Abstract
Hyperphosphorylated tau, which is the major protein of the neurofibrillary tangles in Alzheimer's disease brain, is most probably the result of an imbalance of tau kinase and phosphatase activities in the affected neurons. By using metabolically competent rat brain slices as a model, we found that selective inhibition of protein phosphatase 2A by okadaic acid induced an Alzheimer-like hyperphosphorylation and accumulation of tau. The hyperphosphorylated tau had a reduced ability to bind to microtubules and to promote microtubule assembly in vitro. Immunocytochemical staining revealed hyperphosphorylated tau accumulation in pyramidal neurons in cornu ammonis and in neocortical neurons. The topography of these changes recalls the distribution of neurofibrillary tangles in Alzheimer's disease brain. Selective inhibition of protein phosphatase 2B with cyclosporin A did not have any significant effect on tau phosphorylation, accumulation, or function. These studies suggest that protein phosphatase 2A participates in regulation of tau phosphorylation, processing, and function in vivo. A down-regulation of protein phosphatase 2A activity can lead to Alzheimer-like abnormal hyperphosphorylation of tau.
Highlights
Hyperphosphorylated tau, which is the major protein of the neurofibrillary tangles in Alzheimer’s disease brain, is most probably the result of an imbalance of tau kinase and phosphatase activities in the affected neurons
We found that treatment of brain slices with 0.1 M okadaic acid achieved a 50% inhibition of PP2A (Fig. 1)
This was probably due to a saturated permeability of okadaic acid under 1.0 M into the cells of the tissue, because PP2A activity was almost completely inhibited when the rat brain homogenate was treated with 1.0 M okadaic acid
Summary
Materials—Phosphorylase kinase was purified from the skeletal muscle of New Zealand White rabbits by the method of Cohen [39]. The brain slices were rinsed briefly with the homogenizing buffer containing 50 mM Tris-HCl, pH 7.0, 10 mM -mercaptoethanol, 1.0 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 2.0 mM benzamidine, and 2.0 g/ml each of aprotinin, leupeptin, and pepstatin A. The activities of PP1 and PP2A in the extracts of brain slices were assayed using [32P]phosphorylase a as a substrate as described by us previously [24]. Microtubule Binding Assay and in Vitro Microtubule Assembly—The ability of tau to bind to microtubules was assessed by incubating tau with taxol-stabilized microtubules and quantitating the bound and unbound tau This binding assay was carried out as described previously [53] except that antibody 134d instead of 92e was used to detect. MAP-free tubulin (2.0 mg/ml) was incubated with tau (0.18 mg/ml) and GTP (1.0 mM) in an assembly buffer at 37 °C, and the turbidity of the solution was continuously monitored at 350 nm by a thermostatically controlled Cary 1 recording spectrophotometer
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