Parkinson’s disease (PD) is the second most common neurodegenerative disease affecting approximately 1.8% of the population over 65 years of age. It is characterized by three cardinal symptoms: bradykinesia, muscle rigidity and resting tremor. Symptoms are presented following 50% loss of dopaminergic neurons within the substantia nigra pars compacta (SNc). Neurodegeneration is associated with reactive oxygen species (ROS) production, protein aggregation, mitochondrial dysfunction, ubiquitin-proteasome system (UPS) inhibition and lysosomal malfunction; however it is unclear if a single mechanism or multiple mechanisms lead to disease onset. The primary aim of the studies described in this thesis was to elucidate the interactions between various pathological mechanisms underlying PD pathology. An examination of organelle function during exposure of SH-SY5Y neuroblastoma to a variety of toxins which mimic purported pathological processes in PD reveal mitochondrial membrane potential becomes depolarized, not only following mitochondrial impairment, but also after the UPS and lysosome are inhibited. Given that mitochondrial dysfunction appeared to be central to PD pathology, mitochondrial dysfunction was studied in more detail. Mitochondrial fission and fusion maintains mitochondrial integrity, which is critical to neuronal health. Thus, we examined mitochondrial dynamics in a common genetic variant linked with familial PD, known as leucine-rich repeat kinase 2 (LRRK2). Upon the expression of wild-type and mutant LRRK2, mitochondrial fusion was inhibited causing fragmentation of mitochondria. This inhibition of fusion may be the initial step leading to mitochondrial dysfunction, since inhibition of fusion occurs prior to the induction of cell stress. The findings that mitochondrial dysfunction appears to be central to PD pathology, suggest that mitochondria may be an excellent therapeutic target for PD. Thus, the potential neuroprotective function of a regulator of mitochondrial function, known as SIRT3 was examined. In SH-SY5Y cells, over-expression of SIRT3 protected neurons from degeneration associated with LRRK2 over-expression. The studies described in this thesis provide evidence that multiple sub-cellular mechanisms converge to inhibit mitochondrial function. Furthermore, mitochondrial dynamics which regulate mitochondrial function could be a key mediator in the pathology associated with PD. The work herein suggests therapies which target the mitochondria are likely to be successful in the treatment of PD.

Parkinson’s disease (PD) is the second most common neurodegenerative disease affecting approximately 1.8% of the population over 65 years of age. It is characterized by three cardinal symptoms: bradykinesia, muscle rigidity and resting tremor. Symptoms are presented following 50% loss of dopaminergic neurons within the substantia nigra pars compacta (SNc). Neurodegeneration is associated with reactive oxygen species (ROS) production, protein aggregation, mitochondrial dysfunction, ubiquitin-proteasome system (UPS) inhibition and lysosomal malfunction; however it is unclear if a single mechanism or multiple mechanisms lead to disease onset. The primary aim of the studies described in this thesis was to elucidate the interactions between various pathological mechanisms underlying PD pathology. An examination of organelle function during exposure of SH-SY5Y neuroblastoma to a variety of toxins which mimic purported pathological processes in PD reveal mitochondrial membrane potential becomes depolarized, not only following mitochondrial impairment, but also after the UPS and lysosome are inhibited. Given that mitochondrial dysfunction appeared to be central to PD pathology, mitochondrial dysfunction was studied in more detail. Mitochondrial fission and fusion maintains mitochondrial integrity, which is critical to neuronal health. Thus, we examined mitochondrial dynamics in a common genetic variant linked with familial PD, known as leucine-rich repeat kinase 2 (LRRK2). Upon the expression of wild-type and mutant LRRK2, mitochondrial fusion was inhibited causing fragmentation of mitochondria. This inhibition of fusion may be the initial step leading to mitochondrial dysfunction, since inhibition of fusion occurs prior to the induction of cell stress. The findings that mitochondrial dysfunction appears to be central to PD pathology, suggest that mitochondria may be an excellent therapeutic target for PD. Thus, the potential neuroprotective function of a regulator of mitochondrial function, known as SIRT3 was examined. In SH-SY5Y cells, over-expression of SIRT3 protected neurons from degeneration associated with LRRK2 over-expression. The studies described in this thesis provide evidence that multiple sub-cellular mechanisms converge to inhibit mitochondrial function. Furthermore, mitochondrial dynamics which regulate mitochondrial function could be a key mediator in the pathology associated with PD. The work herein suggests therapies which target the mitochondria are likely to be successful in the treatment of PD.

Inhibition of mitochondrial translation as a therapeutic strategy for acute myeloid leukemia Marko Škrtić Doctor of Philosophy Institute of Medical Science University of Toronto 2012 Abstract Intro: Acute myeloid leukemia (AML) therapies have remained unchanged for 20 years, and thus new therapies are needed. Objective: To identify FDA-approved agents with anti-leukemia stem cell activity, we performed a screen and identified the antimicrobial tigecycline (TIG). Methods: Primary AML mononuclear cells were isolated by Ficoll centrifugation from peripheral blood. Flow cytometry dye; JC-1, Carboxy-H2DCFDA, Mitotracker GreenFM. Leukemia stem cell activity was assayed by human AML engraftment in NOD/SCID mice. Results: TIG induced cell death in primary AML patient samples (LD50, 3-6μM n=14), preferentially over normal hematopoietic cells. Likewise, in colony assays, TIG (5μM) reduced the clonogenic growth of AML samples (n=7) by 93%, demonstrating an effect on leukemia progenitor cells, but not normal hematopoietic cells (34% reduction, n=5). A yeast genome-wide screen identified mitochondrial translation inhibition as the mechanism of tigecycline-mediated cell death in eukaryotic cells. TIG decreased the expression of mitochondrial peptides, enzyme activity and membrane potential preferentially in AML cells over normal hematopoietic cells. ShRNA knockdown of TuFM mitochondrial translation factor in leukemia cells reproduced TIG anti-leukemia target effects previously described. We discovered that primary AML CD34+/CD38- stem cells have greater mitochondrial mass (3-fold, n=5) than normal CD34+ cells (n=4). Higher baseline mitochondrial mass in primary AML samples was predictive for tigecycline sensitivity in vitro (r=-0.71, p<0.05). We assessed the effect of TIG on primary AML stem cells defined by their ability to initiate leukemic engraftment in vivo. NOD/SCID mice treated with TIG had decreased human AML engraftment (n=3 AML patients) compared to control. Conclusions: We identified mitochondrial translation inhibition as a novel therapeutic strategy for AML. Currently, a Phase I clinical trial of tigecycline in hematological malignancies is underway.

Mitochondrial dysfunction is considered as a key mechanism of aging but little is known about the impact of mitochondrial biogenesis. Mitochondrial DNA (mtDNA) copy number control is an important aspect of mitochondrial biogenesis and is highly regulated in eukaryotic organisms. By studying mtDNA copy number, our aim is to gain a better understanding of the relationship between mitochondrial biogenesis and aging. We developed an optimized protocol for measuring mtDNA copy number in Caenorhabditis elegans using quantitative real-time PCR (qPCR). We investigated how mtDNA regulation is affected by a variety of aging-related pathways. We found the insulin/IGF-1 signaling (IIS) pathway regulates mtDNA content in a DAF-16- and UCP-4-dependent manner. By utilizing RNA interference (RNAi) against polg-1, we showed that mitochondrial stress likely modulates lifespan through the IIS pathway. Our work identifies IIS as a communications pathway between mitochondria and the nucleus in modulating mitochondrial biogenesis and lifespan in Caenorhabditis elegans.

This project investigated the effects of exercise and epinephrine on the mRNA expression of peroxisome proliferator activated receptor gamma coactivator-1 alpha (PGC-1 alpha), a master regulator of mitochondrial biogenesis, in rat adipose tissue. Rats that swam 2 hours daily for 4 weeks had increased mitochondrial marker proteins and PGC-1 alpha mRNA expression in epididymal and retroperitoneal adipose tissue (p<0.05). Adipose tissue organ culture treatment with epinephrine increased (p<0.05) PGC-1 alpha mRNA expression in both depots, but only epididymal adipose responded to a supra-physiological dose. Beta blockade attenuated the effects of an acute bout of exercise on PGC-1 alpha mRNA expression in epididymal, but not in retroperitoneal adipose tissue. This is the first study to demonstrate that rat white adipose tissue PGC-1 alpha mRNA expression is increased by acute and chronic exercise and epinephrine. Increases in circulating catecholamine levels might be one potential mechanism mediating exercise induced increases in PGC-1 alpha mRNA expression in rat abdominal adipose tissue. / Nutrition and Metabolism

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1990. / Vita. Abstract. Includes bibliographical references (leaves 171-183). Also available via the Internet.

Thesis (Ph. D.)--University of Adelaide, 1984. / Includes bibliographical references (leaves [i]-xvi).

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.

Thesis (M.Sc.)--University of Adelaide, Dept. of Biochemistry, 1971.