Astrocytes are glial cells located in the central nervous system, where they play a crucial role in maintaining homeostasis and mediating immune responses. Under the influence of various inflammatory and pathological stimuli, astrocytes can become reactivated into either a pro-inflammatory A1 phenotype, which promotes inflammatory changes, or an anti-inflammatory A2 phenotype, which has protective effects in the brain. Some isoforms of the enzyme enolase, also expressed by astrocytes, exhibit neurotrophic properties important for preventing neuroinflammation and neurodegenerative alterations; however, the precise role of enolase in astrocytes has not yet been clarified. In this master’s thesis, we aimed to define the expression levels and determine the cellular localisation of enolase isoforms in reactivated astrocytes. We first established a cellular model of reactive astrocytes using C8-D1A astrocyte cell line, with the goal of demonstrating the effect of stimulation with lipopolysaccharide (LPS) and BV2 microglia-conditioned medium (MCM) on astrocyte reactivation into a pro-inflammatory phenotype. MCM proved to be more effective, as shown by morphological observations of C8-D1A cells and the evaluation of the effect of stimulation on cell survival, proliferation rate, and the release of inflammatory mediators in astrocyte cells. We then assessed the expression levels and intracellular localisation of α- and γ-enolase in reactivated C8-D1A cells. Exposure to MCM, but not to LPS, increased the secretion of enolase isoforms, particularly the γ isoform, while the intracellular levels of enolase did not significantly change, as shown by ELISA analysis. In the final part of the thesis, we examined the effects of the irreversible cathepsin X inhibitor AMS36 and synthetic α- and γ-enolase peptides on astrocyte reactivation. We showed that AMS36, by inhibiting cathepsin X following exposure to MCM, prevented the transition of astrocytes into the pro-inflammatory A1 phenotype, as evidenced by cell morphology and a decrease in caspase-3 activity, while the presence of the inhibitor was associated with an increased level of the active γ-enolase form. Similar results were obtained in experiments with synthetic peptides that mimic the C-terminal region of enolase. The obtained results indicate an important role of enolase isoforms and the regulation of the active form of γ-enolase by cathepsin X in the transition of astrocytes to the pro-inflammatory A1 phenotype, thus the development of compounds that would promote the neuroprotective activity of α- and γ-enolase would contribute to the therapy of inflammatory and neurodegenerative conditions of the central nervous system.
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