The precise control of two major proteolysis systems, the ubiquintin-proteasome system and the autophagy-lysosomal pathway, is important for normal cell function. With aging, the proteasome weakens, causing protein accumulation and the risk of neurodegeneration, while its alternative form, the immunoproteasome, becomes more active. Excessive immunoproteasome activity can eventually damage neurons and promote inflammation, which may contribute to the development of neurodegenerative conditions. However, it is known that cathepsins, which are part of the lysosomal pathway, also play an important role in the course of these conditions. Therefore, as part of the master's thesis, we checked the influence of mutual regulation of the proteasome and cathepsins in activated microglia cells. We started by establishing a cell model of polarized microglia using the BV2 cell line, in which we showed that stimulation with lipopolysaccharide (LPS) and interferon gamma (INF-γ) leads to the polarization of microglia towards the M1 phenotype. The polarization of microglia into a pro-inflammatory phenotype was further confirmed by measuring the amount of released nitric oxide. Next, we studied the influence of stimulation of BV2 cells on the intracellular activity of cathepsin X and S, and we found that both LPS and INF-γ cause a decrease in enzyme activity. At the same time, we also checked the influence of stimulation on the expression of the β5 and β1 subunits of the proteasome and the immunoproteasome. Simultaneous stimulation of BV2 cells with LPS and INF-γ did not significantly affect the expression of the β5 and β1 subunits of the constitutive proteasome, while there was a marked increase of its alternative form. In the last part of the master's thesis, we further evaluated the influence of the simultaneous inhibition of cathepsin X or S and β subunits of the (immuno)proteasome on the polarization of BV2 cells to the M1 phenotype. The most promising results were achieved with the simultaneous use of an irreversible cathepsin X inhibitor, AMS36, and a selective inhibitor of the β5i subunit of the immunoproteasome, KZR-504, as the latter prevented the polarization of microglia towards the M1 phenotype, which was evident from the reduced proportion of NO produced. Reduced levels of NO were also achieved by treating cells with the non-selective proteasome inhibitor, bortezomib, but the latter showed cytotoxicity for mircoglia cells. From the obtained results, we can conclude that inhibiting the activity of cathepsins, such as the immunoproteasome, represents a promising strategy for the treatment and prevention of neurodegenerative diseases associated with excessive inflammation.
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