| Abstract | Amyotrophic lateral sclerosis (ALS) is neurodegenerative disease caused by mutations in more than 30 different genes, which act by different and often still unclear pathogenic mechanisms. Two major features mark all ALS cases: insoluble protein aggregates in neurons and glia, and neuroinflammation. Aggregates in the majority of ALS cases, regardless of genetic cause, comprise TAR DNA-binding protein 43 (TDP-43), an RNA/DNA-binding protein with functions in splicing, mRNA metabolism and miRNA production. In contrast, OPTN gene is a rare cause of ALS. It encodes a multifunctional adaptor protein optineurin (Optn) proposed to regulate many cellular processes like autophagy, inflammation, Golgi maintenance and vesicular trafficking. Recent data from our laboratory have shown that both Optn insufficiency and deficiency caused an increase in endogenous TDP-43 levels in myeloid cells (microglia and macrophages), which was unexpectedly not caused by an autophagy block. Inflammatory stimulation by a lipopolysaccharide (LPS), which was previously shown to stimulate TDP-43 aggregation, elevated TDP-43 levels in WT but could not increase already elevated TDP-43 levels in cells lacking functional optineurin. Notably, no aggregates were formed in neither WT nor optineurin insufficient or deficient cells. Here generated a new mouse model carrying transgenic TDP-43G348C and truncated Optn470T/470T to test if transgenic TDP-43 causes greater proteotoxic stress than endogenous TDP-43 and leads to signs of ALS pathology in vitro or in vivo. We investigated transgenic TDP-43 levels in bone marrow-derived macrophages (BMDM) and observed higher TDP-43 levels in Optn470T/470T/TDP-43G348C compared to WT/TDP-43G348C BMDM. Transgenic TDP-43 thus did accumulate in Optn insufficient cells, but not because of an autophagy block. Also, we observed that transgenic TDP-43 levels cannot be additionally increased upon LPS stimulation in Optn470T/470T/TDP-43G348C BMDM, in contrast to WT cells. Notably, transgenic TDP-43 was not cytotoxic in unmanipulated BMDM but caused apoptosis after LPS stimulation in both optineurin sufficient and
insufficient cells. Immunofluorescent analysis showed that transgenic TDP-43 was not depleted from the nucleus and did not aggregate upon LPS stimulation in BMDM. In addition, we investigated spinal cords of transgenic mice model and observed no signs of ALS pathology (neuronal loss and TDP-43 aggregation) in neither WT nor Optn470T/470T 8-month-old mice. In conclusion, in this study, we showed that transgenic TDP-43 has elevated protein levels in Optn insufficient BMDM, just like it was previously shown for endogenous TDP-43, but this is not caused by an autophagy block and does not result in cytotoxicity. Moreover, optineurin insufficiency did not precipitate the pathology in TDP-43G348C mouse model, at least not at the early stage of disease. Further studies are necessary to investigate pathology in Optn470T/470T/TDP-43G348C model at later stages. |
| Abstract (croatian) | Amiotrofična lateralna skleroza (ALS) je neurodegenerativna bolest koju uzrokuju mutacije u više od 30 gena, koji djeluju prema različitim, i još uvijek nejasnim, patogenim mehanizmima. Dva glavna obilježja označavaju sve slučajeve ALS-a: netopljivi proteinski agregati, u neuronima i glija stanicama, i neuroinflamacija. Agregati u većini ALS slučajeva, neovisno o genetskom uzroku, sadrže TAR DNA-vezujući protein 43 (TDP-43), RNA/DNA-vezujući protein čije su funkcije prekrajanje, metabolizam RNA i miRNA. Suprotno tome, OPTN gen je rijedak uzrok ALS-a. Gen kodira multifunkcionalni adaptor protein optineurin (Optn), za koji se smatra da regulira mnoge stanične procese poput autofagije, upale, održavanja Golgija i vezikularnog transporta. Nedavni rezultati iz našeg laboratorija pokazali su da insuficijencija i deficijencija Optn uzrokuju povećanje razina endogenog TDP-43 u mijeloidnim stanicama (mikrogliji i makrofagima), koje, neočekivano, nije uzrokovano blokadom autofagije. Stimulacija upale sa lipopolisaharidom (LPS), koja je ranije pokazala da stimulira agregaciju TDP-43, povećala je razine TDP-43 u WT, ali nije uspjela povećati već povećane razine TDP-43 u stanicama bez funkcionalnog Optn. Značajno, agregati nisu formirani niti u WT niti u Optn insuficijentnim ili deficijentnim stanicama. U ovom radu proizveli smo novi mišji model sa transgeničnim TDP-43G348C i skraćenim Optn470T/470T kako bismo istražili uzrokuje li transgenični TDP-43 veći proteotoksični stres od endogenog TDP-43 i dovodi li do znakova patologije ALS-a in vitro ili in vivo. Testirali smo razine transgeničnog TDP-43 u makrofagima dobivenim iz koštane srži (BMDM) i uočili smo veće razine TDP-43 u Optn470T/470T/TDP-43G348C nego u WT/TDP-43G348C BMDM. Transgenični TDP-43 se nakupljao zbog insuficijencije Optn, a ne blokade autofagije. Također, uočili smo da se razine transgeničnog TDP-43 ne mogu dodatno povisiti stimulacijom LPS-om u Optn470T/470T/TDP-43G348C BMDM, za razliku od WT stanica. Transgenični TDP-43 nije bio citotoksičan u netretiranim stanicama, ali je uzrokovao apoptozu nakon stimulacije LPS-om u stanicama oba genotipa. Imunofluorescentna analiza nije
pokazala nuklearnu depleciju niti agregaciju transgeničnog TDP-43 nakon stimulacije LPS-om u BMDM. Pored toga, testirali smo leđne moždine transgeničnog mišjeg modela i nismo uočili patološke znakove ALS-a (gubitak neurona i TDP-43 agregati) niti u WT niti Optn470T/470T miševima starim 8 mjeseci. Zaključno, u ovom radu pokazali smo da transgenični TDP-43 ima povećane razine proteina u Optn insuficijentnim BMDM, kao što je to prethodno prikazano za endogeni TDP-43, ali to nije uzrokovano blokadom autofagije i nije rezultiralo citotoksičnošću. Štoviše, Optn insuficijencija nije ubrzala patologiju TDP-43G348C mišjeg modela, barem ne u ranom stadiju bolesti. Dodatna istraživanja su potrebna kako bi se istražila patologija u Optn470T/470T/TDP-43G348C modelu u kasnijim stadijima. |
