Cysteine cathepsins S and X have been recognised as important enzymes in different pathological disorders, including neurodegenerative diseases. Their protein expression, activity and undesired role in the central nervous system has already been studied in different neurodegenerative models, such as Parkinson’s and Alzheimer’s diseases. The goal of our master thesis was to evaluate protein expression and activity of cathepsins S and X in in vitro and in vivo model of neurodegeneration, that mimic pathological features of multiple sclerosis. To design in vitro model, we used HOG cell line. Firstly, we set up a cell differentiation model. Differentiation is a key process to form mature myelinating oligodendrocytes. Observing morphological characteristics of cells stimulated with serum-free medium and serum-free medium with phorbol ester led us to a finding that cells treated with the latter show many neuritogenic processes. In cell proliferation analysis we observed a trend of differentiation in both media. We also showed higher myeline basic protein expression in differentiated cells. On the other hand, we proved that differentiation does not impact on cathepsins S in X activities in cells. Furthermore, we developed in vitro neurodegenerative model with inflammatory cytokines interferon ^ (IFN-^) and tumour necrosis factor ^ (TNF-^). First, we proved their cytotoxic effect on HOG cells with flow cytometry and then we measured cathepsins S and X activities in cell lysates after stimulation with IFN-^ or TNF-^. We proved statistically significant lower cathepsins S and X activities after treatment with IFN-^. TNF-^ treatment showed a trend of higher cathepsin S activity and lower cathepsin X activity, compared to control, which was not so prominent as after IFN-^ treatment. Higher protein expression of cathepsin X in supernatants also indicates on enzyme secretion in extracellular space. We complimented these results with evaluation of in vivo inflammatory animal model of experimental autoimmune encephalomyelitis. We analysed protein expression, activity and localization of cathepsins S and X in homogenates of different brain regions and spinal cord regions. We proved higher cathepsins S and X activities in spinal cord of animals in peak disease. However, the protein expression was the highest at the end of the disease. Immunofluorescent staining also confirmed higher cathepsins S and X expressions in inflammatory cells marked with arginase-1 in peripheral nerve at the end of disease. To conclude, in our master thesis we have confirmed higher cathepsins S and X protein expressions and activities in neurodegenerative processes mediated with inflammation. This finding designates cathepsin S and X as potential targets for treatment of this kind of disorders.
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