Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive loss of motor neurons that results in muscular weakness and fatal paralysis. About 5–10 % of ALS cases are familial and are caused by specific mutations. Many of the genes implicated in familial ALS encode RNA-binding proteins (RBPs), such as fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43). In patients of ALS, FUS and TDP-43 mislocalize in the cytoplasm of motor neurons and form insoluble aggregates called cytoplasmic inclusions (CI), a common pathological hallmark of neurodegenerative diseases. Moreover, increasing evidence supports the involvement of DNA damage and DNA damage response (DDR) in several neurodegenerative diseases, including ALS. The aim of my Master thesis is to understand the mechanistic link between the formation of FUS/TDP-43 CI and the activation of DDR at a single cell level. In our cellular model of FUS and TDP-43 proteinopathies, transient overexpression of the P5255L mutant form of FUS or the wild type form of TDP-43 is sufficient for the formation of FUS and TDP-43 CI, respectively. We discovered that the formation of FUSP525L/TDP-43 inclusions is associated with an impairment of the DNA Damage Response (DDR) activation. The results obtained indicate that FUSP525L/TDP-43 inclusions in the cytoplasm of undamaged cells can induce a chronic activation of DDR which results in a pan-nuclear signal of phosphorylated H2AX (γH2AX). In addition, treatments with DNA damaging agents also demonstrate that these cells present DDR defects. Indeed, cells with FUSP525L/TDP-43 CI exhibit an impairment in the formation of 53BP1 foci, one of the main DDR markers, prompting the idea that these cells are unable to respond properly to exogenous DNA damage. To understand whether these DDR defects are associated specifically with FUSP525L/TDP-43 CI or more generally with the presence of a cytoplasmic aggregate per se, we induced the formation of cytoplasmic inclusions negative for FUS and TDP-43 with Na Arsenite treatment and we concluded that the defective DDR observed in cells with FUSP525L/TDP-43 CI is partially due to the presence of an insoluble structure in the cytoplasm and in part to a specific effect of FUS/TDP-43 sequestration into the CI. To further explore the mechanistic link between FUSP525L/TDP-43 CI and DDR defects, we investigated the involvement of DROSHA, an endoribonuclease that is involved in the initial step of microRNA biogenesis and which was recently shown to be essential for enabling the secondary recruitment of DDR factors and fuel the amplification of DDR signalling. We observed reduced levels of DROSHA upon overexpression of FUSP525L/TDP-43, suggesting that DDR defects associated with the formation of CI may be partially caused by lower levels of DROSHA. The reduction in protein levels is also associated with a strong impairment in global transcription upon formation of FUSP525L/TDP-43 CI. We are currently investigating whether lower DROSHA levels and impaired transcription have an effect on the generation and processing of small non-coding RNAs, termed DNA damage response RNA (DDRNA), which are central players in DDR activation.
Risposta al danno al DNA nella SLA: studio della downregolazione di DROSHA in modelli cellulari di proteinopatie di FUS e TDP-43. La Sclerosi Laterale Amiotrofica (SLA) è una malattia neurodegenerativa caratterizzata dalla perdita progressiva dei motoneuroni risultante in debolezza muscolare e paralisi. Circa il 5-10 % dei casi di SLA sono familiari e sono causati da specifica mutazioni. Molti dei geni implicati della SLA familiare codificano per proteine leganti l’RNA, come FUS e TDP-43. Nei pazienti di SLA, FUS e TDP-43, normalmente nucleari, sono de-localizzati nel citoplasma dei motoneuroni dove formano aggregati insolubili chiamati inclusioni citoplasmatiche (CI), una caratteristica comune a molte malattie neurodegenerative. Inoltre, studi recenti evidenziano il ruolo del danno al DNA e della risposta al danno al DNA (DDR) in molte malattie neurodegenerative, tra cui la SLA. Lo scopo della mia tesi magistrale è capire il legame meccanicistico tra la formazione di inclusioni citoplasmatiche di FUS e TDP-43 e l’attivazione del DDR a livello di singola cellula. Nel nostro modello cellulare di proteinopatie di FUS e TDP-43, l’over-espressione della forma mutata di FUS (FUSP525L) e della forma wild type di TDP-43 sono sufficienti per la formazione di inclusioni di FUS and TDP-43, rispettivamente. Abbiamo osservato che la formazione di inclusioni di FUSP525L/TDP-43 è associata a difetti nell’attivazione del DDR. I risultati ottenuti indicano che le inclusioni di FUSP525L/TDP-43 nel citoplasma di cellule non danneggiate induce attivazione cronica del DDR che risulta in un segnale pan-nucleare dell’istone H2AX fosforilato (γH2AX). Inoltre, trattamenti con agenti in grado di danneggiare il DNA dimostrano che queste cellule presentano difetti nella risposta al danno. Infatti, cellule con inclusioni di FUSP525L/TDP-43 mostrano difetti nella formazione di foci di 53BP1, uno dei principali marker del DDR; questo suggerisce l’ipotesi che queste cellule non siano in grado di rispondere propriamente a danno esogeno al DNA. Per capire se questi difetti nel DDR sono associati specificamente alle inclusioni di FUSP525L/TDP-43 o più in generale alla presenza di aggregati citoplasmatici, abbiamo indotto la formazione di inclusioni citoplasmatiche prive di FUSP525L/TDP-43 tramite trattamenti con Na Arsenite. I risultati indicano che i difetti nel DDR osservati in cellule con inclusioni di FUSP525L/TDP-43 sono dovuti in parte alla presenza di strutture insolubili nel citoplasma, in parte al fatto che FUSP525L/TDP-43 sono sequestrati nelle inclusioni. Infine, abbiamo studiato il coinvolgimento di DROSHA, un’endoribonucleasi necessaria per la biogenesi dei microRNA ed essenziale per il reclutamento di fattori del DDR al sito di danno e per la segnalazione a valle. I ridotti livelli di DROSHA in cellule con inclusioni di FUSP525L/TDP-43 suggeriscono che i difetti del DDR associati alla formazione di inclusioni citoplasmatiche potrebbe essere in parte causata dai minori livelli di DROSHA. La riduzione nei livelli proteici e di trascritto di DROSHA è inoltre associata a una forte riduzione nei livelli di trascrizione globale in cellule con inclusioni di FUSP525L/TDP-43. Attualmente stiamo studiando se i minori livelli di DROSHA possano avere effetto sulla generazione e sul processamento di piccoli RNA non codificanti (DDRNA) che hanno un ruolo centrale nell’attivazione del DDR.
DNA damage response defects in ALS: mechanistic insight into DROSHA downregulation in cellular model system of TDP-43 and FUS proteinopathies
VITALI, GIULIA
2021/2022
Abstract
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive loss of motor neurons that results in muscular weakness and fatal paralysis. About 5–10 % of ALS cases are familial and are caused by specific mutations. Many of the genes implicated in familial ALS encode RNA-binding proteins (RBPs), such as fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43). In patients of ALS, FUS and TDP-43 mislocalize in the cytoplasm of motor neurons and form insoluble aggregates called cytoplasmic inclusions (CI), a common pathological hallmark of neurodegenerative diseases. Moreover, increasing evidence supports the involvement of DNA damage and DNA damage response (DDR) in several neurodegenerative diseases, including ALS. The aim of my Master thesis is to understand the mechanistic link between the formation of FUS/TDP-43 CI and the activation of DDR at a single cell level. In our cellular model of FUS and TDP-43 proteinopathies, transient overexpression of the P5255L mutant form of FUS or the wild type form of TDP-43 is sufficient for the formation of FUS and TDP-43 CI, respectively. We discovered that the formation of FUSP525L/TDP-43 inclusions is associated with an impairment of the DNA Damage Response (DDR) activation. The results obtained indicate that FUSP525L/TDP-43 inclusions in the cytoplasm of undamaged cells can induce a chronic activation of DDR which results in a pan-nuclear signal of phosphorylated H2AX (γH2AX). In addition, treatments with DNA damaging agents also demonstrate that these cells present DDR defects. Indeed, cells with FUSP525L/TDP-43 CI exhibit an impairment in the formation of 53BP1 foci, one of the main DDR markers, prompting the idea that these cells are unable to respond properly to exogenous DNA damage. To understand whether these DDR defects are associated specifically with FUSP525L/TDP-43 CI or more generally with the presence of a cytoplasmic aggregate per se, we induced the formation of cytoplasmic inclusions negative for FUS and TDP-43 with Na Arsenite treatment and we concluded that the defective DDR observed in cells with FUSP525L/TDP-43 CI is partially due to the presence of an insoluble structure in the cytoplasm and in part to a specific effect of FUS/TDP-43 sequestration into the CI. To further explore the mechanistic link between FUSP525L/TDP-43 CI and DDR defects, we investigated the involvement of DROSHA, an endoribonuclease that is involved in the initial step of microRNA biogenesis and which was recently shown to be essential for enabling the secondary recruitment of DDR factors and fuel the amplification of DDR signalling. We observed reduced levels of DROSHA upon overexpression of FUSP525L/TDP-43, suggesting that DDR defects associated with the formation of CI may be partially caused by lower levels of DROSHA. The reduction in protein levels is also associated with a strong impairment in global transcription upon formation of FUSP525L/TDP-43 CI. We are currently investigating whether lower DROSHA levels and impaired transcription have an effect on the generation and processing of small non-coding RNAs, termed DNA damage response RNA (DDRNA), which are central players in DDR activation.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
Per maggiori informazioni e per verifiche sull'eventuale disponibilità del file scrivere a: unitesi@unipv.it.
https://hdl.handle.net/20.500.14239/15137