Utjecaj optineurina na bazalnu i IFN-β induciranu autofagiju u Neuro2a staničnim linijama

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive denervation of voluntary muscles. So far, more than 30 genes have been associated with ALS, which participate in many molecular pathways, including autophagy, neuroinflammation and DNA repair. Autophagy is a dynamic process that eliminates damaged organelles, misfolded proteins and pathogens, and ensures nutrients for cell survival. Since autophagy disposes of protein aggregates, its dysfunction is linked to neurodegenerative diseases. Optineurin is an ALS-linked protein involved in cell signalling and autophagy. For this reason, we hypothesized that optineurin deficiency could disturb autophagy flux. To investigate this, we monitored the levels of several autophagy-associated proteins in wild-type (WT) and optineurin-deficient (OPTN-KO) Neuro2a cell lines under basal conditions and upon IFN-β treatment. We observed that the lack of optineurin resulted in increased inhibitory ULK1 phosphorylation and diminished degradation of phosphorylated TBK1 (pTBK1) via autophagy. However, the lack of optineurin did not impact p62, LC3-II and GABARAP levels in the basal state, suggesting that it does not disturb autophagy flux. IFN-β treatment increased TBK1 phosphorylation, but overall, it did not induce autophagy in Neuro2a cells. Next, we inhibited TBK1 activation by BX795 to determine if optineurin phosphorylation is primarily mediated by pTBK1 in neuronal cells and/or if pTBK1 inhibition will have a greater effect on autophagy than optineurin deficiency. We observed diminished phosphorylation of optineurin, demonstrating that optineurin is a TBK1 target in Neuro2a cells. We also observed decreased ULK1 phosphorylation, which has previously not been described. The latter could be caused by either the BX795 off-target effect or the currently unknown TBK1 effect on ULK1 modification. In conclusion, neither optineurin deficiency nor IFN-β affected autophagy flux in Neuro2a cells. However, this study highlights the possibility that optineurin mayregulate ULK1 phosphorylation and pTBK1 turnover under basal conditions, but further research is needed to confirm this.Amiotrofična lateralna skleroza (ALS) je neurodegenerativna bolest karakterizirana progresivnom denervacijom voljnih mišića. Do sada je više od 30 gena povezano s ALS-om, koji sudjeluju u mnogim molekularnim putevima, uključujući autofagiju, neuroinflamaciju i popravak DNA. Autofagija je dinamičan proces koji eliminira oštećene organele, nepravilno savijene proteine i patogene, te osigurava hranjive tvari za preživljavanje stanica. Budući da autofagija uklanja proteinske agregate, njezina disfunkcija je povezana s neurodegenerativnim bolestima. Optineurin je protein povezan s ALS-om koji sudjeluje u signalnim putevima i autofagiji. Iz tog razloga, pretpostavljamo da bi nedostatak optineurina mogao poremetiti protok autofagije. Kako bismo to istražili, promatrali smo razine nekoliko proteina povezanih s autofagijom u divljem tipu (engl. wild-type (WT)) i optineurindeficijentnim (OPTN-KO) Neuro2a staničnim linijama pri bazalnim uvjetima i nakon IFN-β tretmana. Uočili smo da nedostatak optineurina dovodi do povećane inhibitorne fosforilacije ULK1 i smanjene razgradnje fosforiliranog TBK1 (pTBK1) putem autofagije. Međutim, nedostatak optineurina nije utjecao na razine p62, LC3-II i GABARAP u bazalnom stanju, sugerirajući da ne remeti tok autofagije. IFN-β tretman je povećao fosforilaciju TBK1, ali općenito nije potaknuo autofagiju u Neuro2a stanicama. Zatim smo inhibirali aktivaciju TBK1 pomoću BX795 kako bismo utvrdili je li fosforilacija optineurina primarno posredovana pTBK1 u neuronskim stanicama te hoće li inhibicija pTBK1 imati veći utjecaj na autofagiju u odnosu na nedostatak optineurina. Uočili smo smanjenu fosforilaciju optineurina, pokazujući da je optineurin meta TBK1 u Neuro2a stanicama. Primijetili smo i sniženu fosforilaciju ULK1, što dosada nije bilo opisano. Potonje bi moglo biti uzrokovano nespecifičnim učinkom BX795 ili dosad nepoznatim utjecajem TBK1 na modifikaciju ULK1. Zaključno, ni nedostatak optineurina ni IFN-β nisu utjecali na tok autofagije. Međutim, ovaj rad ističe mogućnost da optineurin može regulirati fosforilaciju ULK1 i promet pTBK1 pri bazalnim uvjetima, ali potrebna su daljnja istraživanja kako bismo to mogli potvrditi

In vitro sustav za detekciju ADAR uređivanja miRNA kod HSV-1

Post-transcriptional changes in the RNA nucleotide sequences, known as RNA editing, include insertion, deletion, and substitution of nucleotides. Editing can be present in both protein-coding and non-coding transcripts, impacting their stability, splicing, ability to regulate gene expression, and it can even lead to the recoding of proteins. Conversion of adenosine to inosine (A→I) in dsRNAs, catalyzed by the adenosine deaminase acting on RNA (ADAR) protein family, is the most prevalent form of RNA editing. Herpes simplex virus 1 (HSV-1), a highly prevalent human pathogen, encodes for over 20 miRNAs; and during latency, the only present transcripts are latency-associated transcripts (LATs) and miRNAs. Our group previously detected A→I editing events in HSV-1 miRNAs from latently infected human terminal ganglia. Highly expressed miR-H2, whose target is ICP0, an immediate early viral gene crucial for viral replication, was most frequently edited. However; the molecular mechanism of editing still needs to be unraveled. Thus, we generated an in vitro system for detecting ADAR editing events in HSV-1 miRNAs, based on the co-transfection of miRNA-expressing constructs with different ADAR-expressing plasmids. The backbone for miRNA expression plasmids (ps-Cassette) was successfully produced by cloning synthetic expression cassette into pEGFP-N1. Furthermore, the miRNA expression plasmid pS-miR-H2 and the editing positive control plasmid pS-miR-376a were generated by cloning miRNA coding fragments into the pS-cassette. Expression of the pS-miR plasmids transfected into HEK293T cells was not confirmed because of plasmid DNA contaminations. Additionally, Western blot analysis revealed high expression of endogenous ADAR1 p110 in HEK293 cells indicating potential background contribution to editing, which is why ADAR1 KO cells need to be used. Our system still has to be validated and there is room for improvement in the future. Nevertheless, the successful production of HSV-1 miRNA-expressing plasmids represents a significant advancement in detecting the molecular mechanism behind ADAR editing of HSV-1 miRNAs.Post-transkripcijske modifikacije u slijedu RNA nukleotida, poznate kao uređivanje RNA, uključuju inserciju, deleciju i substituciju nukleotida. Uređivanje može biti prisutno u protein-kodirajućim i nekodirajućim transkriptima, te posljedično utječe na njihovu stabilnost, prekrajanje, sposobnost regulacije genske ekspresije te čak može dovesti do rekodiranja proteina. Konverzija adenozina u inozin (A→I) u dvolančanim RNA, katalizirana adenozin deaminazama koje djeluju na RNA (ADAR), najučestalija je forma uređivanja RNA. Herpes simplex virus tip 1 (HSV-1), visoko prevalentni humani patogen, kodira više od 20 mikroRNA (miRNA). Zanimljivo je da su tijekom latentne faze jedino prisutni transkripti povezani s latencijom (LAT) te miRNA. Naša istraživačka skupina je nedavno detektirala A→I uređivanja u HSV-1 miRNA u latentno inficiranim humani terminalnim ganglijima. Visoko eksprimirana miR-H2, čija je meta ICP0, neposredno rani viralni gen ključan za viralnu replikaciju, je bila najčešće uređivana. Međutim, molekularni mehanizam ovog uređivanja još uvijek nije otkriven. Obzirom na to, generirali smo in vitro sustav za detekciju ADAR uređivanja u miRNA HSV-1, baziran na kotransfekciji miRNA-eksprimirajućih kostrukata s različitim ADAR-eksprimirajućim plazmidima. Okosnica miRNA ekspresijskog plazmida (ps-Cassette) je uspješno producirana kloniranjem sintetske ekspresijske kazete u pEGFP-N1 plazmid. Nadalje, miRNA ekspresijski plasmidi pS-miR-H2 te pS-miR-376a, koji je ujedno i pozitivna kontrola editiranja, su generirani kloniranjem miRNA kodirajućih fragmenata u pS-Cassette. Ekspresija pS-miR plazmida transfeciranih u HEK293T stanice nije potvrđena zbog kontaminacije izolirane RNA s plazmidnom DNA. Western blot analiza pokazala je kako je ekspresija endogenog ADAR1 p110 u HEK293 stanicama iznimno visoka što ukazuje da bi mogao neposredno doprinijeti RNA uređivanju u našem sustavu. Stoga je u budućnosti potrebno koristiti stanice s utišanim genom za ADAR1. Ovaj in vitro sustav još treba biti validiran te postoji mjesta za napredak u budućnosti. Usprkos tome, uspješna produkcija HSV-1 miRNA-ekspresijskih plazmida predstavlja značajan napredak u detekciji molekularnog mehanizma koji se krije iza ADAR uređivanja HSV-1 miRNA

Aggregation Studies of Protoporphyrin IX, Mesoporphyrin IX and Their Peptide Conjugates by Molecular Dynamics and UV/Vis Spectroscopy

Porfirini, klasa makrocikličkih spojeva, ključni su u biološkim procesima i primjenjuju se u katalizi i fotodinamičkoj terapiji (PDT) zbog svoje sposobnosti da proizvode reaktivne kisikove vrste (ROS), što je učinkovito u liječenju nekih tipova raka. S obzirom na rastući problem multirezistentnih mikroba i izbijanja virusnih infekcija, antimikrobna svojstva porfirina ponovno privlače pažnju. Međutim, njihov terapijski potencijal suočava se s problemom agregacije. Ovaj rad ima za cilj istražiti agregaciju antimikrobnih porfirina, konkretno protoporfirina IX (PPIX) i mesoporfirina IX (MPIX), te njihovih konjugata s peptidom AGILKRW (PepH3). Korištene su metode „all-atom“ simulacije molekularne dinamike i UV-Vis spektroskopije kako bi se razjasnili mehanizmi agregacije i učinci konjugacije s peptidom. Ciljevi UV-Vis spektroskopije uključuju određivanje spektralnih promjena kao indikacije agregacije. Peptid AGILKRW-Am sintetiziran je koristeći se metodom sinteze peptida na nosaču (eng. SPPS) i konjugacija sa PPIX je urađena dok je peptid još na nosaču. Uspješno sintetizirani konjugat PPIX-PepH3 potvrđen je MALDI-TOF-MS i LC- MS analizama. Spektroskopske analize PPIX-a u dimetil sulfoksidu, pokazale su prisutnost Soretov-og pojasa na 408 nm, koji se razdvaja i širi s povećanjem sadržaja vode, što ukazuje na agregaciju. Konjugacija peptida smanjila je ove spektralne promjene na blagi pomak k nižim valnim duljinama, što sugerira da konjugacija peptida može ublažiti agregaciju. Simulacije su otkrile da porfirini primarno agregiraju kroz π-π interakcije, formirajući dimere i veće agregate. Prisustvo peptida promijenilo je ovo ponašanje, dovodeći do formiranja globularnih struktura stabiliziranih dodatnim elektrostatskim i vodikovim vezama

HSV-1 infekcija dovodi do redistribucije jezgrene ADAR1 p110 izoforme

Herpes simplex virus 1 (HSV-1) is a dsDNA virus, which causes many different pathological conditions in the human population. The virus has the ability to modulate host immune response, which enables its replication. Adenosine deaminase acting on RNAs (ADAR) proteins are enzymes, whose main function is deamination of adenosine to inosine on dsRNA molecules, which consequently alters their structure and function. ADAR1 is the member of ADAR family, which is a known modulator of the immune response. Therefore, ADAR1 can exhibit proviral and antiviral effects, during viral infections. ADAR1 possesses two functionally active isoforms: p110 (constitutively expressed) and p150 (expression induced with the interferons (IFNs)). To add new insights to the HSV-1 field of research, we investigated whether ADAR1 proteins exhibit the ability to change their subcellular localisation during the early phase of HSV-1 productive infection. Firstly, by using the immunofluorescence and subcellular fractionation methods, we determined the ADAR1 expression profile of MOCK infected RPE1 cells. Results show that both p110 and p150 isoforms localise mainly in the nucleus of uninfected and unstimulated RPE1 cells. We also used immunofluorescence and subcellular fractionation to examine whether the translocation of ADAR1 proteins happens during first 24 hours after the infection. These results show that ADAR1 p110 isoform translocates from the nucleus to the cytoplasm, at a time point between 7 and 24 hours after infection. On the other hand, p150 isoform depletes in both the nucleus and the cytoplasm during the first day of the infection. Afterwards, we tried to compare cellular expression of ADAR1 isoforms between MOCK and HSV-1 infected RPE1 cells. Also, we analysed expression profile of ADAR1 proteins, during first 24 hours after infection, in several different cell lines. These data, together with the ADAR1 transcriptome analysis, show that the expression of ADAR1 proteins does not elevate during the early phase of HSV-1 productive infection. Nonetheless, increased expression of both ADAR1 isoforms, induced by the IFN-β, does not contribute to increased amounts of ADAR1 proteins in the cytoplasm. This was confirmed by the immunoflurescence and Western blot method. Overall, described results indicate that ADAR1 p110 translocates from the nucleus to the cytoplasm of RPE1 cells, and that this phenotype is not caused by the increase in protein's expression.Herpes simpleks virus 1 (HSV-1) je dvolančani DNA virus, koji uzrokuje više različitih patofizioloških stanja u ljudskoj populaciji. Virus ima sposobnost moduliranja imunosnog odgovora domaćina, što omogućuje njegovu replikaciju. Adenozin deaminaza koja djeluje na RNA (engl. Adenosine deaminase acting on RNAs, ADAR) je grupa proteina s enzimatskom funkcijom deaminacije adenozina u inozin, na dvolančanim RNA molekulama, što posljedično mijenja njihovu strukturu i funkciju. ADAR1 je član ADAR obitelji te znani modulator imunosnog odgovora. Stoga, ADAR1 može pokazivati proviralne i antiviralne učinke, tijekom virusnih infekcija. ADAR1 posjeduje dvije funkcionalno aktivne izoforme: p110 (konstitutivno eksprimiran u stanicama) te p150 (ekspresija potaknuta interferonima (IFN)). Kako bi dodali nova saznanja u područje istraživanja koje se bavi s HSV-1 virusom, istražili smo mogu li ADAR1 proteini pokazivati mogućnost promjene njihovog unutarstaničnog položaja, tijekom rane produktivne faze HSV-1 infekcije. Najprije smo, korištenjem imunofluorescencije te metode unutastaničnoga frakcioniranja , odredili ADAR1 ekspresijski profil u RPE1 stanica, koje nisu bile niti tretirane niti inficrane (engl. „MOCK“). Rezultati ukazuju na to da su obje izoforme ADAR1 proteina ponajviše eksprimirane u jezgri MOCK inficiranih RPE1 stanica. Metode imunofluorescnecije te unutarstaničnoga frakcioniranja koristili smo i za ispitivanje moguće translokacije ADAR1 proteina, unutar prva 24 sata nakon infekcije. Dobiveni rezultati pokazuju da ADAR1 p110 izoforma prelazi iz jezgre u citoplazmu inficiranih stanica, u vremenskom intervalu između 7 i 24 sata nakon infekcije. Nasuprot tome, ekspresija p150 izoforme se smanjuje tijekom prvoga dana infekcije, u jezgri kao i u citoplazmi. Nakon toga, pokušali smo usporediti staničnu ekspresiju ADAR1 proteina između MOCK te inficiranih RPE1 stanica. Također smo analizirali razinu ekspresije proteina, tijekom prva 24 sata infekcije, u različitim staničnim linijama. Svi dobiveni rezultati, uz analizu ADAR1 transkripta, ukazuju na to da se ekspresija ADAR1 proteina ne povećava tijekom rane, produktivne HSV-1 infekcije. No ipak, povećana ekspresija obje ADAR1 izoforme, potaknuta s IFN-β, ne doprinosi povećanoj količini ADAR1 proteina u citoplazmi. Taj rezultat je potvrđen preko imunofluorescencije i „Western blot“ metode. Generano, opisani rezultati ukazuju na translokaciju ADAR1 p110 proteina iz jezgre u citoplazmu RPE1 stanica te da navedeni fenotip nije uzrokovan povećanom ekspresijom proteina

Ekstrakcija kolagena iz meduza: optimizacija metode

Jellyfish are a novel source for collagen extraction, and it is derived from multiple Medusozoa species, including Rhizostoma pulmo. Their collagen is evolutionary older and simpler than mammalian collagens making them compatible with human biology. Its specific response to macrophages - a lower M1 macrophage response and a higher M2 macrophage response - triggers regeneration in tissue, making jellyfish collagen a candidate biomaterial for tissue engineering. To compare the collagen composition, multiple sequence alignment was performed between different types of human collagen and between murine collagen using SALIGN. Sequence alignment of fibrillar collagens showed similarities in characteristic glycine and proline regions, and in lysine sites in the sequence. Amino acid analysis showed several differences in composition between different collagen sources. The extraction of collagen from jellyfish consists of three main steps: sample preparation, extraction, and recovery, and can last up to 7 days. This lengthy procedure can lead to collagen degradation and a decrease in final yield. Current methods consist of tissue cutting and chemical pretreatment during sample preparation, acid and enzymatic extraction, and dialysis. The goal of this research was to see how changes in the extraction steps affect collagen yield, purity, and the methods duration. For this purpose, samples from Rhizostoma pulmo were first lyophilized, followed by acid extraction with 0,5 M acetic acid, salting out with NaCl, and purification with C18 cartridges. The obtained collagen powder was analyzed using MALDI-TOF-MS to confirm the successful extraction of collagen. In addition, FTIR and SDS-PAGE were performed to further confirm the presence of collagen. The collagen yields that were obtained are similar to those recorded in previous studies. Based on these results, it can be concluded that the use of C18 cartridges can be an alternative for dialysis and could shorten the extraction process.Meduze su nov izvor kolagena, a kolagen se dobiva iz nekoliko vrsta Medusozoa, uključujući Rhizostoma pulmo. Kolagen iz meduza je evolucijski stariji i jednostavniji od kolagena sisavaca, što ga čini kompatibilnim s ljudskom biologijom. Njegov specifičan odgovor na makrofage - niži odgovor M1 makrofaga i viši odgovor M2 makrofaga - potiče regeneraciju tkiva, što čini kolagen meduza potencijalnim biomaterijalom u tkivnom inženjerstvu. Da bi se usporedio sastav kolagena, višestruko poravnanje sekvenci (multiple sequence alignment) je izvršeno između različitih tipova ljudskog kolagena i između mišjeg kolagena koristeći SALIGN. Poravnanje sekvenci fibrilarnih kolagena pokazalo je sličnosti u karakterističnim glicinskim i prolinskim regijama te u mjestima na kojima su lizini u sekvencama. Analiza aminokiselina pokazala je nekoliko razlika u sastavu između različitih izvora kolagena. Ekstrakcija kolagena iz meduza sastoji se od tri glavna koraka: pripreme uzorka, ekstrakcije i oporavka, i može trajati do 7 dana. Ovaj dugotrajan postupak može dovesti do degradacije kolagena i smanjenja konačnog prinosa. Trenutne metode uključuju usitnjavanje tkiva i kemijsko tretiranje tokom pripreme uzorka, kiselinsku i enzimsku ekstrakciju, kao i dijalizu. Cilj ovog istraživanja bio je utvrditi kako promjene u koracima ekstrakcije utiču na prinos, čistoću kolagena i trajanje metode. U tu svrhu, uzorci vrste Rhizostoma pulmo su prvo liofilizirani, nakon čega je uslijedila ekstrakcija kiselinom koristeći 0,5 M octenu kiselinu, isoljavanje pomoću NaCl i pročišćavanje uz pomoć C18 kolona. Dobiveni kolagen je analiziran pomoću MALDI-a kako bi se potvrdila uspješna ekstrakcija kolagena. Također, FTIR i SDS-PAGE su odrađeni radi dodatne potvrde prisustva kolagena u izolatu. Prinosi kolagena koji su dobiveni slični su onima zabilježenim u prethodnim studijama. Na osnovu ovih rezultata, može se zaključiti da upotreba C18 kolona može biti alternativa za dijalizu i mogla bi skratiti proces ekstrakcije

Interakcija TDP-43 i optineurina u modleima amiotreofične lateralne skleroze

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.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

Neutralization of mouse cytomegalovirus variants by antibodies

Razvoj cjepiva protiv humanog citomegalovirusa (HCMV) javnozdravstveni je prioritet obzirom da virus globalno uzrokuje ozbiljne posljedice kao što su povećana smrtnost kod imunosuprimiranih pojedinaca i teške neurološke posljedice kod novorođenčadi, poput gubitka sluha i mentalne retardacije. Velik broj cjepiva za citomegalovirus je testiran u pretkliničkim i kliničkim istraživanjima, no trenutno nema odobrenog cjepiva i terapija je usmjerena na suzbijanje virusa i smanjenje simptoma infekcije. Razlog za to se djelomično nalazi u činjenici da je humoralni odgovor na infekciju citomegalovirusom nedovoljno istražen. Infekcija mišjim citomegalovirusom je najčešći korišteni model za infekciju humanim citomegalovirusom. U ovom radu korištene su ženke, 8 – 12 tjedana starosti, imunizirane s divljim tipom virusa (WT MCMV), te njihovo potomstvo, neonatalni miševi inficirani s različitim sojevima i različitim virusnim dozama. Titar sojeva virusa u organima neonatalnih miševa određen je pomoću testa virusnih čistina, a neutralizacijski kapacitet transplacentarno prenesenih protutijela određen je uz pomoć neutralizacijskog testa. Fenotipska analiza mikroglije provedena je pomoću protočne citometrije. Rezultati ovog diplomskog rada pokazuju učinkovitu neutralizaciju različitih varijanti mišjeg citomegalovirusa protutijelima prenesenim od majke inficirane divljim tipom virusa, što za posljedicu ima inhibiciju replikacije virusa u različitim organima. Daljnja istraživanja su potrebna za bolje razumijevanje uloge protutijela u sprječavanju širenja virusa, za njihovu dugoročnu učinkovitost protiv različitih varijanti te usporedbu rezultata između modela i kliničkih istraživanja.The development of a vaccine against human cytomegalovirus (HCMV) is a public health priority, as the virus globally causes serious consequences such as increased mortality in immunosuppressed individuals and severe neurological sequelae in newborns, such as hearing loss and mental retardation. A large number of vaccines for cytomegalovirus have been tested in preclinical and clinical research. However, currently, there is no approved vaccine, and therapy is aimed at suppressing the virus and reducing infec tion symptoms. The reason for this is partly due to the fact that the humoral response to cytomegalovirus infection has not been sufficiently investigated. Infection with murine cytomegalovirus (MCMV) is the most commonly used model for human cytomegalovir us infection. Females, 8 12 weeks old, immunized with the wild type virus (WT MCMV), along with their offspring, were used for this thesis, while neonatal mice were infected with different virus strains and different viral doses. The virus titer was determ ined using the plaque assay, while the neutralization capacity was determined using the neutralization assay in the presence of immune serum or serum from non immunized animals. Phenotypic analysis of the expression of MHC molecules on microglia was conduc ted using a flow cytometer. The results of this thesis show the effective neutralization of different variants of murine cytomegalovirus by antibodies transferred from a mother infected with the wild type virus, which results in the inhibition of viral rep lication in different organs. Further research is required to better understand the role of antibodies in preventing the spread of the virus, their long term effectiveness against different variants, and to correlate results obtained in mouse models and clinical studies

Uloga optineurina u fagocitozi fibrilarnog amiloida b u makrofagima i dendritičkim stanicama proizvedenim iz koštane srži

Optineurin is a multifunctional ubiquitin-binding adaptor protein involved in regulation of cellular processes, for instance vesicle trafficking, autophagy and inflammatory signalling. The mutations of the OPTN gene that encodes for optineurin were linked to several neurodegenerative diseases, the most important being amyotrophic lateral sclerosis (ALS), which leads to destruction of motor neurons. Research has shown that phagocytosis, a process of internalizing and degrading particles larger than 0.5 µm in diameter, is disrupted in various neurodegenerative diseases, including ALS and Alzheimer’s disease (AD). Previous research in our lab showed no effect of optineurin during phagocytosis of dead neurons and pHrodo™ Green E. coli BioParticles™. The main goal of this thesis was to investigate the role optineurin has in phagocytosis of fibrillar amyloid beta (A-beta) . To investigate this, we performed a flow cytometry analysis of the phagocytosis assay of fluorescent A-beta in wild type (WT) and optineurin truncation Optn470T-carrying bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs). Optn470T mice mimic the C-terminal truncations found in a small percentage of ALS patients. Our results showed no difference between BMDM genotypes in the number of phagocytic cells, but there was a decrease of ~20% in the phagocytic capacity of Optn470T cells compared to WT BMDMs. There was also a very significant difference between WT and Optn470T BMDC in both the number of phagocytosing cells and their phagocytic ability. Optn470T BMDCs had a ~15-20% lower number of phagocytosing cells than WT BMDCs. Their phagocytic ability was ~30-40% lower than that of WT cells. The differences between the genotypes did not significantly increase if the cells were pretreated with inflammatory stimuli. In conclusion, this study demonstrates that optineurin has a role in phagocytosis of fibrillar fA-beta in BMDCs, opening a possibility that this is the mechanism by which optineurin contributes to disease pathogenesis.Optineurin je multifunkcionalni adapterski protein koji veže ubikvitin. Uključen je u regulaciju staničnih procesa kao što je upalna signalizacija, promet vezikulama i autofagija. Mutacije u OPTN genu koji kodira za optineurin povezane su s nekoliko neurodegenerativnih bolesti, od kojih je najvažnija amiotrofična lateralna skleroza (ALS), koja dovodi do odumiranja motornih neurona. Istraživanja su pokazala da je fagocitoza, proces internalizacije i razgradnje čestica većih od 0,5 µm u promjeru, poremećena u raznim neurodegenerativnim bolestima uključujući ALS i Alzheimerovu bolest (AD). Prethodna istraživanja u našem laboratoriju nisu pokazala nikakav učinak optineurina tijekom fagocitoze mrtvih neurona i pHrodo™ Green E. coli BioParticles™. Cilj ovog diplomskog rada bio je istražiti ulogu optineurina u fagocitozi fibrilarnog amiloida beta (A-beta). U tu smo svrhu proveli analizu fagocitoze fluorescentnog A-beta na stanicama divljeg tipa (WT) i stanicama s trunkacijom optineurina (Optn470T) na temelju protočne citometrije. Korišteni su makrofagi (BMDM) i dendritičke stanicame (BMDC) porijeklom iz koštane srži. Optn470T miševi oponašaju skraćenja C-terminala pronađena u maloj podskupini pacijenata s ALS-om. Naši rezultati nisu pokazali razliku između genotipova BMDM u broju fagocitnih stanica, ali došlo je do smanjenja od ~20% u fagocitnom kapacitetu Optn470T stanica u usporedbi s WT BMDM-ovima. Nadalje, postojala je značajna razlika između WT i Optn470T BMDC-a u broju fagocitoznih stanica i njihovoj fagocitnoj sposobnosti. Optn470T BMDC-i imali su ~15-20% niži broj stanica koje fagocitiraju od WT BMDC. Njihova fagocitna sposobnost bila je ~30-40% niža nego kod WT stanica. Razlike između genotipova nisu se značajno povećale ako su stanice prethodno tretirane upalnim podražajima. Zaključno, ova studija pokazuje da optineurin ima ulogu u fagocitozi fibrilarnog fA-beta u BMDC, otvarajući mogućnost da je to mehanizam kojim optineurin doprinosi patogenezi bolesti

Validacija novog staničnog modela za mapiranje interaktoma Q398X mutacije optineurina iz ALS bolesnika

Amyotrophic lateral sclerosis (ALS) is a progressive adult-onset neurodegenerative disease of upper and lower motor neurons that leads to a complete paralysis and death usually in 2-5 years from the disease onset. Although decades of research have been invested in understanding the ALS pathogenesis, the extremely heterogenous genetic background, with more than 20 genes being implicated, makes it difficult to pinpoint the exact mechanisms that lead to development of this disease. Optineurin protein, encoded by the OPTN gene, has been linked to ALS in a small subset of patients. It was suggested that OPTN acts solely through a loss-of-function mechanism in contrast to a toxic gain-of-function mechanism that was suggested for more common genes mutated in ALS, such as C9ORF72, SOD1 and TARDBP. We decided to apply the proximity-dependent labeling method to study the interactome of Q398X optineurin, a mutation that has been found in ALS patients. For that purpose, we generated new HEK293 Flp-In cell models that upon doxycycline treatment express constructs that carry BioID2 enzyme linked to the N-terminus of the wild-type or Q398X optineurin. Our results demonstrated that doxycycline successfully induced the expression of inserted constructs and that BioID2 enzyme was able to conjugate biotin to a wide range of proteins. Additionally, we detected interactions between both wild-type or Q398X optineurin and TBK1 demonstrating that linkage of BioID2 and Myc tag to optineurin N-terminus did not disturb its physiological interactions. Unexpectedly, we found that wild-type optineurin interacted with p-p65, but the Q398X optineurin did not. Although this was likely an indirect interaction of wild-type optineurin, it highlighted the possibility that Q398X mutation loses some relevant interactions in the NF-κB pathway, which contributes to the ALS development. On the other hand, we found no interaction between wild-type or Q398X optineurin with RIPK1, which we attributed to a small labeling radius of BioID2 that was likely insufficient for labeling interaction partners binding to the C-terminus of optineurin. This signified the importance of generating additional cell lines with BioID2 linked to optineurin C-terminus. Combined with our existing cell lines, they should enable the mapping of complete interactomes of wild-type and Q398X optineurin using mass spectrometry and hopefully contribute to the understanding of mechanisms by which Q398X optineurin causes ALS.Amiotrofična lateralna skleroza (ALS) je progresivna neurodegenerativna bolest odrasle dobi koja zahvaća gornje i donje motoričke neurone. Vodi do potpune paralize i smrti uglavnom u roku 2-5 godine nakon početka bolesti. Iako su već desetljeća istraživanja posvećena razumijevanju patogeneze ALS-a, izuzetna heterogenost genetske pozadine, s više od 20 gena vezanih s njezinim nastankom, stvara teškoće u razumijevanju točnih mehanizama koji vode ka razvoju ove bolesti. Protein optineurin, kojeg kodira gen OPTN, povezan je s ALS-om u maloj skupini bolesnika. Pretpostavlja se da OPTN djeluje isključivo mehanizmom gubitka funkcije (eng. loss-of-function) za razliku od djelovanja putem dobitka novih toksičnih funkcija (eng. gain-of-function) koji je bio predložen za učestalije mutirane gene u ALS-u poput C9ORF72, SOD1 i TARDBP. Odlučili smo koristiti metodu obilježavanja na temelju bliskosti (eng. proximity-dependent labeling) za istraživanje interaktoma Q398X optineurina, mutacije pronađene u pacijentima oboljelih od ALS-a. U tu smo svrhu napravili nove HEK293 Flp-In stanične modele koji po tretmanu s doksiciklinom eksprimiraju proteinske konstrukte koji sadrže optineurin divljeg soja ili Q398X povezan s BioID2 enzimom na N-kraju. Naši su rezultati pokazali da doksiciklin uspješno izaziva ekspresiju umetnutih konstrukata te da BioID2 enzim biotinilira širok raspon proteina. Nadalje, uočili smo interakcije optineurina divljeg soja i Q398X s TBK1, što je pokazalo da vezanje BioID2 enzima i Myc oznake za N-kraj optineurina nije omelo njegove fiziološke interakcije. Neočekivano, pronašli smo da optineurina divljeg soja ulazi u interakciju s p-p65, no ta interakcija nije pronađena u Q398X mutanta. Iako je posrijedi vjerojatno bila indirektna interakcija s optineurinom divljeg soja, ovo saznanje je istaknulo mogućnost da Q398X mutacija gubi neke značajne interakcije u NF-κB signalnom putu i time doprinosi razvoju ALS-a. S druge strane, interakcije s RIPK1 nisu pronađene u optineurina divljeg soja ni u Q398X mutacije, što smo pripisali malenom radijusu obilježavanja BioID2 koji je vjerojatno bio nedovoljan da obuhvati interakcije s partnerima koji se vežu za C-terminalni dio optineurina. To je istaknulo potrebu za stvaranjem dodatnih staničnih linija sa BioID2 enzimom vezanim za C-kraj optineurina. Te bi stanične linije, u kombinaciji s već postojećima, omogućile mapiranje cjelovitog interaktoma optineurina divljeg soja i Q398X mutacije pomoću masene spektrometrije te, nadajmo se, doprinijele razumijevanju mehanizama kojima Q398X optineurin uzrokuje ALS

The role of NK and ILC1 cells in the control of murine cytomegalovirus during ontogeny

Ljudski citomegalovirus (HCMV) je jedan od najrasprostranjenijih virusa diljem svijeta. Njegova raširenost predstavlja veliki problem u zdravstvenom sektoru zbog toga što može nakon infekcije ostaviti ozbiljne posljedice, pogotovo ukoliko dođe do prijenosa virusa s trudnice na plod, čime uzrokuje prirođenu infekciju HCMV-om koja može imati i letalan ishod. Imunosni sustav u prenatalnom i ranom postnatalnom razdoblju nije razvijen kao u odraslih te je nedostatak zrelosti razlog tome što infekcije u tom periodu mogu uzrokovati ozbiljne posljedice. Mehanizmi kako nerazvijeni imunosni sustav, odnosno prirođena imunost, štiti plod i novorođenčad od infekcija različitim patogenima su slabo istraženi. Za potrebe istraživanja HCMV-a koristi se mišjicitomegalovirus (MCMV) s kojim dijeli mnoge sličnosti u temeljnoj biologiji i patogenezi, te zbog toga infekcija miševa MCMV-om predstavlja najčešće korišteni model infekcije citomegalovirusom. U ovom istraživanju koristili smo transgenične miševe, pristup uklanjanja imunoloških stanica monoklonskim protutijelima, te rekombinantni MCMV kako bi istražili ulogu stanica NK i ILC1 u nadzoru infekcije MCMV-om u novookoćenih miševa. Transgenične miševe smo genotipizirali PCR-om, prisustvo stanica NK i ILC1 odredili smo metodom protočne citometrije, dok smo testom virusnih čistina odredili količinu virusa u ispitivanim organima. Rezultati ovog rada pokazali su kako stanice urođene imunosti, stanice NK i ILC1, imaju važnu ulogu u nadzoru infekcije MCMV-om u jetri novookoćenih miševa. Daljnja istraživanja će pružiti bolje razumijevanje neonatalnog imunosnog sustava.Human cytomegalovirus (HCMV) is one of the most widespread viruses worldwide. It is a major problem in healthcare because the infecion can leaveserious consequences, especially if the virus is transmitted from the pregnant woman to the fetus, i.e., if it causes congenital HCMV infection that can havea fatal outcome. The immune system in the prenatal and early postnatal period is immature as compared to adults, and the immaturity is the reason why infections in that period can result in serious consequences. The mechanisms by which an underdeveloped immune system, i.e. innate immunity, protects the fetus and newborns from infections by various pathogens have been poorly investigated. To study HCMV, murine cytomegalovirus (MCMV) is often used, which shares many biological similarities and pathogenesis; therefore, infection of mice with MCMV is the most commonly used model of cytomegalovirus infection. In this study, we used transgenic mice, an immune cell depletion approach with monoclonal antibodies, and recombinant MCMV to investigate the role of NK cells and ILC1 cells in the control of MCMV infection in newborn mice. The transgenic mice were genotyped by PCR, the presence of NK and ILC1 cells was determinedby flow cytometry method, while the levels of virus in the examined organs was determined by the plaque assay. The results of this work showed that innate immune cells, NK cells and ILC1, play an important role in controlling MCMV infection in the liver of newborn mice. Further research will provide a better understanding of the neonatal immune system