Astrocytes are glial cells in the central nervous system and play a key role in homeostasis by protecting neurons from damage and inflammation. Chronic injury in central nervous system triggers the reactivation of astrocytes, leading to the emergence of an inflammatory phenotype that promotes inflammation and contributes to neurodegeneration. Cysteine cathepsins, including cathepsin X, have been recognized as important factors in neurodegeneration in glial cells. Cathepsin X is secreted from activated microglia, while its role in astrocytes remains unknown. In the Master's thesis, we evaluated the level of expression and activity of cathepsin X in C8-D1A astrocytes. First, we characterized the astrocyte cell line C8-D1A and the effect of the serum in the culture medium of astrocytes. We found that the latter proliferates more at a higher concentration of serum, but the proportion of dead cells increases. Furthermore, on the C8-D1A cells we showed that the higher concentration of serum in the culture medium reduces the activity of cathepsin X and also affects the intracellular lysosomal localization of cathepsin X. Then, astrocytes were reactivated with lipopolysaccharide or amyloid β in a serum free culture medium to stimulate inflammatory processes, and we found changes in the morphology of astrocytes and larger clusters of cells after exposure to amyloid β. Using immunofluorescence staining, we showed that the exposure to amyloid β increases the presence of the astrocyte reactivation marker GFAP. Amyloid β also causes increased cell death and the production of reactive oxygen species that cause oxidative stress in cells. We then evaluated the levels of expression and activity of cathepsin X in the reactivated astrocytes and, contrary to expectations, showed that the activity of cathepsin X in damaged cells decreases after exposure to amyloid β. Using immunofluorescence staining, we showed increased localization of cathepsin X in the lysosomes of reactive cells, subjected to inflammatory changes. Lastly, we observed the effect of cathepsin X inhibition on the reactivation of C8-D1A cells and the presence of oxidative stress, and we additionally evaluated the effect of the cathepsin X inhibitor, AMS36, on C8-D1A cells. We found that inhibition of cathepsin X partially reduces fibrillar amyloid β-stimulated toxicity of reactive astrocytes and its induced oxidative stress. The results indicate an important role of cathepsin X in astrocyte reactivation. By inhibiting cathepsin X, we reduced the toxicity of lipopolysaccharide and amyloid β, which promote neurodegeneration through reactivation. Cathepsin X thus represents a potential therapeutic target for the treatment of neurodegenerative diseases stimulated by inflammation, but its role in astrocytes needs to be further investigated.
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