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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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Several types of animal models have been developed to investigate Alzheimer's disease (AD). Okadaic acid (OA), a potent inhibitor of phosphatases 1 and 2A, induces characteristics that resemble AD-like pathology. Memory impairment induced by intra-hippocampal injection of OA has been reported, accompanied by remarkable neuropathological changes including hippocampal neurodegeneration, a paired helical filament-like phosphorylation of tau protein, and formation of β-amyloid containing plaque-like structures. Rats were submitted to bilateral intrahippocampal okadaic acid-injection (100 ng) and, 12 days after the surgery, behavioral and biochemical tests were performed. Using this model, we evaluated spatial cognitive deficit and neuroglial alterations, particularly astroglial protein markers such as glial fibrillary acidic protein (GFAP) and S100B, metabolism of glutamate, oxidative parameters and alterations in MAPKs. Our results indicate significant hippocampal changes, including increased GFAP, protein oxidation, and phosphorylation of p38(MAPK); and decreases in glutathione content, transporter EAAT2/GLT-1, and glutamine synthetase activity as well as a decrease in cerebrospinal fluid S100B. No alterations were observed in glutamate uptake activity and S100B content. In conclusion, the OA-induced model of dementia caused spatial cognitive deficit and oxidative stress in this model and, for the first time to our knowledge, specific astroglial alterations. Findings contribute to understanding diseases accompanied by cognitive deficits and the neural damage induced by AO administration., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) is a progressive neurodegenerative disease that causes cognitive and behavioral deterioration in the elderly. Neurofibrillary tangles (NFTs) are one of the pathological hallmarks of AD that has been shown to correlate positively with the severity of dementia in the neocortex of AD patients. In an attempt to characterize an in vivo AD tauopathy model, okadaic acid (OA), a protein phosphatase inhibitor, was microinfused into the right lateral dorsal hippocampus area of ovariectomized adult rat. Cognitive deficiency was seen in OA-treated rats without a change in motor function. Both silver staining and immunohistochemistry staining revealed that OA treatment induces NFTs-like conformational changes in both the cortex and hippocampus. Phosphorylated tau as well as cyclin-dependent kinase 5 (cdk5) and its coactivator, p25, were significantly increased in these regions of the brain. Oxidative stress was also increased with OA treatment as measured by protein carbonylation and lipid peroxidation. These data suggest that the unilateral microinfusion of OA into the dorsal hippocampus causes cognitive deficiency, NFTs-like pathological changes, and oxidative stress as seen in AD pathology via tau hyperphosphorylation caused by inhibition of protein phosphatases., (Copyright © 2010. Published by Elsevier B.V.)

One of the pathological hallmarks of Alzheimer's disease (AD) is neurofibrillary tangles (NFTs), which are composed of abnormally hyperphosphorylated tau, but the mechanism of tau hyperphosphorylation in AD is still unclear. To investigate the effects of estrogens on tau phosphorylation, SH-SY5Y cells were treated with okadaic acid (OA), a serine/threonine phosphatase inhibitor, to induce tau phosphorylation and the effects of estrogen were observed by co-treatment with 17beta-estradiol (E2). We found that OA induced in vitro tau hyperphosphorylation, which was prevented by E2 in a dose-dependent manner. This effect of E2 was partially blocked by an estrogen receptor (ER) antagonist, ICI 182,780. In addition to tau hyperphosphorylation, inhibition of serine/threonine phosphorylation induced upregulation of cdk5 levels, which was attenuated by E2 in a manner that was counteracted by ICI 182,780. Our results show that cdk5 is involved in OA-induced tau hyperphosphorylation, and estrogens ameliorate the tau hyperphosphorylation, which may be mediated in part by ER., (Published by Elsevier B.V.)