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Vloga katepsina X v aktivaciji mikroglije, posredovani z agonisti TLR receptorjev
ID Pertoci, Vesna (Avtor), ID Pišlar, Anja (Mentor) Več o mentorju... Povezava se odpre v novem oknu

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Izvleček
Dobro uravnavana aktivacija mikroglije je izjemnega pomena za zaščito centralnega živčnega sistema pred različnimi grožnjami. Slabo uravnavana in prekomerna aktivacija mikroglije privede do nastanka pretiranega vnetnega odziva, ki škoduje nevronom in povzroča nevrodegeneracijo, ki vodi v razvoj življenjsko ogrožajočih bolezenskih stanj, kot je Parkinsonova bolezen (PB). Predhodne raziskave so že pokazale, da aktivirana mikroglija v svojo okolico poleg vnetnih dejavnikov in citokinov sprošča tudi cisteinske katepsine, med njimi tudi katepsin X. Za slednjega je bilo pokazano, da je pomemben regulator nevrodegeneracije, povzročene z vnetnimi procesi, v katerih sodelujejo celice mikroglije. V sklopu magistrske naloge smo podrobneje raziskali vpliv aktivacije mikroglije na vnetni odziv preko sočasne aktivacije »Toll-u« podobnih receptorjev (TLR), in sicer TLR3 in TLR4, obenem pa smo opredelili vlogo katepsina X v tem procesu. Poskuse smo izvedli na celičnem modelu aktivirane mikroglije z uporabo celic BV2, ki smo jih stimulirali z agonistoma receptorjev TLR3 in TLR4, in sicer s poly(I:C) in LPS. S pomočjo spektrofotometrije in pretočne citometrije smo preverili vpliv aktivacije mikroglije, v prisotnosti ali odsotnosti zaviralca katepsina X, AMS36, na celično preživetje in nivo vnetnega odziva. Spektrofotometrično smo določili tudi aktivnost kaspaze-3 in katepsina X, medtem ko smo izražanje katepsina X preverili z uporabo encimsko- imunskega testa na trdni podlagi (ELISA), njegovo znotrajcelično lokalizacijo pa z uporabo imunofluorescenčne konfokalne mikroskopije. Za določanje izražanja inducibilne NO sintaze (iNOS) smo uporabili prenos western. Sočasna aktivacija TLR3 in TLR4 z agonistoma poly(I:C) in LPS je v primerjavi z enostransko stimulacijo s posameznim agonistom izkazovala močnejši, sinergistični učinek na aktivacijo celic mikroglije BV2, opredeljeno preko merjenja koncentracije sproščenih vnetnih dejavnikov, in sicer dušikovega oksida (NO), interlevkina-6 (IL-6) in tumorje-nekrotizirajočega faktorja ? (TNF-?). Meritve izražanja in aktivnosti katepsina X v celičnih lizatih in supernatantih aktivirane mikroglije so pokazale, da sočasna aktivacija TLR3 in TLR4 s poly(I:C) in LPS v približno enaki meri kot enostranska stimulacija z LPS značilno poveča nivo proteina kot tudi aktivnost katepsina X v supernatantih celic BV2, medtem ko se njegova aktivnost v celicah značilno zniža. Z opazovanjem znotrajcelične lokalizacije katepsina X smo pokazali, da ko-aktivacija celic mikroglije značilno poveča lokalizacijo katepsina X ob plazemski membrani in hkrati zmanjša lokalizacijo slednjega v lizosomskih veziklih, kar dodatno nakazuje na znotrajcelično translokacijo katepsina X v aktivirani mikrogliji. Z uporabo ireverzibilnega specifičnega zaviralca katepsina X, AMS36, smo v nadaljevanju potrdili vlogo katepsina X kot mediatorja aktivacije mikroglije, saj je prisotnost AMS36 pomembno zmanjšala koncentracijo sproščenih vnetnih dejavnikov NO, IL-6 in TNF-? iz ko-aktiviranih celic mikroglije, hkrati pa je AMS36 vplival tudi na zmanjšano izražanje iNOS po ko-stimulaciji mikroglije. Zaviranje katepsina X je zmanjšalo tudi delež celične smrti in apoptozo aktivirane mikroglije, spodbujene s sočasno stimulacijo z LPS in poly(I:C), hkrati pa je bil opazen vpliv AMS36 na povišano aktivnost kaspaze-3 v aktivirani mikrogliji. Nenazadnje smo na ko-kulturnem modelu celic mikroglije BV2 in nevronskih celic SH-SY5Y pokazali tudi zaščitno delovanje zaviralca katepsina X na nevronske celice. Dodatek AMS36 pred ko-stimulacijo celic mikroglije z LPS in poly(I:C) je namreč značilno zmanjšal degeneracijo nevronskih celic, ki so bile izpostavljene supernatantu aktivirane mikroglije. Rezultati, pridobljeni v sklopu magistrske naloge, torej nakazujejo na pomembno vlogo katepsina X pri ko-aktivaciji mikroglije in uravnavanju vnetnega odziva, zato katepsin X predstavlja pomembno tarčo pri načrtovanju strategij zdravljenja nevrodegenerativnih bolezni povzročenih z vnetjem, kot je PB.

Jezik:Slovenski jezik
Ključne besede:katepsin X, mikroglija, agonisti receptorjev TLR, vnetni odziv, zaviralec katepsina X
Vrsta gradiva:Magistrsko delo/naloga (mb22)
Organizacija:FFA - Fakulteta za farmacijo
Leto izida:2019
Datum objave v RUL:28.06.2019
Število ogledov:704
Število prenosov:215
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Sekundarni jezik

Jezik:Angleški jezik
Naslov:Role of cathepsin X in microglia activation mediated through TLR agonists
Izvleček:
Well-regulated microglial activation is important for the protection of the central nervous system against various threats. Poorly regulated and excessive microglial activation leads to an exaggerated inflammatory response, which damages the neurons and causes neurodegeneration, which in turn, leads to the development of life-threatening medical conditions, such as Parkinson's disease. Previous research has shown that activated microglia releases in addition to inflammatory factors and cytokines also cysteine cathepsins, including cathepsin X. The latter has already been designated as an important regulator of neurodegeneration, caused by an inflammatory process mediated by microglial cells. In the scope of the Master's thesis, the effect of the microglial activation through simultaneous activation of Toll-like receptors (TLR), TLR3 and TLR4 on the resulting inflammatory response was studied in detail as well as we defined the role of cathepsin X in this process. We performed the experiments on a cellular model of activated microglia using BV2 cells, that were stimulated with TLR3 and TLR4 agonists, respectively with poly(I:C) and LPS. With the help of spectrophotometry and flow cytometry, we examined the effect of microglial activation, in the presence or absence of cathepsin X inhibitor, AMS36 on cell survival and the level of inflammatory response. We also spectrophotometrically determined the activities of caspase-3 and cathepsin X, while the analysis of the cathepsin X expression was performed with the use of enzyme-linked immunosorbent assay (ELISA) and its intracellular localization with the use of immunofluorescent confocal microscopy. To determine the expression of inducible NO synthase (iNOS) we used western blot. Concurrent activation of TLR3 and TLR4 with poly (I:C) and LPS agonists exhibited a stronger, synergistic effect on the activation of microglial BV2 cells, compared to one-sided stimulation with a single agonist, which we determined by measuring the concentrations of released inflammatory factors, namely nitric oxide (NO), interleukin-6 (IL-6) and tumor necrosis factor ? (TNF-?). Measurements of the expression and activity of cathepsin X in cell lysates and supernatants of stimulated BV2 cells showed that co-activation of TLR3 and TLR4, with poly(I:C) and LPS, significantly increased the level as well as the activity of cathepsin X in BV2 cell supernatants, in approximately the same extent as one-sided stimulation with LPS, while the intracellular protein's activity was significantly decreased. Moreover, the intracellular localization analysis of cathepsin X showed that localization of cathepsin X is significantly increased at the plasma membrane after co-activation of microglial cells by LPS and poly(I:C), while the cathepsin X localization within the lysosomal vesicles is decreased, which further indicates the subcellular translocation of cathepsin X in activated microglia cells. Additionally, we confirmed the role of cathepsin X as a mediator of microglial activation by the use of the specific, irreversible inhibitor of cathepsin X AMS36. The presence of the AMS36 significantly reduced concentrations of released inflammatory factors, including NO, IL-6 and TNF-? from activated microglia, while at the same time AMS36 also influenced and reduced the expression of iNOS in microglial cells after co-stimulation with LPS and poly(I:C). Inhibition of cathepsin X also decreased cell death and apoptosis of activated microglia cells mediated by simultaneous stimulation with LPS and poly(I:C), and AMS36 also visibly affected the increased activity of caspase-3 in activated microglia. Last but not least we also demonstrated the protective action of cathepsin X inhibitor on neuronal cells, with the use of a co-culture model using microglial BV2 cells and neuronal SH-SY5Y cells. Indeed, the addition of AMS36 to microglial cells, simultaneously stimulated with LPS and poly(I:C), significantly reduced degeneration of neuronal cells, exposed to supernatants of co-activated microglia. Results, obtained in the scope of research for the Master's thesis, therefore indicate that cathepsin X plays an important role in the co-activation of microglia and regulation of resulting inflammatory response, which defines cathepsin X as an important target in the development of strategies for the treatment of neurodegenerative diseases caused by inflammation, such as Parkinson's disease.

Ključne besede:cathepsin X, microglia, Toll-like receptor agonists, inflammatory response, cathepsin X inhibitor

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