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.
Original language | English |
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Pages (from-to) | 60-68 |
Number of pages | 9 |
Journal | Journal of Neuroscience Methods |
Volume | 319 |
DOIs | |
State | Published - 1 May 2019 |
Keywords
- Alzheimer's disease
- Cell culture
- Immunoblot
- Neurodegeneration
- Okadaic acid
- Oligomerization
- Phosphorylation
- Protein phosphatase
- SH-SY5Y
- Tau protein