Mitochondria are ubiquitous eukaryotic organelles which carry out a range of essential functions, most notably the production of ATP through the process of oxidative phosphorylation. While the main biochemical function of mitochondria was established over 50 years ago, the processes which control mitochondrial morphology are, at present, poorly understood. The thesis aims to add to our knowledge of the factors that control mitochondrial morphology and dynamics in the model plant species, Arabidopsis thaliana. The phenotypic characteristics of two novel mitochondrial morphology mutants, motley mitochondria I (mmtl) and network mitochondria (nmt), were examined and quantified. mmtl has an increased heterogeneity of mitochondrial plan area relative to wild-type, which is matched by a similar chloroplast phenotype. nmt exhibits a reticular mitochondrial morphology, similar to the mitochondria found in yeast and animals. Genetic mapping of the two mutant loci has established that mmtl resides on a short region of chromosome 11w, hile nmt was mapped to a small area of chromosome V. This thesis describes the identification and functional analysis of two novel orthologs of yeast and animal mitochondrial division genes. Using TDNA reverse genetics, it is shown that disruption of the dynamin-like DRP3A or BIGYIN (an Arabidopsis orthologue of yeast FISI) led to an increase in mitochondrial plan area, which is coupled with a decrease in the number of physically discrete mitochondria per cell. Finally, the morphology and behaviour of Arabiclopsis mitochondria is investigated upon the induction of cell death. Abiotic stress treatments that induce cell death led to fast and irreversible changes in mitochondrial morphology. The role of these changes, as possible early indicators of cell death, are discussed.

In order to support rampant cell growth, tumor cells must reprogram metabolism to simultaneously drive macromolecular biosynthesis and energy production. Mitochondrial uncoupling proteins (UCPs) oppose this phenotype by inducing futile mitochondrial respiration that is disengaged from ATP synthesis. We found that uncoupling protein 3 (UCP3) was normally expressed in follicular and epidermal keratinocytes and that its levels were augmented by calcium-induced differentiation in vitro. Over-expression of a UCP3 transgene targeted to the basal epidermis by the keratin-5 promoter (K5-UCP3) led to increased differentiation of both epidermal and bulge stem cells, the progenitors of most squamous carcinomas. Consistent with this phenotype, K5-UCP3 mice were completely protected from chemically induced skin carcinogenesis. To define the mechanisms by which UCP3 conferred such strong tumor resistance, we interbred K5-UCP3 mice with a “pre-initiated” mouse model, and found that UCP3 over-expression blocked tumor promotion. Uncoupled epidermis displayed reduced proliferation after treatment with tumor promoter, along with diminished activation of Akt signaling. This effect corresponded to decreased Akt activation by epidermal growth factor (EGF) in K5-UCP3 cells, along with UCP3 overexpressing primary human keratinocytes. Mechanistic studies revealed that uncoupling drove global lipid catabolism, along with impaired recruitment of Akt to the plasma membrane. Over-expression of wild type Akt rescued tumor promoter-induced proliferation and two-stage chemical carcinogenesis in bi-transgenic mice. Collectively, these findings demonstrate that mitochondrial uncoupling is an effective strategy to limit cell proliferation and tumorigenesis through inhibition of Akt, and suggest a novel mechanism of crosstalk between mitochondrial metabolism and growth signaling. / text

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One of the many recorded adaptive responses in respiratory chain complex I deficient cells is the over-expression of the small metal binding proteins, metallothioneins (MTs). The antioxidant properties of MTs putatively protect the deficient cells against oxidative damage, thus limiting further damage and impairment of enzymes involved in energy production. Moreover, the role of metallothioneins in supplying metal cofactors to enzymes and transcription factors in order to promote energy metabolism was previously proposed, which could accompany their role as antioxidants. This view is supported by the observations that MT knockout mice tend to become moderately obese, implying a lower energy metabolic rate. Hence, the involvement of metallothioneins in mitochondrial function and disease cannot be ignored. However, this association is still very vague due to the diversity of their functions and the complexity of the mitochondrion. The use of systems biology technology and more specifically metabolomics technology was thus employed to clarify this association by investigating the metabolic differences between wild type and MT knockout mice in unchallenged conditions as well as when mitochondrial function (energy metabolism) was challenged with exercise and/or a high-fat diet. The metabolic differences between these mice were also studied when complex I of the respiratory chain was inhibited with rotenone. The metabolome content of different tissues and bio-fluids were examined in an untargeted fashion using three standardized analytical platforms and the data mined using modern metabolomics and related statistical methods. Clear metabolic differences were found between the wild type and MT knockout mice during unchallenged conditions. These metabolic differences were persisted and were often amplified when mitochondrial metabolism was specifically challenged through exercise, high-fat intake or complex I inhibition. The data pointed to an overall reduced metabolic rate in the MT knockout mice and possible insulin resistance after the interventions which imply (and confirm) the involvement of MTs in promoting energy metabolism in the wild type mice. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2012

Rationale and hypothesis: Diabetic sensory neuropathy involves a distal dying-back of nerve fibers. Neuronal mitochondrial function is impaired in diabetes and Sirtuin 2 (SIRT2) is a sensor of redox state that regulates cellular bioenergetics. The role of SIRT2 in regulating the phenotype of adult sensory neurons derived from both control and diabetic rats or wild type and SIRT2 knockout (KO) mice was studied. It was hypothesized that sensory neurons under a hyperglycemic state would have a lowered NAD+/NADH ratio thus deactivating the SIRT2 pathway. It was further hypothesized that the down regulation of SIRT2 would diminish the activity of the AMP-activated protein kinase (AMPK) pathway resulting in mitochondrial dysfunction. This defect would contribute to distal dying-back of axons observed in diabetes. Methodology: Type 1 diabetes was induced in rodents by streptozotocin (STZ). Adult sensory neurons derived from control or STZ-diabetic rats or control and SIRT2 knockout (KO) mice were cultured in defined media with varying doses of neurotrophic factors and D-glucose. Protein levels were determined by quantitative Western blotting and neurite outgrowth quantified by immunocytochemistry. Plasmid transfection was initiated for overexpression of SIRT2 constructs and Seahorse XF24 analyzer was utilized to measure mitochondrial function of cultured neurons. Results: Overexpression of SIRT2 elevated total neurite outgrowth in cultures derived from control and STZ-diabetic rats. Cultures derived from SIRT2 KO mice exhibited diminished neurite outgrowth. The AMPK pathway was inhibited under high glucose treatment through activation of the polyol pathway. Pharmacological inhibition of the polyol pathway improved mitochondrial bioenergetics and neurite outgrowth in sensory neurons. Augmented expression of electron transport proteins and increased mitochondrial mass was associated with enhanced bioenergetic function. Conclusion: SIRT2 is a key component driving mitochondrial function and axon regeneration through the activation of AMPK pathway. In diabetes this pathway is suppressed via elevated polyol pathway activity. / October 2016

Aberrant metabolism has become an increasingly interesting area of cancer biology. In many cancers including lower grade glioma, glioblastomas and some leukemias, a mutation in the metabolic enzyme Isocitrate Dehydrogenase (IDH), has been found in more than 70% of cases and has been shown to lead to a distinct hypermethylator phenotype. IDH commonly converts isocitrate to alpha-ketoglutarate in normal cell metabolism. Three isoforms of this enzyme are found in humans: IDH1, IDH2 and IDH3. Studies on IDH1, the cytosolic isoform, have revealed that mutations in the enzyme’s binding site lead to a novel gain of function: the synthesis of an oncogenic metabolite, 2-hydroxyglutarate (2HG). 2HG competitively inhibits alpha-ketoglutarate dependent enzymes such as the TET 5-methylcytosine (5mC) oxygenases and histone demethylases. These oxygenases are responsible for the hydroxymethylation (5hmC) of cytosine residues, ultimately leading to demethylation and gene re-expression. Thus, mutant IDH may lead to an elevation in 5mC levels producing the hypermethylator phenotype described. A similar gain-of-function mutation in IDH2, the mitochondrial isoform of IDH1, has been associated with leukemias and gliomas lacking IDH1 mutations. Mutations in IDH1, IDH2 and TET2 are mutually exclusive, and each yields a similar hypermethylator phenotype. IDH2, along with IDH3, is primarily involved in the TCA cycle and energy production for the cell. Recently, the Taylor lab has uncovered evidence of 5mC and 5hmC residues in mitochondrial DNA, established and maintained by mtDNMT1 and TET2. Changing levels of mtDNMT1 appears to alter the patterns and levels of mtDNA transcription from the mitochondrial genome. We hypothesized that mutant IDH would produce a similar effect on the mitochondrial genome as that found in the nuclear genome and result in a decrease in the level of 5-hydroxymethylcytosine, as well as a subsequent increase in the level of 5-methylcytosine caused by the competitive inhibition of the TET enzymes by 2-hydroxyglutarate accumulation. Using molecular biology techniques such as Western blots and MeDIP (methylated DNA immunoprecipitation) I aim to uncover the role of IDH mutation on mitochondrial DNA methylation and its effect on energy production in mammalian cells.

Objective. The aim of this study was to determine the effects of interleukin-10 on mast cell development from bone marrow progenitors.Materials and Methods. Unseparated mouse bone marrow cells were cultured in IL-3+SCF, giving rise to mast cells and monocytes/macrophages. The addition of IL-10, and the use of Signal Transducer and Activator of Transcription (STAT)3-deficient bone marrow cells were employed to measure the effects of IL-10 and STAT3 expression on cell viability, proliferation, and differentiation. Bax-deficient and Bcl-2 transgenic bone marrow cells were used to determine the importance of the mitochondria in IL-10-mediated effects.Overview. Mast cells arise from hematopoietic stem cells and continue development in either connective tissue or mucosa. Th2 cytokines have been implicated in the regulation of mast cell development and subsequent function. Mast cells have also been shown to be essential players in many Th2 immune responses. In the following study we investigate the effects of the Th2 cytokine IL-10 on mast cell development from isolated bone marrow progenitors. The addition of IL-10 to whole murine bone marrow greatly reduced cell numbers and altered the phenotype of the developing progenitor cells. The reduction in cell numbers was due to apoptosis, as judged by DNA fragmentation and caspase activation. The apoptosis observed included alteration in mitochondrial membrane potential. Furthermore, apoptosis could be reduced by the overexpression of Bcl-2 or by ablating p53 expression. Utilizing a flox/cre system we found that IL-10 mediated apoptosis required expression of Stat-3, since Stat-3 deficient bone marrow cells did not undergo apoptosis in response to IL-10. In this study we also observed significant alterations in the mast cell growth factor receptors IL-3R and c-kit. The loss of these growth factor receptors may explain the apoptosis induced by IL-10. These data demonstrate the potent regulatory capabilities of Th2 cytokines on mast cells, a central effector in the Th2 response.

Cytosine methylation of mitochondrial DNA (mtDNA) was first described several decades ago, but neither the mechanism generating this modification nor its functional significance was known. Because mitochondrial dysfunction is a hallmark characteristic of numerous human diseases, including neurological and cardiovascular disease, aging and cancer, this dissertation addressed whether epigenetic modification of mtDNA regulates mitochondrial function. We show that mtDNA contains not only 5-methylcytosine (5mC), but also 5-hydroxymethylcytosine (5hmC), suggesting that previous reports likely underestimated the degree of epigenetic modification within the mitochondrial genome. We questioned how these modifications were generated by looking for mitochondrial isoforms of the nuclear-encoded DNA methyltransferases. We found that an isoform of the most abundant mammalian methyltransferase, DNA methyltransferase 1 (DNMT1) translocates to mitochondria, driven by an in-frame mitochondrial targeting sequence (MTS) located upstream of the nuclear DNMT1 translational start site. This MTS is highly conserved across mammalian species, and directs a heterologous protein to the mitochondria. To investigate the function of mitochondrial DNMT1 (mtDNMT1), we created a cell line that carries a tandem-affinity purification (TAP) tag at the C-terminus of a single endogenous human DNMT1 allele. Using the DNMT1-TAP cell line, we showed that mtDNMT1 specifically binds mtDNA in a manner that is proportional to CpG density, proving its presence in the mitochondrial matrix. mtDNMT1 exhibits CpG-specific methyltransferase activity in vitro that is resistant to trypsin-treatment of intact mitochondria, but moderately susceptible to pharmacologic inhibition by the nucleoside analog 5-aza-2’-deoxycytidine (5-aza-dC). NRF1 and PGC1α, transcription factors that activate nuclear-encoded mitochondrial proteins in response to oxidative stress, were observed to up-regulate expression of mtDNMT1. Loss of p53, a tumor suppressor gene known to help control mitochondrial metabolism, also results in a striking increase in mtDNMT1 expression, and this up-regulation of mtDNMT1 appears to modify mitochondrial transcription in a gene-specific fashion. Our data suggests roles for mtDNMT1 in both the establishment and maintenance of cytosine methylation (from which 5hmC is presumably derived) and in the regulation of mitochondrial transcription. We propose that the enzymes responsible for epigenetic modification of mtDNA have potential as therapeutic targets, with relevance to a broad spectrum of human disorders.