Vpliv mutacije v genu c9orf72 na izražanje proteinov pri boleznih amiotrofična lateralna skleroza in frontotemporalna demenca

Čerček, Urša (avtor), Rogelj, Boris (mentor)

Amiotrofična lateralna skleroza (ALS) in frontotemporalna demenca (FTD) sta neozdravljivi nevrodegenerativni bolezni. Z njunim razvojem so povezani številni proteini, vključno s proteini Tdp-43, Sod1 in Fus. Najpogostejša mutacija pa je razširitev ponovitve heksanukleotida (G4C2) v genu c9orf72. Patološka razširitev se prepiše v toksično smerno (G4C2)n in protismerno (C4G2)n RNA, ki interagira z RNA vezavnimi proteini. Naša skupina je pokazala, da protismerne (C4G2)n RNA interagirajo z alfa podenoto fenilalanin-tRNA sintetaze (Farsa), ki omogoča polnjenje fenilalaninske tRNA. Namen doktorske naloge je raziskati povezavo med mutacijo C9orf72 in funkcijo aminoacil-tRNA sintetaz ter preučiti njihov vpliv na sintezo proteinov in razvoj ALS ter FTD. Osredotočili smo se na novo odkrito interakcijo med protismernimi (C4G2)n ponovitvami in proteinom Farsa, ki smo jo odkrili v naši raziskovalni skupini in preučili učinek na globalno izražanje proteinov ter aktivacijo stresnega odziva. Razvili smo metodo bližnje ligacije RNA-RNA, ki nam omogoča opazovanje lokalizacije protismernih RNA znotraj celice, vključno s citoplazmo, kar do sedaj večinoma ni bilo mogoče. Pokazali smo, da se protismerne ponovitve razporejajo po celotni celici in so prisotne tudi v citoplazmi. Za preučevanje vpliva na izražanje proteinov in aktivacijo celičnega stresa smo uporabili reakcijo »click«, prenos western, RNA »pull-down«, imunocitokemijo in analizo z masno spektrometrijo. Pri tem smo uporabili različne bolezensko pomembne celične modele, vključno z limfoblastoidnimi celičnimi linijami, pridobljenimi od pacientov z mutacijo C9orf72 in diferencirane motoričnim nevronom podobne celice NSC-34. V sklopu doktorske naloge smo pokazali, da je interakcija med (C4G2)n RNA in proteinom Farsa odvisna od dolžine ponovitev in vpliva na vključevanje fenilalanina, kar povzroča nepravilnosti v translaciji. Pokazali smo, da je izražanje proteinov, bogatih s fenilalaninom, zmanjšano v celičnih linijah, pridobljenih od pacientov z mutacijo v genu c9orf72, in v post mortem možganskem tkivu pacientov s C9orf72 ALS ter FTD. Pokazali smo, da so ponovitve RNA (C4G2) zmožne aktivirati kinazo Gcn2 v HEK293 celicah, vendar ne sprožijo akutnega stresnega odziva v bolezensko relevantnih celičnih modelih. Raziskava ponuja pomembne vpoglede v mehanizme ALS in FTD ter zagotavlja nove terapevtske tarče za bolezni, povezane z mutacijo v genu c9orf72.

• Jezik: Slovenski jezik
• Ključne besede: ALS, FTD, C9orf72, aminoacyl-tRNA sintetaze, translacija, celični stres
• Vrsta gradiva: Doktorsko delo/naloga
• Organizacija: MF - Medicinska fakulteta
• Leto izida: 2025
• PID: 20.500.12556/RUL-167722
• Datum objave v RUL: 09.03.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Effects of C9orf72 mutation associated with amyotrophic lateral sclerosis and frontotemporal dementia on protein expression

Izvleček

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are devastating neurodegenerative diseases. Many genes, including TDP-43, SOD-1 and FUS are linked to their development, but the most common mutation is the (G4C2) hexanucleotide repeat expansion in the C9orf72 gene. Repeat expansion is transcribed into toxic sense (G4C2) and antisense (C4G2) repeat RNA which interact with several RNA-binding proteins. Our group has previously shown that antisense (C4G2) RNA interacts with alpha subunit of phenylalanine-tRNA synthetase (FARSA) which is a protein crucial for proper phenylalanine-tRNA aminoacylation. The aim of this doctoral thesis is to investigate the connection between C9orf72 mutation and aminoacyl-tRNA synthetase function, exploring their impact on protein synthesis and ALS and FTD development. Specifically, we examined the influence of antisense (C4G2)n on the function of FARSA and effect on global protein expression and integrated stress response. We developed an RNA-RNA proximity ligation assay (PLA) that enables us to observe the subcellular localisation of the repeats which we showed are also present in the cytoplasm of C9orf72 mutation positive cells. To study the effect of interaction on protein expression and cellular stress activation we used click-chemistry reaction, western blot analysis, RNA pull-down, immunocytochemistry, and mass spectrometry analysis. We used disease relevant cellular models, including C9orf72 patient-derived lymphoblastoid cell lines and differentiated motor neuron like NSC-34 cells. In the scope of this doctoral thesis we showed that the (C4G2)n: FARSA interaction is length dependant and inhibits phenylalanine incorporation, which leads to translational irregularities. We observed a decreased expression of phenylalanine-rich proteins in C9orf72-patient derived cell lines and post-mortem brain tissue of C9orf72 patients. We also investigated the activation of integrated stress response and observed that both (G4C2)n and (C4G2)n repeats activate GCN2 kinase in transfected HEK293 cells but the activation of integrated stress response was not observed in disease-relevant cell models. This suggests that other stress response mechanisms might be activated in C9orf72 ALS and FTD patients. Our research offers significant insights into ALS and FTD mechanisms, providing new therapeutic targets for C9orf72 mutation-related diseases.

• Ključne besede: ALS, FTD, C9orf72, aminoacyl-tRNA synthetase, translation, cellular stress