Nivo izražanja in aktivnosti katepsina X v procesu diferenciacije nevroblastomske celične linije

Kladnik, Jana (author), Pišlar, Anja (mentor)

Katepsin X je cisteinska peptidaza, ki s proteolitično cepitvijo C-končnega dipeptida glikolitičnega encima γ-enolaze onemogoča njeno nevrotrofično delovanje. S tem se posredno vključuje v mehanizme diferenciacije nevronskih celic. V sklopu magistrske naloge smo z eksperimentalnim delom na celicah SH-SY5Y ugotavljali nivo izražanja in aktivnosti katepsina X v procesu diferenciacije nevroblastomske celične linije ter opredelili vlogo katepsina X v procesu diferenciacije. V prvem delu magistrske naloge smo postavili model diferenciacije celic SH-SY5Y, pri čemer smo s pogoji diferenciacije želeli spodbuditi nastanek posameznega nevronskega fenotipa. Najprej smo pokazali, da stimulacija celic SH-SY5Y z retinojsko kislino (RA) v koncentraciji 10 in 20 µM poveča izraščanje nevritov, ki so značilni za diferencirane celice. Ravno tako je izraščanje nevritov povečano pri stimulaciji celic z RA v kombinaciji s forbol 12-miristat 13-acetatom (PMA) oz. nevrotrofičnim dejavnikom možganskega izvora (BDNF) z namenom diferenciacije v posamezen nevronski fenotip, in sicer v dopaminergični oz. holinergični fenotip. Pokazali smo, da RA samostojno oz. v kombinaciji z BDNF spodbudi polimerizacijo F-aktina, kar vpliva na reorganizacijo aktinskega citoskeleta, ki je nujna za diferenciacijo, medtem ko dodatek PMA zavira z RA spodbujeno polimerizacijo. Z uporabo specifičnih nevronskih označevalcev smo delno potrdili nastanek posameznega nevronskega fenotipa. V nadaljevanju smo v diferenciranih celicah pokazali, da je aktivnost katepsina X povečana v celicah, tretiranih z RA samostojno kot tudi z RA v kombinaciji s PMA ali BDNF. Ravno tako je v celicah, diferenciranih v smeri dopaminergičnega in holinergičnega fenotipa, povečan proteinski nivo izražanja katepsina X. Skladno z nivojem izražanja je v celicah diferenciranih z RA v kombinaciji s PMA oz. BDNF povečana intenziteta fluorescence katepsina X, določena z uporabo konfokalnega mikroskopa. Opazna je lokalizacija katepsina X v izrastkih, ki nastanejo tekom diferenciacije celic SH-SY5Y, obenem pa je vidna ko-lokalizacija z γ-enolazo v rastnih conah nevritov. V zadnjem sklopu naloge smo vpletenost katepsina X v diferenciacijo potrdili z določanjem vpliva zaviranja delovanja katepsina X na proces diferenciacije. Uporabili smo ireverzibilni zaviralec katepsina X AMS36. Zaradi vidnih sprememb v morfologiji celic in povečanja izraščanja nevritov v prisotnosti zaviralca lahko sklepamo, da z zaviranjem delovanja katepsina X spodbujamo nadaljnjo diferenciacijo, kar nakazuje na pomembnost proteolitičnega uravnavanja γ-enolaze v procesu diferenciacije nevronov.

• Language: Slovenian
• Keywords: Diferenciacija, celice SH-SY5Y, rast nevritov, katepsin X, zaviralec katepsina X
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-144263
• Publication date in RUL: 08.02.2023

Secondary language

• Language: English
• Title: Protein level and activity of cathepsin X in the process of neuroblastoma cell differentiation

Abstract

Cathepsin X is a cysteine peptidase that by proteolytic cleavage of the C-terminal dipeptide of the glycolytic enzyme γ-enolase, abolishes its neurotrophic activity. In this way, it is indirectly involved in the mechanisms of neuronal cell differentiation. As part of the Master's thesis, we determined protein level and activity of cathepsin X in the process of neuroblastoma cell differentiation, and defined the role of cathepsin X in the differentiation process. In the first part of the Master's thesis, we set up a model of differentiation of SH-SY5Y cells, whereby we wanted to stimulate the formation of a specific neuronal phenotype with the differentiation conditions. First, we showed that stimulation of SH-SY5Y cells with retinoic acid (RA) at a concentration of 10 and 20 µM increases neurite outgrowth, which is characteristic of differentiated cells. Neurite outgrowth is also increased when cells are stimulated with RA in combination with phorbol 12-myristate 13-acetate (PMA) or brain-derived neurotrophic factor (BDNF), which induce differentiating into a specific neuronal phenotype, dopaminergic or cholinergic, respectively. We showed that RA independently or in combination with BDNF promotes F-actin polymerization, which affects actin cytoskeleton reorganization, a process that is crucial for neuronal differentiation, while addition of PMA inhibits RA-stimulated polymerization. With the use of specific neuronal markers, we partially confirmed the formation of an individual neuronal phenotype. In the following, we showed that the activity of cathepsin X is increased in differentiated cells treated with RA alone, as well as with RA in combination with PMA or BDNF. Likewise, the protein level of cathepsin X is increased in cells differentiated towards to dopaminergic and cholinergic phenotypes. In accordance with the protein level, in cells differentiated with RA in combination with PMA or BDNF increased cathepsin X fluorescence intensity was also observed. Localization of cathepsin X was noticeable in the growth cones and extensions that are formed during the differentiation of SH-SY5Y cells, as well as co-localization of cathepsin X with γ-enolase in the growth zones of neurites in differentiated cells was determined. In the last part, we confirmed the involvement of cathepsin X in differentiation by determining the effect of cathepsin X inhibition on the differentiation process. We used the irreversible cathepsin X inhibitor AMS36. Due to the visible changes in cell morphology and the increase in neurite outgrowth in the presence of the inhibitor, we can conclude that inhibition of cathepsin X promotes further differentiation, which indicates the importance of proteolytic regulation of γ-enolase in the differentiation process of neuronal cells.

• Keywords: Differentiation, SH-SY5Y cells, neurite outgrowth, cathepsin X, cathepsin X inhibitor