Human neuroblastoma SH-SY5Y cells treated with okadaic acid express phosphorylated high molecular weight tau-immunoreactive protein species

Boban, Mirta and Babić Leko, Mirjana and Miškić, Terezija and Hof, Patrick R. and Šimić, Goran (2019) Human neuroblastoma SH-SY5Y cells treated with okadaic acid express phosphorylated high molecular weight tau-immunoreactive protein species. Journal of Neuroscience Methods, 319. pp. 60-68. ISSN 0165-0270

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Abstract

Background: Early stages of Alzheimer's disease (AD) are characterized by high phosphorylation of microtubule-associated protein tau, which may result from the downregulation of protein phosphatases. ----- New method: In order to model phosphatase downregulation and analyze its effect on tau aggregation in vitro, we treated neuroblastoma SH-SY5Y cells with okadaic acid (OA), a protein phosphatase inhibitor, and examined high molecular weight phospho-tau species. ----- Results and comparison with existing methods: OA treatment led to the appearance of heat-stable protein species with apparent molecular weight around 100 kDa, which were immunoreactive to anti-tau antibodies against phosphorylated Ser202 and Ser396. As these high molecular weight tau-immunoreactive proteins (HMW-TIPs) corresponded to the predicted size of two tau monomers, we considered the possibility that they represent phosphorylation-induced tau oligomers. We attempted to dissociate HMW-TIPs by urea and guanidine, as well as by alkaline phosphatase treatment, but HMW-TIPs were stable under all conditions tested. These characteristics resemble properties of certain sodium dodecyl sulfate (SDS)-resistant tau oligomers from AD brains. The absence of HMW-TIPs detection by anti-total tau antibodies Tau46, CP27 and Tau13 may be a consequence of epitope masking and protein truncation. Alternatively, HMW-TIPs may represent previously unreported phosphoproteins cross-reacting with tau. ----- Conclusions: Taken together, our data provide a novel characterization of an OA-based cell culture model in which OA induces the appearance of HMW-TIPs. These findings have implications for further studies of tau under the conditions of protein phosphatase downregulation, aiming to explain mechanisms involved in early events leading to AD.

Item Type: Article
MeSH: Alzheimer Disease / enzymology ; Antibodies ; Cell Line, Tumor ; Enzyme Inhibitors / administration & dosage ; Humans ; Models, Biological ; Okadaic Acid / administration & dosage ; Phosphoprotein Phosphatases / antagonists & inhibitors ; Phosphoprotein Phosphatases / metabolism ; Phosphorylation ; Radioimmunoprecipitation Assay ; tau Proteins / immunology ; tau Proteins / metabolism
Departments: Hrvatski institut za istraživanje mozga
Katedra za neurologiju
Depositing User: Kristina Berketa
Status: Published
Creators:
CreatorsEmail
Boban, MirtaUNSPECIFIED
Babić Leko, MirjanaUNSPECIFIED
Miškić, TerezijaUNSPECIFIED
Hof, Patrick R.UNSPECIFIED
Šimić, GoranUNSPECIFIED
Date: 1 May 2019
Date Deposited: 05 Oct 2020 09:31
Last Modified: 05 Oct 2020 09:31
Subjects: /
Related URLs:
URI: http://medlib.mef.hr/id/eprint/3679

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