Ovrednotenje fosforilacije RNA-vezavnih proteinov v nevrosferoidih

Matić, Iva (author), Motaln, Helena (mentor), Rogelj, Boris (comentor)

FUS in TDP-43 sta RNA-vezavna proteina, vključena v številne celične procese, katerih motnje so povezane z nevrodegenerativnimi boleznimi, kot sta amiotrofična lateralna skleroza (ALS) in frontotemporalna demenca (FTD). Posttranslacijske modifikacije, zlasti fosforilacija, vplivajo na njuno celično lokalizacijo in funkcijo. V magistrski nalogi smo uporabili tridimenzionalni model nevrosferoidov, ki vsebujejo nevronom podobne celice, diferencirane v petih tednih iz mišjih induciranih pluripotentnih matičnih celic (iPSC). Nevrosferoide smo analizirali v štirih časovnih točkah diferenciacije (2., 3., 4. in 5. teden), s pripravo zamrznjenih rezin in imunocitokemijskim označevanjem s protitelesi proti FUS in TDP-43 ter njunim fosforiliranim oblikam (FUS$^{p-Y526}$, TDP-43$^{p-S369}$, TDP-43$^{p-S375}$, TDP-43$^{p-S409/410}$). Za dodatno validacijo rezultatov smo uporabili dvodimenzionalni model diferencirane človeške nevroblastomske celične kulture SH-SY5Y, kjer smo spremljali izražanje izbranih proteinov med 7-dnevno diferenciacijo. Analiza izražanja in celične lokalizacije omenjenih proteinov je bila izvedena s pomočjo konfokalne fluorescenčne mikroskopije. Rezultati so pokazali, da se lokalizacija proteinov FUS in TDP-43 ter njunih fosforiliranih oblik spreminja med diferenciacijo, pri čemer je fosforilacija FUS na Tyr526 povezana z zgodnjimi fazami diferenciacije in prisotnostjo proteina v citoplazmi nevronskih celic. Kolokalizacija FUS$^{p-Y526}$ s kinazami iz družine Src, zlasti s p-Abl, potrjuje njihovo vlogo pri fosforilaciji FUS. V nevrosferoidnem modelu smo z L-glutamatom kot induktorjem kinaz družine Src analizirali njegov vpliv na fosforilacijo FUS ter možen posreden vpliv na fosforilacijo in lokalizacijo TDP-43, pri čemer smo potrdili učinek na oba proteina. Za potrditev diferenciacije celic v specifične nevronske in glialne podtipe smo uporabili protein TUBB3 kot označevalec za nevrone ter protein GFAP kot označevalec za astrocite. Skupno ti rezultati prispevajo k boljšemu razumevanju dinamike lokalizacije fosforiliranih oblik FUS in TDP-43 med diferenciacijo živčnih celic ter potrjujejo uporabnost izbranih celičnih modelov za preučevanje molekularnih mehanizmov, povezanih z nevrodegenerativnimi boleznimi.

• Language: Slovenian
• Keywords: FUS, TDP-43, fosforilacija, nevrosferoidi, SH-SY5Y, imunocitokemija
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2025
• PID: 20.500.12556/RUL-174745
• COBISS.SI-ID: 258983683
• Publication date in RUL: 09.10.2025

Secondary language

• Language: English
• Title: Evaluation of phosphorylation of RNA-binding proteins in neurospheroids

Abstract

FUS and TDP-43 are RNA-binding proteins involved in numerous cellular processes, dysfunction of which has been linked to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Post-translational modifications, particularly phosphorylation, influence their intracellular localization and function. In this master's thesis, we employed a three-dimensional neurospheroid model derived from mouse-induced pluripotent stem cells (iPSCs), which differentiated over five weeks into neuron-like cells. Neurospheroids were collected at four time points of differentiation (weeks 2, 3, 4, and 5), followed by cryosectioning and immunocytochemical labeling with antibodies against total FUS, TDP-43, and their phosphorylated forms (FUS$^{p-Y526}$, TDP-43$^{p-S369}$, TDP-43$^{p-S375}$, TDP-43$^{p-S409/410}$). To further support our findings, we also employed a two-dimensional model of differentiated human neuroblastoma SH-SY5Y cells, in which we monitored the expression of selected proteins during a 7-day differentiation. Protein expression and localization were analyzed using confocal fluorescence microscopy. The results showed that the localization of FUS and TDP-43, as well as their phosphorylated forms, changes during differentiation, with FUS phosphorylation at Tyr526 linked to the early stages of differentiation and cytoplasmic localization in neuronal cells. Colocalization of FUS$^{p-Y526}$ with Src family kinases, particularly with p-Abl, supported their role in FUS phosphorylation. In the neurospheroid model, L-glutamate was used as an inducer of Src family kinases to analyze its effect on FUS phosphorylation and a possible indirect effect on TDP-43 phosphorylation and localization, confirming its impact on both proteins. To confirm differentiation of cells into specific neural and glial subtypes, TUBB3 and GFAP were used as markers for neurons and astrocytes, respectively. Taken together, these results contribute to a better understanding of the localization dynamics of phosphorylated FUS and TDP-43 during neuronal differentiation and demonstrate the applicability of the chosen cell models for investigating molecular mechanisms involved in neurodegeneration.

• Keywords: FUS, TDP-43, phosphorylation, neurospheroids, SH-SY5Y, immunocytochemistry