Neuroprotective Effects Of Green Tea Polyphenols Against Okadaic Acid-induced Neurotoxicity And Tau Hyperphosphorylation In Cultured Primary Hippocampal Neurons

Objectives:Alzheimer's disease (AD) is a progressive neurodegenera- tive disease and the most common form of dementia. The neuropathological hallmarks of AD include extensive neuronal loss, the presence of numerous neurofibrillary tangles (NFTs), and senile plaques (SP) in the brain. The major constituent of a senile plaque is Aβand the NFTs are composed of hyperphosphorylated tau protein. So far, the etiology and mechanisms of AD still have been unknown clearly, and no objective and specific biomarkers can be used for early diagnosis and efficient cure. Accordingly, strategies that oppose proteolytic processing of APP into Aβpeptides and ensuing cerebral amyloidosis have remained a primary focus of AD research. Clinical researches found that the formation of NFTs is closely correlated with the temporal and spatial progression of AD pathology and other tauopathies. In the case of AD and various other tauopathies, tau has been found to be abnormally phosphorylated or dephosphorylated at specific residues by several possible neuronal kinases/phosphotases both in vitro and in vivo, which is hypothesized to ultimately lead to conformational abnormality, dysfunction, and aggregation. Okadaic acid (OA) an inhibitor of protein phosphotase 2A (PP-2A) and PP-1, has been a useful tool to study AD. Treatment neurons with OA in vitro or in vivo induced aberrant hyperphosphorylation of tau or neuronal death or both. Previous studies showed that apoptosis was involved in neuronal death induced by OA or other inhibitors of PP in vitro.Tea polyphenols are natural plant flavonoids present in the leaves and stem of tea plant. The green tea polyphenols comprise (-)-epigallocatechin- 3-gallate (EGCG), (-)-epigallocatechin, (-)-epicatechin, (-)-gallocatechin, and catechin. Many biological functions of tea polyphenols have been reported, including anti-inflammatory, anticarcinogenic, and antioxidant effects. However, whether green tea polyphenol can reduce the OA induced toxicity and inhibit the tau hyperphosphorylation has been unknown. Our previous studies (unpublished) indicated that OA can induce tau protein hyperphosphorylation and cause memory impairment, while GTPs can reduce the hyperphosphorylation and attenuate the impairment. Therefore, according to our previous data and references, the aim of present study is to explore the effects of green tea polyphenols (GTPs) on Okadaic acid induced toxicity and tau phosphorylation in cultured primary hippocampal neurons.Methods: Hippocampal cell cultures were prepared from newly born(less than 24 hours) Sprague-Dawley rats and neurons were cultured in CO2 incubator with 37℃and 5% CO2 .The experiments were performed using 7-day-old cultures, at which time the hippocampal neurons are fully different- tiated. On the 7th day, hippocampal neurons were fixed in cold acetone and identified by thionine staining. For experiments, neurons were divided into 6 groups which respectively added normal culture medium, culture medium contained DMSO(<0.01%), 10 nmol/L OA, and OA co-treatment with three different concentration GTPs(5μg/ml, 15μg/ml, 25μg/ml). Neurons were visualized and photographed by using phase-contrast microscopy. Cell viability was evaluated 12 h later using the MTT colorimetric assay, whereas the extent of neuronal injury was measured by using lactate dehydrogenase (LDH) release as a marker for membrane leakage and cell death. Immuno- cytochemistry was used to detect the distribution of hyperphosphorylated tau protein, while Western blot were used to analyze the expression of hyperpho- sphorylated tau protein.Results:1. Hippocampal neurons were approximately 90% of the total cells in the cultured system.2. Normal healthy neurons were defined as having a cell body that was phase bright, with a smooth contour, round to oval shape, and multiple neuritic processes. After exposure for 12 h to OA(10 nmol/L) treatment, degenerating neurons were characterized by marked fragmentation and degeneration of neurites and a round irregular cell body, whereas OA co- treatment with GTPs exhibited less impairment, especially the OA+GTPs 15μg/ml and OA+GTPs 25μg/ml groups.3. The MTT colorimetric assay and LDH release revealed that a 12 h exposure to OA(10 nmol/L) is neurotoxic to hippocampal cells(about 30% decrease in MTT values vs. control and approximately 50% increase in LDH release vs. control).Both assays revealed that the toxic effect of OA was reduced, in a dose-dependent manner, by a co-treatment with GTPs. These effects are significant at 15μg/ml and 25μg/ml.4. Immunocytochemistry staining showed that tau protein hyperpho- sphorylation at Ser 396 (PHF-13)-positive neuritic processes appeared very clearly and PHF-13 immunoreactivity was marked increased in cell bodies after exposure to OA(10 nmol/L) for 12 h. Whereas, the groups co-treated with GTPs was obviously reduced the PHF-13 immunoreactivity, especially at the dose of 15μg/ml and 25μg/ml. Moreover, examination of the tau in a total extract of cells by using Western blot suggested that average relative density was obviously increased in the OA group, while the groups of OA co-treated with GTPs decreased obviously when compared with OA group, especially at 15μg/ml and 25μg/ml (P<0.01).Conclusions:Our results demonstrate that treatment of cultured primary hippocampal neurons with OA (10 nmol/L) induced cell neurotoxicity and hyperpho- sphorylation of tau, while GTPs can attenuate these neurotoxic effects.