The mitochondrial serine protease ClpXP regulates the integrity of the respiratory chain by degrading damaged and/or misfolded proteins. This protease is over-expressed in multiple malignancies including acute myeloid leukemia (AML). Inhibiting or hyperactivating it kills malignant AML cells in vitro and in vivo. Yet, it is unknown how the mitochondrial ClpXP achieve substrates specificity. In Bacillus subtilis, the bacterial ClpXP homologue recognizes proteins tagged with phospho-arginine for degradation. Therefore my project aims to investigate whether protein phosphorylation marks proteins for degradation by human ClpXP. We will also characterize how these markers impact the mitochondrial protein function in the context of AML.

Supervisors :

Dr. Aaron Schimmer: Research Director and Senior Scientist, Princess Margaret Cancer Centre; Professor, Departments of Medical Biophysics, University of Toronto

Dr. Andrei Yudin: Professor, Department of Chemistry University of Toronto

Patients with mitochondrial disease due to mtDNA mutations are frequently heteroplasmic, where both wild-type and mutant mitochondrial DNA (mtDNA) exists inside the same cell. Due to its guanine-rich nature, the heavy strand of mtDNA has an increased tendency to form a type of secondary structure called guanine quadruplexes (GQs). Some disease-causing mutations, including the m.10191C variant associated with maternally inherited Leigh’s Syndrome, may increase GQ formation. Thus, heteroplasmy shifting presents a potential therapeutic solution, where mtDNA replication is biased away from pathogenic variant. My project aims identify small molecule compounds that bind and stabilize mitochondrial GQs to promote wild-type
mtDNA expression. With our collaborators, we have screened over 20,000 compounds using a small molecule microarray to identify compounds with high affinity for mitochondrial GQs. We will validate these compounds through in vitro primer extension assays as well as cellular assays for mtDNA depletion and heteroplasmy shift. Our goal is to create a novel, highly specific, and mutation-tailored therapy for mitochondrial disease.

Supervisors :

Dr. Neal Sondheimer: Associate Professor of Paediatrics and Molecular Genetics · The Hospital for Sick Children

Dr. Dr. Brett Kaufman: Associate Professor, Faculty of Medicine, University of Pittsburgho

Growing evidence has demonstrated that metabolic and mitochondrial dysfunction are involved in type 2 diabetes. However, due to the variability in study findings and the unique biochemical modifications underlying both mitochondrial dysfunction and diabetes, there are many questions that remain unanswered. Further, it has yet to be investigated how dietary interventions affect mitochondrial stress in diabetic patients and whether there is a relationship with aging in a study using an adequate sample size. Therefore, the overall goal of our study is to gain a further understanding of mitochondrial dysfunction in type 2 diabetes and its relationship with dietary interventions and aging. To achieve this goal, we will investigate two peripheral markers of mitochondrial stress – lactate and circulating cell-free mitochondrial DNA (ccf-mtDNA) in 75 patients with type 2 diabetes and 75 healthy matched controls by age and sex. Demographic correlations will be explored to investigate if diet plays an evident role in mitochondrial function and type 2 diabetes.

Supervisors :

Dr. David Jenkins: Professor, Departments of Nutritional Sciences and Medicine, Faculty of Medicine, University of Toronto

Dr. Ana Andreazza: Professor, Department of Pharmacology & Toxicology and Psychiatry, University of Toronto

The project aims to examine mitochondrial dysfunction with circulating cell-free mitochondrial DNA (ccfmtDNA) in mild cognitive impairment (MCI) and mild Alzheimer’s disease (AD) patients from our Exercise as a Primer for
Excitatory Stimulation Study (EXPRESS). EXPRESS investigates whether aerobic exercise may be an effective, yet previously unexplored primer for AtDCS, a non-invasive type of brain stimulation, to improve cognition in MCI and mild AD. Mitochondrial dysfunction is a key contributor to AD pathogenesis and has been associated with cognitive impairment; it has also been suggested to be impacted by exercise and AtDCS. Hence, examining the association between ccf-mtDNA levels and cognitive response to exercise-primed AtDCS may identify a peripheral biomarker for this novel combined intervention, helping to define new targets for personalized treatments in MCI and mild AD. This project can enhance our understanding of the mechanistic correlates of exercise-primed AtDCS intervention in MCI and mild AD that are critical periods for treatment to slow cognitive losses.

Supervisors :

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

Metabolic dysfunction is a hallmark of a failing heart, involving a shift from effectual fatty acid oxidation to poorly efficient aerobic glycolysis. Transitioning the failing heart back to an ATP-rich oxidative metabolism would improve cardiomyocyte function, and represent a novel therapy for cardiac dysfunction. The biomolecules secreted by mesenchymal stromal cells (the secretome) have shown a transferable reparative effect when injected into a diseased heart. However, the mechanisms by which the secretome improves myocardial performance have not been elucidated. Considering the higher intrinsic reparative potential of a pediatric heart, this study seeks to reveal whether administration of secretomes derived from pediatric cardiac progenitor (stem) cells will improve organ function by improving the efficiency of metabolism in cardiomyocytes. The aim of the project is to determine the mechanisms by which these treatments are beneficial, advancing their clinical implementation by developing specific metabolic therapies for heart failure.

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

Mutations in the gene encoding C-terminus of HSP70 Interacting Protein (CHIP) have been recently identified as the cause of two cerebellar ataxias, spinocerebellar ataxia autosomal dominant type 48 (SCA48) and spinocerebellar ataxia autosomal recessive type 16 (SCAR16). Both SCA48 and SCAR16 are incurable debilitating neurodegenerative diseases involving significant loss of cerebellar Purkinje neurons, ataxia, cognitive impairment, and a wide range of variable additional symptoms. The mechanism(s) underlying CHIP-associated diseases are currently unknown. We have demonstrated that CHIP plays a role in the regulation of mitochondrial quality control pathways including mitochondrial autophagy (mitophagy), a process by which cells can remove damaged/dysfunctional mitochondria, indicating that mitochondrial dysfunction may play a role in CHIP-associated neurodegeneration. In this project, we will investigate how disease-associated CHIP
mutations influence mitochondrial quality control pathways both in vitro using cell culture models and in vivo in neurons of Caenorhabditis elegans, microscopic nematodes which are frequently utilized to model neurodegenerative diseases. We will also use these systems to screen for compounds which may protect against CHIP-mediated mitochondrial dysfunction and neurodegeneration.

Supervisors :

Dr. Suneil Kalia: Associate Professor, Department of Surgery, University of Toronto

Dr. Joel Watts: Associate Professor, Department of Biochemistry University of Toronto

N-methyl-D-aspartate receptor (NMDAR) is an ionotropic glutamate receptor that plays an important role in brain development. Activation of NMDAR leads to calcium influx and can cause mitochondrial dysfunction. GRIN disorder is a rare genetic condition with mutations in one of the seven GRIN genes encoding NMDAR subunits. Patients with GRIN disorder present neurological phenotypes associated with mitochondrial dysfunction. Our goal is to characterize mitochondrial morphology and function in the brain of mice possessing patient GRIN variants. Two mouse lines carrying heterozygous missense mutation in the Grin1 gene are generated by CRISPR/Cas9 through pronuclear injection. We aim to measure mitochondrial respiration in live neurons and astrocytes dissociated from mice with Grin1 patient variants. We also plan to look at the number and morphology of mitochondria in these mice. The proposed study reveals whether mitochondrial dysfunction is present in GRIN disorder and may lead to development of novel treatments for GRIN disorder.

Supervisors :

Dr. Amy Ramsey: Associate Professor, Department of Pharmacology & Toxicology, University of Toronto

Dr. Margaret Hahn: Assistant Professor, Department of Psychiatry, University of Toronto

Assessing mitochondrial dysfunction due to SARS-CoV-2 infection in a heart-on-a-chip platform Using a novel heart-on-a-chip platform, the Biowire II, the effects of SARS-CoV-2 on mitochondrial function will be investigated. The Biowire II is an organ-on-a-chip model that uses human induced pluripotent stem cell (hiPSC) derived cardiomyocytes and cardiac fibroblasts to measure force, calcium transients and electrical activity. Recent studies have shown how the virus affects critical mitochondrial processes, and thus may play a role in disrupting healthy cardiac function. This will be accomplished by infecting Biowire II cardiac tissues with SARS-CoV-2 and monitoring their function and health over a multi-week timeline. Assays will be used to investigate mitochondrial metabolism by measuring extracellular acidification rate, oxygen consumption rate, and ROS production.

Supervisors :

Dr. Milica Radisic: Professor, Department of Department of Chemical Engineering & Applied Chemistry, University of Toronto

Dr. Ana Andreazza, Professor, Department of Pharmacology & Toxicology and Psychiatry, University of Toronto

The overall objective of this study is to understand the role of mitochondrial quality control (MQC) in the pathophysiology of ischemia/reperfusion injury (IRI), particularly exploring the effect of mitochondrial transplantation on MQC. Acute IRI causes the dysregulation of MQC, including fission, fusion, mitophagy and biogenesis. These systems are regulated by a dynamic, complex, integrated, context- and time-dependent network of pathways. Recently, mitochondrial transplantation (MT) has emerged as a potential biotherapeutic to reduce IRI and protect cells/tissues/organs. Whereas, MT has been shown to be efficacious in multiple animal models of disease, the underlying molecular mechanisms and translational capability remain largely unaddressed. This study aims to fill these knowledge gaps using in vitro, ex vivo and in vivo models of disease. Using in vitro hypoxia/reoxygenation (AR) of human hepatocytes, we will demonstrate if mitochondrial transfer into hepatocytes is possible and can reduce cellular death, injury, and inflammation. We will utilize an in vivo surgical murine model of liver IRI to test the efficacy of MT via direct infusion into the portal system. Precision-cut liver slices (PCLS) will be used as an ex vivo platform to further validate the translatable efficacy of MT in living human tissue. To gain mechanistic insight into MT, knockout mice and siRNA studies will be conducted to establish which protein components are required for the protective effects of MT (including mitochondrial internalization/endocytosis) in cells, tissues, and animals. In this manner, this proposal aims to show the utility of MT in the hepatic system and investigate its potential effects on MQC pathways.

Supervisors :

Dr. Ori Rotstein, Professor and Associate Chair of the Department of Surgery, University of Toronto

Dr. Ana Andreazza , Professor, Department of Pharmacology & Toxicology and Psychiatry, University of Toronto

Mitophagy is a mitochondrial quality control pathway that removes damaged mitochondria from the cell. Disruption of mitophagy is related to the development of neurodegenerative disorders like Parkinson’s disease. Thus, the identification of mitophagy agonists harbors great therapeutic potential against neurodegenerative diseases. Recent studies have suggested that probiotics, which are microorganisms that carry beneficial effects on the host, have positive implications on mitochondrial health. In this study, we aim to identify probiotic strains that promote mitophagy. To date, we have developed cell-based screening methods and successfully found probiotic strains/blends that can positively regulate mitophagy. In the next step, we will decipher their mechanisms of action. We will also utilize Drosophila Parkinson’s disease models to assess the effects of dietary supplementation of the probiotics. Our study will provide valuable insight into the health benefits of probiotics and lay a foundation for further exploring their therapeutic potential.

Supervisors :

Angus McQuibban , Department of Biochemistry, University of Toronto

Elena Comelli, Department of Nutritional Science, University of Toronto

It is not yet clear why 30-40% of healthy, euploid IVF embryos fail to implant. An area requiring further investigation is the impact of mitochondrial DNA (mtDNA) heteroplasmy. The research I am conducting is focused on studying the dynamics of mtDNA heteroplasmy in the inner cell mass (gives rise to the fetus) and in the trophectoderm (gives rise to the placenta) of human pre-implantation embryos, and how it influences IVF outcomes (on-going pregnancy, no implantation, miscarriage). Part of this research will also involve assessing the interaction between the nuclear and mitochondrial genome and how it affects mtDNA heteroplasmy distribution. Gaining further insight into the transmission of heteroplasmy from mother to the pre-implantation embryo, as well as the rate of de novo mutations, can help to improve pre-implantation genetic testing (PGT) for the detection of deleterious mtDNA mutations. This research also aims to identify potential biomarkers for developmentally more competent euploid embryos, which can significantly impact the success of fertility treatment for patients undergoing IVF.

Supervisors :

Dr. Clifford Librach, Associate Professor, Department of Physiology, University of Toronto

Dr. Svetlana Madjunkova. Adjunct Lecturer. Department of Laboratory Medicine & Pathobiology, University of Toronto

Primary Graft Dysfunction (PGD) is one of the early complications that may occur following lung transplantation (LTx). Despite its high incidence, there has been limited knowledge on the mechanisms of disease making it difficult to diagnose, treat, and prevent. The mitochondria’s many roles in the lungs, such as energy production, oxygen consumption, and surfactant production make them valuable targets of investigation since dysfunctional mitochondria can lead to poor lung function. Thus, in our project, we aim to evaluate the relationship between mitochondrial quality and LTx outcomes. To achieve this, we will be identifying patients’ mitochondrial haplogroups and conducting mitochondrial quality assessments to evaluate whether they can be used as screening tools for the risk of PGD development. Further, we will be investigating the relevance of matching haplogroups between donor and recipient to prevent PGD. Finally, our overall goal is to provide clinically relevant screening tools for PGD and open doors for study of novel therapeutic interventions to improve LTx outcomes.

Supervisors :

Dr. Marcelo Cypel: Associate Professor Department of Surgery University of Toronto

Dr. Ana Andreazza: Professor, Department of Pharmacology & Toxicology and Psychiatry, University of Toronto

Endothelial cells form a layer lining all blood vessels known as the endothelium. During diabetes, high blood glucose induces mitochondrial dysfunction in endothelial cells, which plays a central role in commonly associated microvascular complications such as nephropathy and retinopathy. However, mitochondrial dysfunction in endothelial cells is not well studied across many tissues. This includes the pancreatic islets, where the insulin-secreting β-cells are located. The endothelium of islet microvessels are highly specialized to support rapid glucose and insulin exchange between β-cells and the blood circulation. Islet endothelial cells are also imperative for proper β-cell function. Therefore, mitochondrial dysfunction in islet endothelial cells could interfere with glucose homeostasis and exacerbate diabetic disease progression. In this project, we will investigate the effects of hyperglycemia on mitochondrial function in islet endothelium by integrating in vitro cell culture techniques, single cell transcriptomics and microfluidics. Our goal is to elucidate the role of mitochondrial dysfunction in islet endothelial cells during diabetes and discover treatments to alleviate this dysfunction. This could facilitate the development of future therapy for diabetes.

Supervisors :

Dr. Sara Vasconcelos: Associate Professor, Department of Laboratory Medicine and Pathobiology, University of Toronto

Dr. Edmond Young: Associate Professor, Mechanical Engineering, University of Toronto

Dr. Rashidi-Ranjbar is a clinician-scientist with experience in clinical and cognitive neuroscience.

MITO2i has partnered with TFOM initiative, the Toronto Dementia Research Alliance (TDRA) to support the training of a clinical fellow investigating the connection between dementia and mitochondria.

The TDRA will co-fund the fellow with a contribution of $30K for a total of $60K towards the postdoc salary.

The call was announced in August 2021. Through a competitive process, we are pleased to introduce the 2022 MITO2i-TDRA fellow, Dr. Neda Rashidi-Ranjbar. Under the co-supervision of Dr. Tom Schweizer and Dr. Corinne Fischer at Unity Health.

Dr. Rashidi-Ranjbar will be leading a study that investigates the efficacy of photobiomodulation (PBM), a form of therapy that uses lights, in the treatment of early Alzheimer’s disease (AD).