| Abstract [eng] |
Over the last few years, various studies have expanded our knowledge of the molecular mechanisms underlying the pathogenesis of neurodegenerative diseases and have revealed the importance of microglial cells in the development of these diseases. A growing body of evidence suggests that changes in microglial metabolism in response to pathological factors are crucial in regulating immune and inflammatory responses in the brain. Nevertheless, the mechanisms of neurotoxicity of amyloid protein aggregates and endotoxin lipopolysaccharide linked to Parkinson's disease remain to be elucidated. It is still unknown what kind of metabolic changes in microglial cells are induced by these factors and how this contributes to the regulation of their functions and the development of proinflammatory phenotype during neurodegenerative processes. This final degree project aimed to investigate the effects of the amyloid proteins α-synuclein and S100A9 on the microglial energy metabolism and to compare these results with the effect of bacterial endotoxin. To achieve this aim, the following objectives have been set: 1) to assess the respiration of intact BV-2 cells in response to α-synuclein, S100A9 and bacterial endotoxin; 2) to assess the respiration of permeabilized BV-2 cells in response to α-synuclein, S100A9 and bacterial endotoxin; 3) to assess glycolytic activity in response to α-synuclein, S100A9 and bacterial endotoxin; 4) to determine the production of various proinflammatory factors in cell growth media in response to α-synuclein, S100A9 and bacterial endotoxin. The object of investigation was immortalized mouse microglial BV-2 cells. BV-2 cells were exposed to 100 nM and 500 nM α-synuclein and S100A9 oligomers and 100 ng/ml, 500 ng/ml and 1 μg/ml concentrations of lipopolysaccharide for 24 h. Changes in microglial energy metabolism were assessed by measuring respiration rates and glycolytic activity using a high-resolution respirometer Oroboros O2k and O2k-pH ISE-Module. Production of extracellular nitric oxide was measured spectrophotometrically using the Griess Reagent Kit. Quantitative ELISA analysis was performed to determine the production of proinflammatory cytokine TNF-α in cell growth media. The generation of reactive oxygen species was evaluated by measuring efflux of H2O2 into the extracellular media using a fluorometric assay. Statistical analysis was performed using SigmaPlot 14.0 software. We have found that α-synuclein, S100A9 and lipopolysaccharide do not affect the basal cellular respiration rate of the intact BV-2 cells and have no impact on the leak state of respiration in permeabilized cells after 24 h of incubation period with BV-2 cells. However, 24 h incubation of BV-2 cells with 100 nM and 500 nM α-synuclein inhibits oxidative phosphorylation, oxidizing respiratory chain complex I-linked substrates pyruvate and malate. For the first time, we have demonstrated that 100 nM and 500 nM S100A9 suppress oxidative phosphorylation through decreased oxidation of the mitochondrial complex I- and complex II-linked substrates pyruvate and malate, and succinate after 24 h incubation with BV-2 cells. Moreover, S100A9 lowers the overall efficiency of mitochondrial respiratory chain complexes when oxidative phosphorylation is uncoupled. A similar effect was observed when BV-2 cells were exposed to 500 ng/ml lipopolysaccharide; however, incubation with 100 ng/ml and 1 μg/ml lipopolysaccharide for 24 h exerted no significant impact on the respiration of microglial cells across various metabolic states. We have also found that the glycolysis was markedly enhanced in microglial cells when exposed to 500 nM α-synuclein, 500 nM S100A9, as well as to 500 ng/ml and 1 μg/ml lipopolysaccharide for 24 h. However, 100 nM α-synuclein and 100 nM S100A9 oligomers, as well as 100 ng/ml lipopolysaccharide, did not appear to affect the glycolytic activity of BV-2 cells after 24 h incubation. Besides, out of all pathological factors investigated, only 500 nM α-synuclein increased the maximal glycolytic activity of BV-2 cells. We have determined that incubation of BV-2 cells with 500 nM S100A9 and 1 μg/ml lipopolysaccharide considerably increased TNF-α secretion and its concentration in cell growth media; however, 500 nM α-synuclein did not produce this kind of effect after 24 h incubation with BV-2 cells. Additionally, the obtained results revealed that 24 h incubation with 500 ng/ml and 1 μg/ml concentrations of lipopolysaccharide dramatically increased extracellular H2O2 production, whereas α-synuclein, S100A9 and 100 ng/ml lipopolysaccharide did not alter the concentration of this reactive oxygen compound in cell growth media. Moreover, 24 h incubation with α-synuclein, S100A9 and lipopolysaccharide had no impact on extracellular NO production. Overall, our results suggest that α-synuclein and S100A9 oligomers, as well as lipopolysaccharide, may contribute to neurodegenerative processes by triggering energy metabolism changes in microglial cells and inducing distinct inflammatory responses, depending on the pathological factor itself and its concentration. |
