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

Vascular mild cognitive impairment (VaMCI) and mild cognitive impairment due to Alzheimer’s disease (MCI-AD) are both prodromal stages to dementia. Both conditions are characterized by underlying symptoms such as cognitive decline. Nevertheless, there are several key differences between the two conditions. VaMCI is the mild form of vascular cognitive impairment (VCI). It is defined as early cognitive decline in the presence of cerebrovascular disease, where the cognitive decline is not severe enough to meet dementia criteria. VaMCI has been linked to increased risk of progression to many forms of dementia, particularly vascular dementia. MCI-AD on the other hand, particularly amnestic MCI, is referred as the precursor to Alzheimer’s disease (AD), where it is considered as the transitional state between normal cognition and AD. In both forms of MCI, it is common to identify declines in executive function and higher-order cognition like working memory. It is known that deficits in working memory is one of the most common cognitive impairments, where those deficits involve difficulties in both storing and manipulating information. Although mitochondrial dysfunction and oxidative stress (OS) are associated with AD, these relationships have not been fully explored in VaMCI, and compared to that in MCI-AD.

Mitochondria produce reactive oxygen species (ROS) as byproducts of cellular reactions, and OS occurs when there is imbalance between production of ROS and antioxidants. Unlike nuclear DNA, mitochondrial DNA (mtDNA) is especially susceptible to OS due to its proximity to ROS production, limited DNA repair mechanisms, and lack of protective histones. Consequently, excessive ROS levels can trigger further mitochondrial damage through disruptions of the electron transport chains. This series of events eventually triggers cell death, which results in fragments of mtDNA being released into the blood circulation as circulating cell-free mtDNA (ccf-mtDNA).

Therefore, I aim to 1) Identify if the association of OS levels and working memory at baseline is mediated by ccf-mtDNA in patients with VaMCI vs MCI-AD. 2) Evaluate relationship of baseline ccf-mtDNA and working memory in VaMCI vs. MCIAD patients. 3) Investigate and compare baseline ccf-mtDNA levels in VaMCI vs. MCI-AD patients.

Ana Konvalinka, Department of Nephrology, University of Toronto and University Health Network

Ana Andreazza, Department of Pharmacology & Toxicology, University of Toronto

Kidney transplantation is the optimal treatment for patients with end-stage kidney disease. Transplantation significantly prolongs life compared to dialysis, incentivizing the use of marginal kidneys to offset organ shortage. Additionally, ischemia-reperfusion injury (IRI) harms all transplanted kidneys and can damage the mitochondria. Normothermic ex vivo kidney perfusion (NEVKP) is a storage method that results in superior graft outcomes when compared to static cold storage. Repairing grafts prior to transplantation would increase the number of viable kidneys, improving graft outcomes. In our previous study, we determined that NEVKP may be repairing kidneys by preserving their mitochondrial protein expression and metabolism. We identified a potential regulator of these mitochondrial proteins, hepatocyte nuclear factor 4a (HNF4A), which may present a novel target for kidney repair. The goal of this project is to determine whether a novel HNF4A agonist, N-trans caffeoyltyramine (NCT), protects kidneys from IRI. My hypothesis is that NCT preserves mitochondrial function and proximal tubular cell viability in vitro and ameliorates kidney injury in a model of bilateral renal IRI in vivo.

Susan R. George, Departments of Medicine, Pharmacology & Toxicology

Ana Andreazza, Department of Pharmacology & Toxicology, University of Toronto

Significant concern exists regarding the rising prevalence of cannabis use among adolescents, given that human brain development remains incomplete until age 25. Increased risk of psychiatric disorders is associated with adolescent exposure to THC, the main psychoactive, cannabinoid type-1 receptor (CB1R)-activating component of cannabis. The amygdala brain region is a key regulator of fear/anxiety behaviours, maturing during adolescence, and is implicated in psychiatric disorders associated with cannabis use. Our laboratory has found increased amygdala oxidative stress and astrocyte-derived neuroinflammation following adolescent exposure to THC, using rodent and primate administration models with proteomic and immunohistochemical techniques. This project will investigate potential THC-activated mitochondrial mechanisms of pro-inflammatory oxidative stress in amygdala astrocytes, and whether this impairs the maturation of neurons. Techniques employed will include in vitro and in vivo calcium imaging, metabolomics, with behavioural testing to investigate functional amelioration with neurometabolic supplementation. Novel mitochondrial astrocyte-neuron crosstalk mechanisms identified may underlie clinical associations between adolescent cannabis use and psychiatric disorders, revealing novel therapeutic targets to reverse enduring harm following adolescent cannabis use.

Anthony Gramolini, Department of Physiology, University of Toronto

Phospholamban (PLN) regulates cardiac relaxation by inhibiting the function of sarco-endoplasmic reticulum Ca2+ – ATPase, type 2a (SERCA2a)1. In-frame deletion of Arginine 14 in PLN (PLNR14d) has been associated with cardiomyopathy in both human patients and transgenic animal models2-3. Perinuclear aggregates are frequently detected in PLNR14d cardiomyocytes (CM), although the underlying etiology remains unclear4-5. My pilot data has uncovered FK506-binding protein 8 (Fkbp8), a sarcoplasmic-reticulum/mitochondria protein, as a novel PLN-interacting partner. Interestingly, miRNA mediated Fkbp8 reduction results in perinuclear aggregates in cultured mouse neonatal CM6. My project aims to characterize mitochondrial abnormalities in PLNR14d CM, elucidate the role of Fkbp8 in PLNR14d – mediated perinuclear aggregation, and evaluate a novel therapeutic approach for alleviating the cytotoxic effects of PLNR14d in vivo.

Cheryl H. Arrowsmith, Department of Medical Biophysics, University of Toronto/University Health Network

Rachel Harding, Department of Pharmacology and Toxicology, University of Toronto

Huntington’s disease (HD) is a devastating autosomal dominant neurodegenerative disease caused by an expansion of ≥36 CAG trinucleotide repeats (encodes polyglutamine) in the huntingtin gene (Htt). HD is characterized by loss of neurons in the striatal and cortical areas of the brain, particularly medium spiny neuronal (MSNs) cells. However, the mechanism by which expression of mutant huntingtin leads to neuronal cell death remains the subject of intense research focus. To maintain normal synaptic communication, neurons heavily rely on mitochondrial-ATP production and are extremely sensitive to alterations in energy metabolism. Mitochondrial dysfunction is an early pathological mechanism thought to play an important role in selective neurodegeneration in HD. Mitochondrial dysfunction by mutant huntingtin is accompanied by release of mitochondrial RNAs (MT-RNAs) into the cytoplasm of the MSNs of human HD and mouse models of HD. This release of MT-RNAs was shown to trigger the innate immune signaling pathway within MSNs leading to cell death. MT-RNAs have been reported as cross-regulators of paraspeckle biogenesis by regulating long non-coding RNA (lncRNA) NEAT1 (nuclear paraspeckle assembly transcript 1) expression. LncRNA NEAT1 is a scaffold for paraspeckle formation, a nuclear sub-compartment that acts as a sensor of mitochondrial-stress and is required for mitochondrial homeostasis. NEAT1 levels have been previously reported to be altered in human HD tissues and HD mouse models.

In my PhD research, I have found that both wild-type and mutant huntingtin interact with specific RNAs. Specifically, RIP-seq experiments of immunoprecipitated huntingtin from normal and HD fibroblasts and isogenic neuronal progenitor cells, showed significant enrichment of lncRNA NEAT1. Gene ontology analysis of huntingtin RIP-seq data shows enriched transcripts are involved in various mitochondrial pathways such as ATP metabolic process, oxidative phosphorylation as well as in cytokine regulatory processes. Integrating these findings, we hypothesize that huntingtin-NEAT1 interactions are altered in HD cells contributing to a reduced ability to deal with mitochondrial stress, causing mitochondria-fragmentation, thus leading to MT-RNA release and innate immune activation. Aiming to understand the role of huntingtin-NEAT1 in managing mitochondrial stress, my research involves examining mitochondrial bioenergetics in wild-type and HD cell models after huntingtin and/or NEAT1 knockdown. My proposed studies will uncover a completely novel role for huntingtin in regulating paraspeckle biogenesis via interaction with lncRNA NEAT1 and provide a novel perspective on how mutant huntingtin may affect lncRNA metabolism and impact the cellular responses to mitochondrial stressors.