Differential Hyperphosphorylation of Tau-S199, -T231 and -S396 in Organotypic Brain Slices of Alzheimer Mice. A Model to Study Early Tau Hyperphosphorylation Using Okadaic Acid

Abstract

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder of the brain, characterized by extracellular aggregation of beta-amyloid (Aβ) and hyperphosphorylation of tau causing intraneuronal neurofibrillary tangles (NFTs). There is urgent need to study the interactions between Aβ and tau, especially to solve the question of the pathological cascade. In the present study, we aim to develop a model of organotypic brain slices in which both plaque and tau pathology can be examined. Organotypic brain slices (150 μm thick, coronal, at the hippocampal level) from adult (9 month) wildtype (WT, C57BL/6N) and transgenic AD mice (TG, APP_SweDI) were cultured for 2 weeks. To induce tau hyperphosphorylation 100 nM okadaic acid (OA), 10 μM wortmannin (WM) or both were added to the slices. Hyperphosphorylation of tau was tested at tau-S199, tau-T231 and tau-S396 using Western blot. Our data show that in TG mice with plaques a 50 kDa fragment of tau-S396 was hyperphosphorylated and that OA induced hyperphosphorylation of tau-S199. In WT mice (without plaques) OA caused hyperphosphorylation of a 50 kDa and a 38 kDa tau-T231 form and a 25 kDa sdftau-S396 fragment. The N-methyl-D-aspartate (NMDA) antagonist MK801 (1 μM) did not block these effects. Immunohistochemistry showed diffuse increased tau-S396 and tau-T231-like immunoreactivities at the hippocampal level but no formation of NFTs. Confocal microscopy indicated, that pTau-T231 was preferentially located in cytoplasma surrounding nuclei whereas pTau-S396 was found mainly in nerve fibers and strongly associated with plaques. In conclusion we provide a novel in vitro model to study both plaque and tau hyperphosphorylation but not NFTs, which could be useful to study pathological processes in AD and to screen for drugs.

Preparation of organotypic brain slice cultures for the study of Alzheimer’s disease

“…There was also a notable shift in the apparent molecular weight of tau in lysates from LiCl treated slice cultures, which is characteristic of reduced tau phosphorylation 16 . Of interest, others have shown that okadaic acid induces hyperphosphorylation of tau in organotypic brain slice cultures prepared from TG APP_SweDI mice (SweDI; expressing APP harboring the Swedish K670N/M671L, Dutch E693Q, and Iowa D694N mutations; C57BL/6-Tg(Thy1-APPSwDutIowa)BWevn/Mmjax) 17 . Thus, this platform is very amenable for the study of treatments that modulate tau phosphorylation.…”

“…Slice cultures can be pharmacologically or genetically modified using a number of methodologies. These methods are out of the scope of this publication but have previously been published by ourselves and others (for example, 9 – 11 , 14 , 16 , 17 ).…”

“…Alternatively, tissue can be lysed for subcellular fractionation and/or biochemical analysis as described by us and others 10 , 11 , 14 , 16 , 17 . To prepare lysates for immunoblotting, slice culture medium is aspirated and slices washed once with ice-cold PBS.…”

Electroacupuncture at GV24 and bilateral GB13 improves cognitive ability via influences the levels of Aβ, p-tau (s396) and p-tau (s404) in the hippocampus of Alzheimer’s disease model rats

“…So the level of phosphorylated tau protein increases will affect the stability of microtubules, eventually resulting in the formation of NFTs and leading to Alzheimer’s disease [ 31 ]. At present, various phosphorylation sites have been identified on the tau protein, with s396 and s404 identified as key sites involved in the regulation of microtubule binding to tau protein and strongly associated with plaques [ 32 , 33 ]. So the detection of the levels of Aβ, p-tau (s404) and p-tau (s396) in the hippocampus is key to evaluating the effectiveness of any Alzheimer’s disease treatment.…”

Platelet and Plasma Phosphatidylcholines as Biomarkers to Diagnose Cerebral Amyloid Angiopathy

“…Western blot analysis was performed as previously described by us (21). Platelet samples (−80 • C) were thawed and tubes dissolved in 100 µL ice-cold PBS containing a protease inhibitor FIGURE 1 | Characterization of the novel sporadic mouse model of cerebral amyloid angiopathy (CAA).…”