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

J Neurosci Methods. 2019 May 1;319:60-68. doi: 10.1016/j.jneumeth.2018.09.030. Epub 2018 Sep 29.

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.

Keywords: Alzheimer’s disease; Cell culture; Immunoblot; Neurodegeneration; Okadaic acid; Oligomerization; Phosphorylation; Protein phosphatase; SH-SY5Y; Tau protein.

MeSH terms

  • 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*

Substances

  • Antibodies
  • Enzyme Inhibitors
  • MAPT protein, human
  • tau Proteins
  • Okadaic Acid
  • Phosphoprotein Phosphatases