The role of ketone bodies in autophagic flux, cellular energetics and injury-induced neurotoxicity

Wurz, Jana (2016-03)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

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.

AFRIKAANSE OPSOMMING: Inleiding. Alzheimersiekte is die mees algemene neurodegeneratiewe siekte en wêreldwyd toemend die vernaamste oorsaak van demensie. Ontwikkelende lande soos Suid-Afrika sal veral beïnvloed word. Huidiglik bestaan daar geen enkele behandeling nie. Die patologiese aspekte van die fisiologie van Alzheimersiekte is kompleks, maar die vernaamse oorsaaklike faktor behels foutiewe proteolitiese weë, insluitend makroautofagie, en toksiese amyloid beta opeenhoping. Daarbenewens word metaboliese versteurings sowel as ‘n ontwrigte mitochondriese dinamiek geïmpliseer. Hierdie versteurings veroorsaak uiteindelik die dood van neurone. Dit is onlangs bewys dat ketoonliggame voordelige gevolge het in die konteks van neurodegeneratiewe siektes, insluitend Alzheimersiekte waar verbeterings in beide molekulêre versteurings sowel as kognisie waargeneem is. Nieteenstaande die rol van autofagie om proteïn opeenhoping te verlig rapporteer in die literatuur, is die meganisme van aksie steeds onduidelik. Doel. Die doel van hierdie projek was dus om die uitwerking van die ketoonliggaam beta-hidroksie butiraat te bepaal onder basis toestande en in ‘n beseringsmodel. Ons doel was om vier bepalers wat nou gekoppel is met sellulêre oorlewing in die twee modelle te assesseer: seloorlewing, autofagiese stroming, mitochondriese netwerk morfologie en intrasellulêre ATP verskaffing. Ons hipotese was dat ketoonliggame neurone sal beskerm van paraquat-induseerde neurotoksisiteit, deur ATP vlakke te vermeerder deur die toename van autofagiese stroming. Metodes. GT1-7 selle was deur middel van selkultuur geweek met behulp van DMEM verryk met 10% FBS onder standaard toestande (5% CO2, 37 °C). Western blot analise van APP, Aβ, BACE, p62 en LC3-II proteïnvlakke was onderneem om die effek van ketoonliggame onder basis toestande te bepaal. Fluoressensie-gebaseerde mikroskopie was onderneem deur fluorochrome te gebruik gerig aan APP, Aβ en BACE. Kwantitatiewe evaluering van neuronale ATP was voltooi met behulp van ‘n lusiferaas-baseerde toets. Kwalitaties evaluering van neuronale ATP verspreiding was uitgevoer deur selle met ‘n FRET-gebaseerde ATP wyser, ATeam, te transfekteer en af te beeld met fluoresserende mikroskopie. Bafilomycin, ‘n inhibeerder van autofagosomale/lisosomale samesmelting, was gebruik om selle te behandel in alle eksperimente van die eerste model om die autofagiese stroming te assesseer. Selle is blootgestel aan paraquat, ‘n onkruiddoder, om die effek van ketoonliggame te bepaal in reaksie tot skade, en dan is Western blot analise van LC3 en gekliefde PARP, ‘n apoptotiese proteïen, onderneem. ‘n WST-1 toets, wat reduktiewe kapasitieit aandui, is voltooi en mitochondriese morfologie is geassesseer deur die mitochondriese polarisasie-afhanklike fluorokroom, tetrametielrodamien etielester (TMRE) te gebruik, beelde te neem en te analiseer met behulp van ImageJ sagteware. Verder was transmissie elektron mikroskopie onderneem om neuronale ultrastruktuur aan te dui, sowel as ATP kwantifikase en kwalitatiewe assessering en vloeisitometrie om reaktiewe suurstof spesies (ROS) aan te dui deur gebruik van beide dichlorofluoresien (DCF) vir algemene ROS en TMRE as aanduiding van mitochondriese polarisasie. Resultate. Onder basis toestande is daar ‘n verskil in ATP verspreiding waargeneem, met ketoonliggaam en bafilomycin behandeling wat die ATP verspreiding tot groot areas binne selle verander het. Geen beduidende verskille in the ATP vlakke van die behandelings groepe was teenwoordig nie. Die Western blot metode het aangetoon dat bafilomycin behandeling ‘n sterk neiging tot toegenome proteïen vlakke van BACE en APP tot gevolg het, asook ‘n beduidende afname in Aβ vlakke. Ketoonliggaam behandeling het ‘n sterk tendens tot die afname in p62 proteïn uitdrukking tot gevolg gehad, en bafilomycin behandeling het verder ‘n sterk tendens tot die afname van LC3-II proteïnvlakke veroorsaak. Fluoressensie mikroskopie het uitgewys dat bafilomycin behandeling die opeenhoping van APP, BACE and Aβ veroorsaak en die selkern sein van BACE en APP verhoog. Die WST-1 toets resultate het aangedui data paraquat ‘n beduidende afname in reduktiewe vermoë tot gevolg het, wat ketoonliggaam aanvullings beredder het deur die reduktiewe kapasiteit weer beduidend te herstel. Western blot analise het ‘n sterk tendens vir die afname in LC3-II proteïen uitdrukking in reaksie op bafilomycin behandeling aangetoon. Daar is geen verskille tussen groepe vir gekliefde PARP waargeneem nie. Mitochondriale morfologiese assessering het ‘n hoogs geïntegreerde netwerk vir die kontrole en ketoonliggaam groep uitgewys, en ook dat ‘n hoogs gefragmenteerde toestand voorkom in reaksie tot paraquat behandeling, wat tot ‘n meer geïntegreerde toestand verbeter het met ketoonliggaam mede-behandeling. Geen beduidende verskille is waargneem in die vloeisitometrie data nie, alhoewel dieselfde tendens vir hoë fluoressensie intensiteit in reaksie op gesamentlike ketoonliggaam en paraquat behandeling in selle wat gekleur is met DCF en TMRE aangewys is. Paraquat behandeling het ATP konsentrasie en verspreiding beduidend beïnvloed, asook die ATP vlakke en sein verminder. Ketoonliggaam behandeling het ‘n verandering in ATP verspreiding tot gevolg gehad wat voorkom het as groot ‘hotspots’. Transmissie elektron mikroskopie het aangedui dat verlengde mitochondria, en goed gedefinieerde vakuoolstrukture in die kontrole en ketoonliggaam groepe voorgekom het. Hierdie strukture het vermeerder nadat ketoonliggame toegedien is. Paraquat blootstelling het mitochondriale disrupsie tot gevolg gehad, soos aangedui is deur geswelde mitochondria met afgenome integriteit en swakker gedefinieerde vakuool strukture. Minder vakuool strukture is waargeneem in die gesamentlike behandeling groep. Bespreking en gevolgtrekking. Die resultate verskaf ‘n aanduiding dat ketoonliggame ‘n toename in autofagiese stroming veroorsaak en ‘n afname in die voorkoms van spesifieke amyloid-geassosieerde proteïene. Verder verleen ketoonliggame ook beskerming teen neurotoksisiteit en verbeter mitochondriale netwerk verbindingsstatus. Ons dui verder daarop dat ketoonliggame ‘n rol speel in plaaslike ATP beskikbaarheid. Om saam te vat; ketoonliggame het beduidende potensiaal as ‘n toegevoegde terapie in die konteks van neurodegenerasie.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/98841
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