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Cisteinska katepsina S in X sta bila prepoznana kot pomembna encima pri številnih patoloških stanjih, med drugim tudi pri nevrodegenerativnih boleznih. Njuno spremenjeno izražanje, aktivnost in neželen vpliv na centralni živčni sistemu je bil raziskan že na različnih modelih nevrodegeneracije, kot je na primer model Parkinsonove ali Alzheimerjeve bolezni. Cilj naše magistrske naloge pa je bil ovrednotenje nivoja izražanja in aktivnosti katepsinov S in X na in vitro in in vivo vnetnem modelu nevrodegeneracije, ki oponašata procese pri multipli sklerozi. In vitro model smo postavili s pomočjo celične linije oligodendrocitov HOG. Najprej smo postavili celični model diferenciacije, ki je nujen proces za nastanek oligodendrocitov, sposobnih mielinizacije. Pri spremljanju morfoloških lastnosti celic, stimuliranih z brez-serumskim in brez-serumskim gojiščem z dodanim forbolnim estrom smo ugotovili, da celice tretirane s slednjim izražajo številne izrastke, hkrati pa je analiza proliferacije celic pokazala trend diferenciacije pri celicah stimuliranih z obema gojiščema. Opazili smo tudi povišan nivo izražanja mielinskega bazičnega proteina v diferenciranih celicah. Hkrati pa smo pokazali, da diferenciacija nima vpliva na aktivnosti katepsinov S in X. V nadaljevanju smo postavili in vitro model nevrodegeneracije z uporabo vnetnih citokinov interferona ^ (IFN-^) in tumor nekrotizirajoč faktorja ^ (TNF-^). Najprej smo s pretočno citometrijo potrdili njune citotoksične učinke na celicah HOG, nato pa izmerili aktivnosti katepsinov S in X v celičnih lizatih po stimulaciji celic. Dokazali smo, da IFN-^ vodi v statistično značilno znižano aktivnost katepsina S in X. Tretiranje s TNF-^ pa je pokazalo rezultate, ki kažejo trend, da se aktivnost katepsina S rahlo zviša, katepsina X pa zniža, vendar v manjši meri kot pri IFN-^. Povišan proteinski nivo katepsina X v supernatantih nakazuje tudi na njegovo izločanje iz celic v zunajcelični prostor. Rezultate smo dopolnili še z vrednotenjem in vivo živalskega modela eksperimentalnega avtoimunega encefalomielitisa. V homogenatih različnih regij možganov in hrbtenjače smo preverili nivo izražanja, aktivnost in lokalizacijo katepsinov S in X. Dokazali smo značilno povišano aktivnost katepsinov S in X v hrbtenjači živali na vrhuncu bolezni, medtem ko je bil proteinski nivo katepsinov najbolj povišan ob koncu bolezni. Z imunofluorescenčnim označevanjem pa smo še dodatno pokazali, da je izražanje katepsinov S in X v perifernem živcu povišano ob koncu bolezni, in sicer v vnetnih celicah, kar smo pokazali z uporabo označevalca arginaza-1. V magistrski nalogi smo potrdili, da sta proteinski nivo izražanja in aktivnost katepsinov S in X v nevrodegenerativnih procesih, posredovanih z vnetjem, povišana, kar označuje oba katepsina kot potencialni tarči za zdravljenje tovrstnih stanj. 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.

Oligodendrocyte precursor cell (OPC) migration is a mechanism involved in remyelination, these cells migrate from niches in the adult CNS. However, age and disease reduce the pool of OPCs, as a result, the remyelination capacity of the CNS decreases over time. Several experimental studies have introduced OPCs to the brain via direct injection or intrathecal administration. In this study, we used the nose-to brain pathway to deliver oligodendrocyte lineage cells (human oligodendroglioma (HOG) cells), which behave similarly to OPCs in vitro. To this end, we administered GFP-labelled HOG cells intranasally to experimental animals, which were subsequently euthanised at 30 or 60 days. Our results show that the intranasal route is a viable route to the CNS and that HOG cells administered intranasally migrate preferentially to niches of OPCs (clusters created during embryonic development and adult life). Our study provides evidence, albeit limited, that HOG cells either form clusters or adhere to clusters of OPCs in the brains of experimental animals.