%0 Journal Article %A Mirta Boban %A Terezija Miškić %A Mirjana Babić Leko %A Patrick R. Hof %A Goran Šimić %T Human neuroblastoma SH-SY5Y cells treated with okadaic acid express phosphorylated high molecular weight tau-immunoreactive protein species %D 2018 %R 10.1101/284265 %J bioRxiv %P 284265 %X Recent data suggest that early stages of Alzheimer’s disease (AD) are characterized by an abnormally high phosphorylation of microtubule-associated protein tau and truncation of its C-terminus. Tau hyperphosphorylation may result from the downregulation of phosphatases, especially protein phosphatase 2A. In an attempt to model and analyze these molecular events we treated SH-SY5Y cells with okadaic acid (OA), an inhibitor of protein phosphatases. In addition to the low molecular weight tau, such treatment lead to the appearance of heat-stable protein species with apparent high molecular weight around 100 kDa, which were immunoreactive to tau antibodies against phosphorylated Ser202 and phosphorylated Ser396. Based on the observation that high molecular weight tau-immunoreactive proteins (HMW-TIP) correspond to the predicted size of two monomers of tau, one possibility is that HMW-TIP represent tau oligomers. The absence of HMW-TIP detection by anti-total tau antibodies used may be a consequence of epitope masking, or a combination of epitope masking and protein truncation. We noted the stability of HMW-TIP in the presence of strong denaturing agents, such as urea and guanidine, as well as upon partial dephosphorylation by the alkaline phosphatase. Moreover, as HMW-TIP did not dissociate the presence of β-mercaptoethanol, it was also independent from disulfide bonds. Taken together, these data show that OA treatment of SH-SY5Y cells induces the appearance of HMW-TIP, which may represent tau oligomer or tau-crossreactive phospho-proteins. Our findings have implications for further studies of tau under the conditions of protein phosphatase downregulation.ADAlzheimer’s diseaseBDNFbrain-derived neurotrophic factorCP13antibody against phosphorylated tau Ser-202CP27antibody against total tau (aa 130-150)DMEMDulbecco’s modified Eagle mediumDMSOdimethyl sulfoxideEDTAethylenediaminetetraacetic acidFBSfetal bovine serumGAPDHglyceraldehyde 3-phosphate dehydrogenaseGSK-3βglycogen synthase kinase 3βHMW-TIPhigh molecular weight tau-immunoreactive proteinHSheat-stable (fraction)MAPmicrotubule-associated proteinMTBmicrotubule binding (repeat regions)NFTneurofibrillary tanglesOAokadaic acidPBSphosphate-buffered salinePP2Aprotein phosphatase 2ARIPAradioimmunoprecipitation assay bufferSDS-PAGEsodium dodecyl sulfate – polyacrylamide gel electrophoresisTau5antibody against the central part of tau (aa 210-241)Tau13antibody against the N-terminal part of tau (aa 2-18)Tau46antibody against the C-terminal part of tau (aa 404-441)TBSTris-buffered saline %U https://www.biorxiv.org/content/biorxiv/early/2018/03/19/284265.full.pdf