Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive degeneration of motor neurons in patients’ brain and spinal cord. About 10% of cases are familiar ALS (fALS), which are almost all transmitted in an autosomal dominant manner, while the remaining 90-95% are sporadic ALS (sALS), with no connection with family history. One ALS-linked gene is the TARDBP which encodes for TAR DNA binding-protein TDP-43, an RNA/DNA binding protein usually located in the nucleus, where it is involved in several functions such as splicing, transcription, miRNA biogenesis and mRNA transport. A hallmark of both fALS and sALS is the formation of TDP-43 cytoplasmic inclusions, and for this reason ALS is also known as TDP-43 proteinopathy. ALS neurons have also been shown to accumulate oxidative DNA damage and DNA breaks, events that can activate a set of molecular mechanisms known as DNA-damage Response (DDR). My laboratory recently discovered that the overexpression of TDP-43 WT leads to the formation of TDP-43 positive cytoplasmic inclusions (CI) associated with an impairment of DDR activation. Thus, I began my project by evaluating whether the overexpression of different mutated forms of TDP-43 could induce the generation of TDP-43 CI and hamper the proper DDR activation. The results obtained indicate that TDP-43 inclusions in the cytoplasm of undamaged cells can induce a chronic activation of ATM, leading to a wide phosphorylation of H2AX, which results in a pan-nuclear signal of γH2AX. In addition, treatments with DNA damage agents also demonstrate that these cells present DDR defects. Indeed, cells with 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. Studies conducted in our laboratory on the DNA/RNA binding protein FUS, which is another protein prone to aggregation in ALS pathology, have shown that overexpression of its P525L mutant form mislocalizes the protein to the cytoplasm, leading to a concomitant DDR impairment, as observed for TDP-43 overexpression. Furthermore, mutant FUS P525L has been related to autophagic impairment and accumulation of the cargo protein p62 is observed in cytoplasmic bodies. In the context of cancer, p62 interacts with and inhibits the activity of the E3 Ubiquitin ligase RNF168 , a key factor in DNA damage signaling and repair. Inactivation of p62 in mutant FUS overexpressing cells can lead to a restoration of DDR. Given the great similarity between these two proteins, we wondered if also TDP-43 can cause p62 accumulation and as a consequence damper RNF168 activity. We observed that p62 accumulates in the cytoplasm of cells with TDP-43 CI and the same cells also exhibit reduced nuclear signal of RNF168, which appears accumulated in the cytoplasm in proximity to p62 bodies. Therefore, we inactivated p62 to evaluate if, similarly to what was observed in cells with mutant FUS CI, the loss of this factor can restore nuclear RNF168 levels and DDR functions in cells with TDP-43 CI. Intriguingly, differently to the expectations, p62 inactivation did not rescue 53BP1 foci. Furthermore, I conducted other studies with primary pig fibroblasts harboring TDP-43 A382T mutation. We observed that this mutation alone is not able to induce the formation of TDP-43 aggregates in the cytoplasm of these cell type. We also evaluated the DDR of these cells, and we discovered that pig fibroblasts with mutant TDP-43 A382T show higher levels of γH2AX, with respect to the WT cells. These levels might be partially related with replication stress and accumulation of R-loop in the nucleus of these cells. Altogether, these results support a model suggesting that TDP-43 plays a pivotal role in DNA damage response activation since its CI formation leads to DNA damage accumulation, while its mutation in skin fibroblasts causes loss of genome integrity.
La SLA è una patologia neurodegenerativa caratterizzata dalla degenerazione progressiva dei motoneuroni del cervello e del midollo spinale dei pazienti.Circa il 10% dei casi di SLA sono familiari (fALS) trasmessi in maniera autosomica dominante, i restanti 90-95% sono sporadici (sALS) e non presentano connessione con la storia familiare.Uno dei geni correlati con la SLA è TARDBP che codifica per la proteina TDP-43 una RNA/DNA binding protein, che si localizza principalmente nel nucleo, dove regola diverse funzioni.Un segno dei casi di fSLA e di sSLA è la formazione di inclusioni citoplasmatiche(CI) di TDP-43, per questo la SLA è definita anche come proteinopatia indotta da TDP-43.È stato visto che i neuroni dei pazienti affetti da SLA presentano un accumulo di danno ossidativo al DNA e rotture della doppia elica, eventi che attivano meccanismi molecolari conosciuti come Risposta al Danno al DNA (DDR).Nel mio laboratorio si è scoperto che l’overespressione di TDP-43 WT porta alla formazione di CI positive per TDP-43, associate con un difetto nell’attivazione del DDR.Perciò, ho cominciato il mio progetto valutando se l’overespressione di forme mutate di TDP-43 potesse indurre la formazione di aggregati di TDP-43 nel citoplasma, ostacolando la corretta attivazione del DDR.I risultati indicano che le CI di TDP-43 in cellule non danneggiate inducono un’attivazione cronica di ATM, portando ad segnale pan-nucleare di γH2AX.Inoltre, trattamenti con agenti che inducono danno al DNA dimostrano che queste cellule presentano dei difetti nel DDR.Infatti, le cellule con le CI di TDP-43 mostrano dei deficit nella formazione dei foci di 53BP1, uno dei mediatori del DDR, suggerendo che queste cellule non sono in grado di rispondere al danno esogeno.Studi condotti nel nostro laboratorio sulla proteina FUS, una RNA/DNA binding-protein in grado di formare aggregati nei pazienti dei SLA, hanno mostrato che l’overespressione della sua forma mutata, la P525L, induce l’uscita della proteina nel citoplasma portando a difetti di attivazione del DDR.Inoltre FUS P525L è stata correlata con difetti dell’autofagia e con l’accumulo della proteina p62 in corpi citoplasmatici. Nel cancro p62 interagisce e inibisce l’attività dell’Ubiquitina Ligasi RNF-168, uno dei principali attori del DDR. L’inattivazione di p62 nelle cellule che overesprimono FUS mutato conduce ad una ripresa del DDR.Date le molte similitudini tra TDP-43 e FUS, abbiamo studiato se anche l’overespressione di TDP-43 può causare un accumulo di p62 e danneggiare l’attività di RNF-168.Abbiamo osservato che p62 si accumula nel citoplasma delle cellule che presentano CI di TDP-43 e che queste mostrano anche una diminuzione del segnale nucleare di RNF-168, che appare nel citoplasma in prossimità di p62. Perciò abbiamo inattivato p62 per valutare se, come osservato per le cellule con CI di FUS mutato, l’assenza di p62 potesse ristabilire i livelli nucleari di RNF-168 e la normale risposta al danno al DNA, nelle cellule con le CI di TDP-43.Diversamente da quanto atteso, l’inattivazione di p62 non ristabilisce la formazione dei foci di 53BP1.Inoltre, ho condotto altri studi su fibroblasti primari di maiale con la mutazione A382T nel gene di TDP-43.Abbiamo osservato che in questo tipo cellulare la mutazione da sola non induce la formazione degli aggregati di TDP-43.Abbiamo poi caratterizzato il DDR di queste cellule, e abbiamo scoperto che i fibroblasti mutati per TDP-43 mostrano livelli di γH2AX più alti rispetto alle cellule WT.Questi livelli sembrerebbero essere parzialmente correlati con uno stress replicativo e con l’accumulo di R-loop nel nucleo.Questi risultati supportano un modello dove TDP-43 gioca un ruolo cruciale nell’attivazione del DDR, mentre la presenza di una mutazione di TDP-43 nei fibroblasti derivati da maiali causa una perdita dell’integrità genomica.
Caratterizzazione della Risposta al Danno al DNA in modelli cellulari di proteinopatie indotte da TDP-43.
BATTISTONI, MARTINA
2019/2020
Abstract
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive degeneration of motor neurons in patients’ brain and spinal cord. About 10% of cases are familiar ALS (fALS), which are almost all transmitted in an autosomal dominant manner, while the remaining 90-95% are sporadic ALS (sALS), with no connection with family history. One ALS-linked gene is the TARDBP which encodes for TAR DNA binding-protein TDP-43, an RNA/DNA binding protein usually located in the nucleus, where it is involved in several functions such as splicing, transcription, miRNA biogenesis and mRNA transport. A hallmark of both fALS and sALS is the formation of TDP-43 cytoplasmic inclusions, and for this reason ALS is also known as TDP-43 proteinopathy. ALS neurons have also been shown to accumulate oxidative DNA damage and DNA breaks, events that can activate a set of molecular mechanisms known as DNA-damage Response (DDR). My laboratory recently discovered that the overexpression of TDP-43 WT leads to the formation of TDP-43 positive cytoplasmic inclusions (CI) associated with an impairment of DDR activation. Thus, I began my project by evaluating whether the overexpression of different mutated forms of TDP-43 could induce the generation of TDP-43 CI and hamper the proper DDR activation. The results obtained indicate that TDP-43 inclusions in the cytoplasm of undamaged cells can induce a chronic activation of ATM, leading to a wide phosphorylation of H2AX, which results in a pan-nuclear signal of γH2AX. In addition, treatments with DNA damage agents also demonstrate that these cells present DDR defects. Indeed, cells with 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. Studies conducted in our laboratory on the DNA/RNA binding protein FUS, which is another protein prone to aggregation in ALS pathology, have shown that overexpression of its P525L mutant form mislocalizes the protein to the cytoplasm, leading to a concomitant DDR impairment, as observed for TDP-43 overexpression. Furthermore, mutant FUS P525L has been related to autophagic impairment and accumulation of the cargo protein p62 is observed in cytoplasmic bodies. In the context of cancer, p62 interacts with and inhibits the activity of the E3 Ubiquitin ligase RNF168 , a key factor in DNA damage signaling and repair. Inactivation of p62 in mutant FUS overexpressing cells can lead to a restoration of DDR. Given the great similarity between these two proteins, we wondered if also TDP-43 can cause p62 accumulation and as a consequence damper RNF168 activity. We observed that p62 accumulates in the cytoplasm of cells with TDP-43 CI and the same cells also exhibit reduced nuclear signal of RNF168, which appears accumulated in the cytoplasm in proximity to p62 bodies. Therefore, we inactivated p62 to evaluate if, similarly to what was observed in cells with mutant FUS CI, the loss of this factor can restore nuclear RNF168 levels and DDR functions in cells with TDP-43 CI. Intriguingly, differently to the expectations, p62 inactivation did not rescue 53BP1 foci. Furthermore, I conducted other studies with primary pig fibroblasts harboring TDP-43 A382T mutation. We observed that this mutation alone is not able to induce the formation of TDP-43 aggregates in the cytoplasm of these cell type. We also evaluated the DDR of these cells, and we discovered that pig fibroblasts with mutant TDP-43 A382T show higher levels of γH2AX, with respect to the WT cells. These levels might be partially related with replication stress and accumulation of R-loop in the nucleus of these cells. Altogether, these results support a model suggesting that TDP-43 plays a pivotal role in DNA damage response activation since its CI formation leads to DNA damage accumulation, while its mutation in skin fibroblasts causes loss of genome integrity.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.
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https://hdl.handle.net/20.500.14239/12667