Abstract | Optineurin is a multifunctional polyubiquitin-binding protein implicated in autophagy and inflammatory signalling, processes that have been described as pathogenic mechanisms in neurodegenerative diseases. Notably, more than 40 mutations in the OPTN gene, which encodes for optineurin, were linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), neurodegenerations marked with excessive motor neurons loss, loss of neurons from frontal and temporal lobes, chronic inflammation, and protein aggregation in the central nervous system (CNS). However, the pathogenic role of optineurin mutations is still largely unclear. The autopsies of ALS and FTD patients carrying the optineurin mutations show TAR DNA-binding protein 43 (TDP-43) aggregates, which could elicit its nuclear depletion and loss-of-function. Since optineurin acts as an adaptor protein in autophagy and inflammatory signalling, and chronic inflammation could exacerbate TDP-43 aggregation, here we tested if optineurin ALS-mimicking mutations could lead to impaired TDP-43 proteostasis, excessive inflammation, and/or inefficient immune responses. Moreover, as ageing is a major risk factor for ALS and FTD, and untreated young mice carrying optineurin ALS-linked mutations do not develop the disease, here we investigated whether ageing could trigger neurological, neuropathological, and/or immunological alterations. To this end, we used (1) Optn470T mouse model, that mimics loss-of-function Q398X truncation found in ALS patients both of which express a lower level of truncated optineurin (henceforth termed optineurin insufficiency) and (2) optineurin deficient microglial BV2 cells made using CRISPR/Cas9 technology (BV2 Optn KO). We found elevated basal TDP-43 protein levels in primary mouse Optn470T myeloid cells and cortical neurons, which were post-translationally regulated. Moreover, we demonstrated that optineurin deficiency did not sensitize cells to apoptosis upon autophagy inhibition and that TDP-43 accumulation in Optn470T primary microglia was not caused by an autophagy block. In contrast, we showed that optineurin insufficiency caused an altered TDP-43 turnover, which was unaffected by experimental block in autophagy in both bone marrow-derived macrophages (BMDMs) and primary neurons. To further evaluate the role of optineurin in inflammation and TDP-43 accumulation, we stimulated BV2 cell lines and primary microglia with lipopolysaccharide (LPS) to mimic bacterial infection. We observed a significant increase in TDP-43 protein levels in WT cells without changes in optineurin levels. However, LPS failed to increase already elevated TDP-43 levels in untreated optineurin-deficient and-insufficient myeloid cells, suggesting that they already reached a plateau at basal conditions. Moreover, we demonstrated no nuclear depletion or aggregation of TDP-43 in Optn470T primary microglia in basal state or with LPS. Characterization of aged Optn470T mice did not show motor or cognitive defects, or differential TDP-43 insolubility in the whole-brain lysates compared to WT mice. In addition, we demonstrated enhanced expression of certain cytokines and chemokines in the CNS during ageing, but with neglectable differences between old WT and Optn470T mice. Moreover, spleen immunophenotyping uncovered signs of ageing of the immune system (inflammageing and immunosenescence) in old Optn470T mice that were comparable to WT mice. These included an increase in memory and regulatory T lymphocytes, a drop in naïve T lymphocytes, and an increase in the number of macrophages and neutrophils. However, we showed that macrophages and conventional dendritic cells (cDC) exhibited increased expression of activation markers in old Optn470T mice, although we could not link it to any phenotype. Altogether, a combination of ageing and optineurin insufficiency did not induce ALS and/or FTD-like immune imbalance and neuropathology in mice. To further evaluate crosstalk between optineurin insufficiency and TDP-43 we established a new two-hit ALS and/or FTD model by crossing Optn470T mice with the transgenic mice carrying a human TDP-43 patient mutation (G348C) but did not observe an ALS-like phenotype either. In conclusion, we showed TDP-43 accumulation in optineurin-insufficient neurons and microglia. In microglia, the accumulation was not caused by an autophagy block, and it was unresponsive to inflammation, while in neurons it was likely caused by a block in autophagy. Furthermore, the Optn470T mouse model during ageing, even when crossed to mutant transgenic TDP-43 did not show motor or cognitive defects, TDP-43 aggregation, or immunological alterations typical for ALS and/or FTD. Thus, further research is necessary to elucidate the mechanistic links between optineurin mutations and TDP-43-mediated pathology. |
Abstract (croatian) | Optineurin je multifunkcionalni ubikvitin-vezujući protein koji ima ulogu u upalnoj signalizaciji i autofagiji, procesima koji su opisani kao patološki mehanizmi u neurodegenerativnim bolestima. Više od 40 mutacija u genu OPTN, koji kodira za optineurin, je povezano s amiotrofičnom lateralnom sklerozom (ALS) i frontotemporalnom demencijom (FTD), neurodegenerativnim bolestima koje karakterizira prekomjeran gubitak neurona u motoričkom, temporalnom i frontotemporalnom korteksu, kronična upala i agregacija proteina. Međutim, uloga mutacija optineurina u patogenezi ALS-a još je uvelike nejasna. Autopsije pacijenata s ALS-om i FTD-om koji nose mutacije optineurina pokazale su agregaciju TAR DNA-vezujućeg proteina 43 (TDP-43) u središnjem živčanom sustavu koja je usko povezana s njegovom deplecijom u jezgri i gubitkom funkcije. Budući da optineurin djeluje kao adaptorski protein u autofagiji i upalnoj signalizaciji, a kronična upala može pogoršati agregaciju TDP-43-a, u ovom radu smo testirali dovode li mutacije optineurina u ALS-u do poremećene proteostaze TDP-43-a, pretjerane upale i/ili neučinkovitog imunosnog odgovora. Štoviše, budući da je starenje glavni čimbenik rizika za ALS/FTD spektar neurodegenerativnih bolesti, te uz činjenicu da netretirani mladi miševi s mutacijama optineurina povezanih s ALS-om ne razvijaju bolest, ovdje smo istraživali ukoliko starenje može potaknuti neurološke, neuropatološke i imunsne promjene. U tu svrhu smo koristili (1) mišji Optn470T model, koji oponaša Q398X mutaciju optineurina pronađenu u ALS pacijenatima koju nazivamo proteinskom insuficjencijom zbog manjka mutiranog proteina i (2) mikroglijalnu BV2 staničnu liniju s nedostatkom optineurina dobivenu pomoću CRISPR/Cas9 tehnologije (BV2 Optn KO). Utvrdili smo povišene bazalne razine TDP-43 proteina u primarnim mišjim Optn470T mijeloidnim stanicama i kortikalnim neuronima čije su razine bile posttranslacijski regulirane. Nadalje, pokazali smo da nedostatak funkcionalnog optineurina nije povećao osjetljivost stanica na apoptozu nakon inhibicije autofagije te da blokada autofagije ne izaziva nakupljanje TDP-43 u primarnoj mikrogliji i BV2 staničnoj liniji. Međutim, pokazali smo da nedostatak optineurina uzrokuje nakupljanje TDP-43 putem autofagije u primarnim neuronima i makrofagima. Kako bismo dodatno ispitali ulogu optineurina u upali i nakupljanju TDP-43, stimulirali smo primarnu mikrogliju i BV2 staničnu liniju s lipopolisaharidom (LPS) koji oponaša bakterijsku infekciju i uočili smo značajno povećanje razine TDP-43 u WT stanicama. Međutim, LPS nije uspio povećati već nakupljeni TDP-43 u netretiranim mijeloidnim stanicama s insuficijencijom optineurina. Štoviše, pokazali smo da TDP-43 ne pokazuje depleciju u jezgri niti agregaciju u Optn470T mikrogliji. Karakterizacija starih Optn470T miševa nije pokazala motoričke ili kognitivne promjene, niti razliku u topljivosti TDP-43 u lizatima cijelog mozga u usporedbi s WT miševima. Osim toga, pokazali smo pojačanu ekspresiju citokina i kemokina u mozgu i leđnoj moždini bez značajnih razlika između dvogodišnjih WT i Optn470T miševa. Štoviše, imunofenotipizacija slezene otkrila je znakove upale povezane sa starenjem u Optn470T koji su bili usporedivi s WT miševima, kao što je povećanje broja memorijskih i regulacijskih T limfocita i pad broja naivnih, te povećan broj makrofaga i neutrofila. Međutim, pokazali smo da makrofagi i konvencionalne dendritičke stanice (cDC) pokazuju povećanu ekspresiju aktivacijskih markera u dvogodišnjim Optn470T miševima. Zaključno, kombinacija nefunkcionalnog optineurina i starenja nije izazvala imunosnu neravnotežu i neuropatologiju sličnu ALS/FTD-u kod miševa. Kako bismo dodatno istražili vezu između nefunkcionalnog optineurina i TDP-43 proteina, uspostavili smo novi model ALS/FTD-a križanjem Optn470T miševa s transgeničnim miševima koji nose ljudsku TDP-43 mutaciju (G348C) pronađenu u pacijentima, ali bez uočenog ALS fenotipa do dvije godine starosti. Zaključno, pokazali smo akumulaciju TDP-43 u neuronima i mikrogliji s nedostatkom funkcinonalnog optineurina. Akumulacija u mikrogliji nije bila uzrokovana blokadom autofagije niti je TDP-43 reagirao na upalu, dok je u neuronima i makrofagima uzrok akumulacije vjerojatno blok u autofagiji. Nadalje, mišji model Optn470T, čak ni nakon križanja s mutiranim transgeničnim TDP-43 mišjim modelom, nije pokazao motoričke ili kognitivne promjene, niti imunosne promjene vezane uz ALS/FTD spektar bolesti. Stoga su daljnja istraživanja potrebna kako bi se razjasnile mehanističke veze između mutacija optineurina i patologije posredovane TDP-43-om. |