Browsing by Subject "mitophagy"
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Item Open Access GAT inhibition preserves cerebral blood flow and reduces oxidant damage to mitochondria in rodents exposed to extreme hyperbaric oxygen.(Frontiers in molecular neuroscience, 2022-01) Demchenko, Ivan T; Suliman, Hagir B; Zhilyaey, Sergey Y; Alekseeva, Olga S; Platonova, Tatyana F; Makowski, Matthew S; Piantadosi, Claude A; Gasier, Heath GOxygen breathing at elevated partial pressures (PO2's) at or more than 3 atmospheres absolute (ATA) causes a reduction in brain γ-aminobutyric acid (GABA) levels that impacts the development of central nervous system oxygen toxicity (CNS-OT). Drugs that increase brain GABA content delay the onset of CNS-OT, but it is unknown if oxidant damage is lessened because brain tissue PO2 remains elevated during hyperbaric oxygen (HBO2) exposures. Experiments were performed in rats and mice to measure brain GABA levels with or without GABA transporter inhibitors (GATs) and its influence on cerebral blood flow, oxidant damage, and aspects of mitochondrial quality control signaling (mitophagy and biogenesis). In rats pretreated with tiagabine (GAT1 inhibitor), the tachycardia, secondary rise in mean arterial blood pressure, and cerebral hyperemia were prevented during HBO2 at 5 and 6 ATA. Tiagabine and the nonselective GAT inhibitor nipecotic acid similarly extended HBO2 seizure latencies. In mice pretreated with tiagabine and exposed to HBO2 at 5 ATA, nuclear and mitochondrial DNA oxidation and astrocytosis was attenuated in the cerebellum and hippocampus. Less oxidant injury in these regions was accompanied by reduced conjugated microtubule-associated protein 1A/1B-light chain 3 (LC3-II), an index of mitophagy, and phosphorylated cAMP response element binding protein (pCREB), an initiator of mitochondrial biogenesis. We conclude that GABA prevents cerebral hyperemia and delays neuroexcitation under extreme HBO2, limiting oxidant damage in the cerebellum and hippocampus, and likely lowering mitophagy flux and initiation of pCREB-initiated mitochondrial biogenesis.Item Open Access Inhibition of Sphingosine Kinase 2 Results in PARK2-Mediated Mitophagy and Induces Apoptosis in Multiple Myeloma.(Current oncology (Toronto, Ont.), 2023-03) Wu, Jian; Fan, Shengjun; Feinberg, Daniel; Wang, Xiaobei; Jabbar, Shaima; Kang, YubinMitophagy plays an important role in maintaining mitochondrial homeostasis by clearing damaged mitochondria. Sphingosine kinase 2 (SK2), a type of sphingosine kinase, is an important metabolic enzyme involved in generating sphingosine-1-phosphate. Its expression level is elevated in many cancers and is associated with poor clinical outcomes. However, the relationship between SK2 and mitochondrial dysfunction remains unclear. We found that the genetic downregulation of SK2 or treatment with ABC294640, a specific inhibitor of SK2, induced mitophagy and apoptosis in multiple myeloma cell lines. We showed that mitophagy correlates with apoptosis induction and likely occurs through the SET/PP2AC/PARK2 pathway, where inhibiting PP2AC activity may rescue this process. Furthermore, we found that PP2AC and PARK2 form a complex, suggesting that they might regulate mitophagy through protein-protein interactions. Our study demonstrates the important role of SK2 in regulating mitophagy and provides new insights into the mechanism of mitophagy in multiple myeloma.Item Open Access Role of Mitochondrial Dynamics and Autophagy in Removal of Helix-Distorting Mitochondrial DNA Damage(2012) Bess, Amanda SmithMitochondria are the primary energy producers of the cell and play key roles in cellular signaling, apoptosis and reactive oxygen species (ROS) production. Mitochondria are the only organelles that contain their own genome which encodes for a small subset of electron transport chain (ETC) proteins as well as the necessary tRNAs and ribosomal subunits to translate these proteins. Over 300 pathogenic mitochondrial DNA (mtDNA) mutations have been shown to cause a number of mitochondrial diseases emphasizing the importance of mtDNA maintenance and integrity to human health. Additionally, mitochondrial dysfunction and mtDNA instability are linked to many wide-spread diseases associated with aging including cancer and neurodegeneration. Mitochondria lack the ability to repair certain helix-distorting lesions that are induced at high levels in mtDNA by important environmental genotoxins including polycyclic aromatic hydrocarbons, ultraviolet C radiation (UVC) and mycotoxins. These lesions are irreparable and persistent in the short term, but their long-term fate is unknown. Degradation of mitochondria and mtDNA is carried out by autophagy. Autophagy is protective against cell stress and apoptosis resulting from exposure to mitochondrial toxicants suggesting that it plays an important role in removal of unstable mitochondria that can serve as a source of ROS or initiate apoptotic cell death. Furthermore, dysfunctional mitochondria can be specifically targeted for degradation by the more specific process of mitophagy influenced in part by the processes of mitochondrial dynamics (i.e., fusion and fission).
The goals of this dissertation were to investigate the long-term fate of helix-distorting mtDNA damage and determine the significance of autophagy and mitochondrial dynamics in removal of and recovery from persistent mtDNA damage. Removal of irreparable mtDNA damage and the necessity of autophagy, mitophagy, fusion and fission genes in removal of this damage were examined using genetic approaches in adult Caenorhabditis elegans. In order to investigate the significance of autophagy, fusion and fission genes in recovery from mtDNA damage-induced mitochondrial dysfunction in vivo, an experimental method was developed to specifically induce persistent mtDNA damage and mitochondrial dysfunction without persistent nDNA damage in developing C. elegans. Additionally, the effect of persistent helix-distorting DNA damage on mitochondrial morphology, mitochondrial function and autophagy was investigated in C. elegans and in mammalian cell culture. The rate and specificity of mitochondrial degradation was further examined in cell culture using live-cell fluorescence microscopy and transmission electron microscopy.
Removal of UVC-induced mtDNA damage was detectable by 72 hours in C. elegans and mammalian cell culture, and required mitochondrial fusion, fission and autophagy, providing genetic evidence for a novel mtDNA damage removal pathway. UVC exposure induced autophagy with no detectable effect on mitochondrial morphology in both systems; mitochondrial function was inhibited in the C. elegans system but not in the cell culture system in which the degree of mtDNA damage induced was less. Furthermore, mutations in genes involved in these processes as well as pharmacological inhibition of autophagy exacerbated mtDNA damage-mediated larval arrest, illustrating the in vivo relevance of removal of persistent mtDNA damage. Mutations in genes in these pathways exist in the human population, demonstrating the potential for important gene-environment interactions affecting mitochondrial health after genotoxin exposure.
Item Open Access Toxicant-Induced Mitochondrial Dysfunction and Dopaminergic Neurodegeneration in Caenorhabditis elegans(2017) GonzalezHunt, Claudia PatriciaMitochondrial dysfunction appears to be a hallmark of many neurodegenerative diseases. The link between mitochondrial and neuronal health is particularly strong in Parkinson’s disease (PD). Genes that have been shown to cause PD when mutated are known to interact with mitochondria to varying degrees; particularly, two of the genes associated with mitochondria-specific autophagy (mitophagy), PINK1 and PARK2, have been shown to cause autosomal recessive PD. Deletions and mutations in mitochondrial DNA (mtDNA) have been detected in the substantia nigra of PD patients. Furthermore, toxicants used to model PD in the laboratory are known to exert their toxicity by disrupting mitochondrial function. Despite all the advances in PD research, the precise mechanisms through which this disease arises are still not well understood.
The central goal of this dissertation was to investigate the role that mtDNA damage and mitochondrial dysfunction play in causing dopaminergic neurodegeneration in the context of PD. This work was performed with a particular focus on mitochondrial toxicants: their mechanism of action and effect on neuronal health, and how these toxic exposures might exacerbate genetic susceptibilities. Another goal was to identify windows of susceptibility to mtDNA damage, with early life and old age being of particular interest. To explore these ideas, the nematode Caenorhabditis elegans was utilized as a model organism.
To characterize age-related sensitivity to mtDNA damage, worms at different life stages were exposed to ultraviolet C (UVC) to accumulate mtDNA damage and the effect of this damage on mitochondrial genome copy number and dopaminergic neuron health was assessed. To evaluate the role of autophagy in maintaining mitochondrial and neuronal health after toxicant exposure, mitophagy gene knockouts (KOs) and wild type worms were exposed to 6-hydroxydopamine (6-OHDA) and UVC; to study non-selective macroautophagy, starvation was used to induce it, and nematodes were subsequently subjected to a toxic exposure. Dopaminergic neuron morphology, targeted gene expression, and mitochondrial morphology were evaluated. Lastly, the mechanism of toxicity of rotenone in C. elegans was investigated; specifically, mitochondrial respiration (with the Seahorse Bioanalyzer), targeted metabolomics, ATP levels, and targeted gene expression were evaluated.
Due to technical complications with our UVC exposure model and PCR-based evaluation of mtDNA damage in C. elegans eggs, the windows of susceptibility to mtDNA damage hypothesis could not be properly tested. However, our current results do not support this idea. Our studies on the role of autophagy in the context of toxicant-induced dopaminergic neurodegeneration suggest that both non-selective macroautophagy and mitophagy have a protective effect. We saw protection from 6-OHDA-induced neurodegeneration after starvation treatment, suggesting that macroautophagy could be playing a protective role. In the context of mitophagy mutations, we saw different responses to UVC and 6-OHDA. Both pink-1 and pdr-1 mutants were more sensitive to UVC-induced dopaminergic neurodegeneration than the control strain; however, only the pdr-1 strain was more sensitive than the control strain to 6-OHDA. The pink-1 mutant was actually protected from neurodegeneration caused by 6-OHDA. We proceeded to evaluate antioxidant responses in these strains (since 6-OHDA exerts its toxicity mainly via oxidative stress) but saw no differences across strains. We are currently testing the hypothesis that changes in the mitochondrial network in the pink-1 mutant after 6-OHDA exposure allow it to better withstand its toxic effect. Our studies into the mechanism of action of rotenone in C. elegans gave us surprising results: we saw no major alterations in oxygen consumption or ATP levels. However, various metabolites were altered, signaling a metabolic restructuring similar to that observed in C. elegans complex I mutants. Also similar to complex I mutants, it appears that the glyoxylate pathway is upregulated after rotenone exposure; this alternate pathway parallel to the citric acid cycle might be allowing the nematodes to acclimate to rotenone exposure, explaining our respiration and ATP results.
Overall, this dissertation work supports the link between mitochondrial dysfunction and dopaminergic neurodegeneration, and provides new hypotheses for research in this field.