Protective Effect Of Chitosan Oligosaccharide On Okadaic Acid-induced Tau Protein Hyperphosphorylation In Cultured Hippocampal Neurons In Rats

Objective: Alzheimer's disease (AD) is a widespread, neuron- degenerative disorder of the central nervous system of elderly population. The clinical manifestations of AD is progressive hypophrenia, cognition and memory impairment. With the increasing of the population aging and incidence of AD, heavy burden was brought to society and family due to poor life quality in AD patients. AD is characterized by loss of neurons and several pathologic hallmarks including extracellular deposits ofβ- amyloid in senile plaques and intracellular neurofibrillary tangles (NFTs). NFTs is composed of bundles of paired helical filaments (PHF), the major protein subunit of which is the microtubule-associated protein Tau. Tau in PHF is abnormally hyperphosphorylated, aggregated into filaments, and does not bind to microtubules or stimulates microtubule assembly. Evidence has indicated that the hyperphosphorylation of Tau probably plays a key role in neurofibrillary degeneration in AD and the numbers of NFTs correlate closely with the degree of dementia in AD. Previous studies have shown that Tau protein phosphatase activity is significantly reduced in AD brain and three types of protein phosphatase (PP-1, PP-2A and PP-2B) are involved in dephosphorylation of Tau.Chitooligosaccharide (also named Chitosan oligosaccharide, COS) is a kind of oligosaccharides which is composed of 2~10 glucoseamine connecting byβ-1,4- glucosidic bond. COS comes from the degradation of chitosan. Chitosan is a deacetylated polymer of N-acetyl glucosamine, which is obtained after alkaline deacetylation of the chitin derived from the exoskeletons of crustaceans and arthropods. Recent studies on chitosan have attracted interest for converting chitosan to its oligosaccharides COS, because COS is not only water soluble but also reported to have special functional properties such as antitumor activity, immunostimulating effect, antimicrobial activity, radical scavenging activity. Some references suggest that chitooligosaccharide have the ability to promote neuronal differ- entiation of neurospheres. Chitooligosaccharides and water-soluble chitosan derivatives are a kind of BACE1(theβ-site APP-cleaving enzyme) inhibitor. However, it is still unclear whether COS has a protective effect in the hyperphosphorylation of Tau protein of AD. The aim of this study is to explore the function of chitooligosaccharide (COS) on the Tau protein hyperphosphorylation in cultured hippocampal neurons in rats, which is induced by okadaic acid (OA).Methods: Hippocampal neurons were separated and cultured in CO2 incubator with 37℃and 5% CO2 from newborn(less than 24 hours) Sprague-Dawley rats. After cultured for 7 days, hippocampal neurons were fixed in cold acetone and identified by thionine staining. We choose OA as the inducer of Tau protein hyperphosphorylation. The experiment included six groups: normal group, vehicle-control group, model group treated with OA, and three COS groups, pretreated with COS in 10ug/ml,20ug/ml and 40ug/ml respectively. Added three different concentrations of COS(10ug/ml,20ug/ml and 40ug/ml) to the hippocampal neurons for 2 days and fostered together to 7 days. At the 7th day of cultivation, OA group and COS-pretreated groups were added 10nmol/L OA (soluble in 10% DMSO, the final concentration of DMSO was no greater than 0.01% DMSO) and fostered together for 12 hours. The same concentration of DMSO was added to vehicle-control group. The toxicity of OA to the neurons and protective effective of COS was determined by MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] and the release of LDH (lactate de- hydrogenase). Changes of hyperphosphorylation of Tau protein were analyzed by immunocytochemistry and Western blot. Quantified analysis of Western blot was performed later.Results:1. Identification of cultured hippocampal neurons in rats.Hippocampal neurons were approximately 90% of the total number of cells in the cultured.2. Protective effect of COS on okadaic acid-induced damage in cultured hippocampal neurons in rats.10nmol/L OA induce degeneration of neuritis, rounding of cell bodies, detachment from the substratum, and eventual neuronal death. With COS pretreated the morphous of cells improved, especially in 20ug/ml and 40ug/ml COS groups.The survival rate of neurons was quantitated by MTT. OA induced a significant decrease in MTT activity which was prevented by COS treatment, especially in 20ug/ml and 40ug/ml COS groups (n=5, P<0.01).Measurements of LDH activity into the culture medium indicated that COS can reduce the LDH leakage induced by OA, especially in 20ug/ml and 40ug/ml COS groups (n=5, P<0.05).3. COS attenuates the expression of hyperphosphorylation of Tau induced by OA.Immunocytochemistry staining indicated that there is strong positive hyperphosphorylation of Tau granules in the OA group and abundant brown positive hyperphosphorylation of Tau granules were detected in the cultured hippocampal neurons of COS pretreated groups, but weaker than OA group, especially in 20ug/ml and 40ug/ml COS groups.Furthermore, Western blot analysis showed that the average relative photodensity was obviously increased in the OA group compared with the normal group. With COS pretreated, hyperphosphorylation of Tau ex- pression was decreased, and a significant difference between COS and OA group still could be seen (P <0.01).Conclusion: Our data suggested that OA can induce the damage of cultured hippocampal neurons in rats and Tau hyperphosphorylation, and COS can improve the impairment and decrease the hyperphosphorylation of Tau.