Neurodegenerative diseases represent a major public health issue with no effective treatment methods. They are characterized by the gradual degeneration of neurons, in which neuroinflammation with microglial activation play a significant role. In response to stimuli, microglial cells polarize into either the pro-inflammatory M1 phenotype, which promotes the secretion of inflammatory factors, or the neuroprotective M2 phenotype, which increases the secretion of neurotrophic factors. The latter's similar function is also exhibited by γ-enolase—a glycolytic enzyme that exists in several isoforms, with the γ-isoform promoting neuronal survival and neurite growth. As part of this master's thesis, we aimed to evaluate the expression of enolase isoforms in individual microglial phenotypes. First, we established a cellular model of polarized microglia using the BV2 cell line. Polarization into the M1 phenotype was induced with lipopolysaccharide (LPS) or interferon-gamma (IFN-γ), and into the M2 phenotype with interleukin-4 (IL-4) or interleukin-13 (IL-13). Polarization was confirmed by the expression of characteristic phenotypic markers. We next determined the enolase glycolytic activity of polarized microglia and found that it decreased in cells stimulated with IFN-γ. We then evaluated the expression levels of enolase isoforms in polarized microglial cells using western blot and enzyme-linked immunosorbent assay. The results were inconsistent but suggested that enolase isoform expression is regulated depending on the polarization of microglia into specific phenotypes. We also studied the level of the active form of γ-enolase and its secretion from stimulated BV2 cells. The level of active γ-enolase synthesis increased in BV2 cells polarized to the M1 phenotype, while its secretion is also elevated in this phenotype. Finally, we examined the regulation of the active form of γ-enolase during microglial polarization using a cathepsin X inhibitor. A decrease in the amount of secreted nitric oxide confirmed that the inhibitor impairs polarization into the M1 phenotype. At the same time, we demonstrated that the inhibitor increases the level of secreted active γ-enolase from M1 polarized microglia. The obtained results suggest that cathepsin X inhibitors could have potential therapeutic value in preventing the progression of inflammation-associated neurodegenerative diseases, as they inhibit polarization into the pro-inflammatory microglial phenotype and simultaneously increase synthesis levels of the active form of γ-enolase, which promotes neuronal survival.
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