Browsing by Author "Wurz, Jana"
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- ItemThe role of ketone bodies in autophagic flux, cellular energetics and injury-induced neurotoxicity(Stellenbosch : Stellenbosch University, 2016-03) Wurz, Jana; Loos, Benjamin; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Introduction. Alzheimer’s disease is the most common neurodegenerative disease and leading cause of dementia globally, with an increasing prevalence especially in developing countries such as South Africa. Currently, no single disease modifying treatment exists.. Alzheimer’s disease has a complex pathophysiology with the main causative factor involving defective proteolytic pathways, including the process of macroautophagy, and toxic amyloid beta aggregation. In addition, metabolic perturbations as well as disrupted mitochondrial dynamics are implicated. These disruptions culminate in neuronal cell death. Recently, ketone bodies have shown beneficial effects in the context of neurodegenerative diseases including Alzheimer’s disease, indicating improvements in both molecular disruptions as well as cognition. However, although literature implicates a role for autophagy in alleviating protein accumulation, the mechanism of action remains largely unclear. Aims. The aim of this project was therefore assess the effects of the ketone body beta-hydroxybutyrate in neuronal cells under basal conditions and in an injury model. We aimed to assess four parameters intimately linked to cellular survival in these two models: cellular viability, autophagic flux, mitochondrial network morphology and intracellular ATP supply. We hypothesized that ketone bodies will protect neurons from paraquat-induced neurotoxicity by increasing ATP levels through increasing autophagic flux. Methods. GT1-7 cells were cultured using DMEM supplemented with 10% FBS under standard conditions (5% CO2, 37 °C). To assess the role of ketone bodies under basal conditions, Western blot analysis of amyloid precursor protein (APP), amyloid beta (Aβ), beta site APP cleaving enzyme (BACE), p62 and LC3-II proteins was performed. Fluorescence microscopy was performed utilizing fluorochromes targeting APP, Aβ and BACE. Quantitative assessment of neuronal ATP was completed using a luciferase-based assay. Qualitative assessment of neuronal ATP distribution was performed by transfecting cells with a FRET-based ATP indicator, ATeam, and by capturing images with fluorescence microscopy. For all the experiments in the first model, treatment with the autophagosomal/lysosomal fusion inhibitor, bafilomycin A1, was included to assess autophagic flux. To assess the role of ketone bodies in response to injury, cells were exposed to the herbicide paraquat and Western blot analysis of LC3 and cleaved PARP, an apoptotic protein, was performed. A WST-1 reductive capacity assay was completed and mitochondrial morphology assessed by means of the mitochondrial polarization-dependent fluorochrome, tetramethylrhodamine ethyl ester (TMRE), capturing and analysing images with ImageJ software. In addition transmission electron microscopy was performed to indicate neuronal ultrastructure, ATP quantification and qualitative assessment as well as flow cytometry to indicate reactive oxygen species (ROS) by using both dichlorofluorescein (DCF) to indicate general ROS and TMRE to assess mitochondrial polarization. Results. Under basal conditions, it was observed that the ATP distribution within cells changed to large areas of detected signal when treating cells with ketone bodies and bafilomycin. There were no significant differences in detected ATP levels between treatment groups. Western blot results revealed that bafilomycin treatment resulted in a strong trend towards increased protein levels of BACE and APP, and a significant decrease in Aβ levels. Decreased p62 protein expression was observed upon ketone body treatment as well as a strong trend for decreased LC3-II protein levels upon bafilomycin treatment. Fluorescence microscopy revealed that bafilomycin treatment caused accumulation of APP, BACE and Aβ, and increased nuclear signal of BACE and APP. For the injury-induced model the WST-1 assay results reveal that paraquat caused a significant decrease in reductive capacity, which ketone body supplementation rescued by significantly increasing reductive capacity. Western blot analysis revealed a strong trend for decreased LC3-II protein expression upon bafilomycin treatment. No differences between groups were observed for cleaved PARP. Mitochondrial morphological assessment indicated a highly fused network in the control and ketone body group, and a highly fragmented state upon paraquat treatment, which was improved to a more fused state upon ketone body co-treatment. No significant differences were observed in the flow cytometry data, however a similar trend for increased fluorescence intensity upon ketone body and paraquat co-treatment was present in cells stained with DCF and TMRE. ATP concentration and distribution was severely affected by paraquat treatment, which decreased detected ATP levels and signal. Ketone body treatment caused a change in ATP distribution apparent as large ‘hotspots’, however, did not significantly increase ATP concentration in the co-treatment group. Transmission electron microscopy indicated intact, elongated mitochondria in the control and ketone body groups, as well as well-defined vacuolar structures. They were increased upon ketone body supplementation. Paraquat exposure caused mitochondrial disruption as indicated by swollen mitochondria with decreased integrity as well as less well defined vacuolar structures. Fewer vacuolar structures were observed in the co-treatment group. Discussion and conclusion. Our results suggest that ketone body exposure increases autophagic flux and decreases the presence of amyloid-associated proteins. In addition, ketone bodies confer protection from neurotoxicity and improve mitochondrial network connectivity. Our results further indicate a role for ketone bodies in localized ATP supply. Taken together, ketone body exposure may hold great potential as an adjuvant therapy in the context of neurodegeneration.