repair compared to the adult heart. Consistent with this observation, the secreted (paracrine) biomolecules produced by cardiac progenitor cells procured from pediatric hearts can more potently induce repair to damaged tissue4 . Collectively known as the cell’s secretome, these biomolecules can include diverse constituents including exosomes, proteins, RNA, and metabolites, reflecting their cell of origin and playing an important role in inter-cellular signalling and crosstalk5 . The effect of these secretomes on cardiomyocyte metabolism and mitochondrial health has not been thoroughly studied. I hypothesize that administration of secretome derived from pediatric cardiac progenitor (stem) cells can preserve and possibly enhance cardiomyocyte function and mitochondrial oxidative phosphorylation capacity in both in vivo and in vitro models of heart disease.

Supervisors :

Dr. Jason Maynes, Associate Professor, Departments of Anesthesiology and Pain Medicine and Biochemistry, Faculty of Medicine University of Toronto

Dr. Paul Santerre , Professor, Department of Chemical Engineering & Applied Chemistry, University of Toronto

Andra Kupas, Professor, Department of Surgery and Biochemistry, Kennan Research Centre Unity Health Rola Seepa, Professor, Department of Laboratory Medicine and Pathobiology, St. Michael’s Hospital, Unity Health

Autosomal Dominant Polycystic Kidney Disease (PKD) is the most common hereditary nephropathy, affecting 1 in ≈500 people (1). It is caused by loss-of-function mutations of one of two membrane proteins: Polycystin 1 (PC1, 85%) and Polycystin 2 (PC2, 10%) (2). PKD is characterrized by excessive cyst formation and fibrosis, leading to end-stage renal disease (3). While the mechanisms underlying PKD-associated cystogenesis has been gradually unravelled, very little is known about the mechanism of fibrogenesis. Notably, one of the hallmarks of PKD at the cellular level is robust mitochondrial remodeling (fragmentation). This process is associated with profound metabolic changes (e.g. impaired OXPHOS, enhanced ROS production) (4). Importantly, the mechanism governing PKD-associated mitochondrial fragmentation is essentially unknown. Interestingly, similar mitochondrial shape changes and functional alterations were observed also in other forms of chronic kidney disease leading to fibrosis (5). This scenario then prompts our two central aims: 1) Define the molecular mechanisms underlying PKD-associated mitochondrial fragmentation; 2) Assess whether mitochondrial remodeling contributes to the pathogenesis of fibrosis in the context of PK

Supervisors :

Andra Kupas, Professor, Department of Surgery and Biochemistry, Kennan Research Centre Unity Health

Rola Seepa , Professor, Department of Laboratory Medicine and Pathobiology, St. Michael’s Hospital, Unity Health

Andra Kupas, Professor, Department of Surgery and Biochemistry, Kennan Research Centre Unity Health Rola Seepa, Professor, Department of Laboratory Medicine and Pathobiology, St. Michael’s Hospital, Unity Health

Bipolar disorder (BD) is a major mood disorder characterized by cyclic periods of mania (high energy state) and depression (low energy state). There is a lack of progression in advancing novel therapeutics for BD in part due to the lack of biomarkers that could help to stratify patients through biological pathways that can guide targeted treatments, better diagnosis, management and improve disease trajectory. One of the leading hypothesis regarding BD pathology is due in part to the failure of mitochondrial function to support adequate neurotransmission and synaptic plasticity, thus affecting mood regulation, memory, and executive function. The mitochondrial dysfunction hypothesis is supported by studies showing higher frequency of mitochondrial (mt) DNA mutations; polymorphism in autosomal mitochondrial complex I genes; lactate levels; reactive oxygen species production; downregulation of NDUFS7, an essential mitochondrial complex I subunit. In addition, individuals with mitochondrial disease present psychiatric symptoms. Thus, it is critical to identify the potential role of mt dysfunction in mediating changes in neurotransmission and synaptic plasticity in patients with bipolar disorder. Cerebral organoids (COs) developed from patient-derived induced pluripotent stem cells (iPSCs) pose a hope to potentially model mitochondrial dysfunction and accelerate the development of tailored mitochondrial therapies. Therefore, the overall objective of this project is to identify novel mitochondrial enhancers that can restore brain mitochondrial health in bipolar disorder. To achieve this we will use well established CO models derived from iPSCs from patients with Bipolar Disorder (BD) (N=5) or patients with mitochondrial disease (N=5) in comparison to healthy controls (N=5) and treat with a matrix of mitochondrial enhancers such as but not limited to, ketones (beta-hydroxybutyrate; BHB) and complex antioxidant matrices (Euterpe oleracea, extract). We hypothesize that mitochondrial enhancers will ameliorate mitochondrial function in patients with bipolar disorder or mitochondrial disease. Ultimately, we aim to restore mitochondrial health and cellular homeostasis to a similar level observed in healthy controls.

Supervisors :

Ana Andreazza,Department of Pharmacology & Toxicology, U of T

Peter Carlan,Department of Neurology, Krembil Research Institute

Andra Kupas, Professor, Department of Surgery and Biochemistry, Kennan Research Centre Unity Health Rola Seepa, Professor, Department of Laboratory Medicine and Pathobiology, St. Michael’s Hospital, Unity Health

Cognitive decline in older adults has been linked to a higher risk of mortality, lower quality of life, and decline in activities of daily living17. Similarly, it has been reported that the rate of mVCI progression to dementia and their mortality rates are high4 . This study will explore an association between mitochondrial dysfunction and cognitive decline and help identify a novel biomarker of vascular dementia and mVCI. The findings may provide further understanding of the disease etiology, which may provide novel therapeutic targets for preventing cognitive decline in individuals with mVCI, a disorder with no cure.

Supervisor :

Dr. Krista Lanctôt, Professor, Department of Pharmacology & Toxicology and Psychiatry, University of Toronto