| Alzheimer's disease is the most prevalent neurodegenerative disease,characterized by the presence of extensive extracellular amyloid plaques,intracellular neurofibfillary tangles(NFTs), neuronal death and synaptic loss in cerebral cortex and hippocampus,along with progressive impairment of cognition and emotional activities.It has been well known that tau is abnormally hyperphosphorylated and is accumulated as intraneuronal tangles of paired helical filaments (PHF),twisted ribbons and or straight filaments.This hallmark brain lesion of these diseases directly correlates with dementia in these patients.Despite the mechanisms leading to the formation of NFTs is not very clear,hyperphosphorylated tau protein is the major pathological change in neurodegeneration of AD.To date,many studies indicated that the phosphorylation level of tau protein is regulated by protein kinases and protein phosphatases.Thus,the state of phosphorylation oftau protein is a function of the balance between the activities of the protein kinases and the protein phosphatases that regulated its phosphorylation.Tau is a substrate for several protein kinases,among these kinases,glycogen synthase kinase-3(GSK-3),cyclin dependent protein kinase-5(cdc-5),protein kinase A(PKA)have been most implicated in abnormal hyperphosphorylation of tau.Okadaic acid(OA)has been widely used as a selective protein phosphatase inhibitor including PP2A,PP1 and PP2B in vitro as well as in animals.In cultured neurons,okadaic acid-treated induced amyloiddeposition,neuronal degeneration, synaptic loss and impairment of cognition.Which mimics the pathological changes of AD.Humanin(HN)is a newly identified 24 residues peptide from occipital lobe of AD brains, which has been demonstrated to suppress the neuronal death initiated by AD-relacant insults including familial AD(FAD)-linked mutant genes,amyloid-β(Aβ)peptides(full length Aβpeptide and Aβ25-35 fragment).And previous studies in our lab also indicated that HN protect cultured cortical neurons against apoptosis induced by Aβ31-35,an even shorter Aβfragment. However no evidence showed whether HN could against tau hyperphosphorylation.Thus,we designed this experiment in which okadaic acid-induced neurotoxicity was used as a damage model to verify whether HN could against okadaic acid-induced abnormal hyperphosphorylation of tan.The result showed that:1.The phosphorylation level of tau protein treated with okadaic acid(10nM,20nM)assayed by western-blot tau was significantly increased respectively(P＜0.05),while there was no change of phosphorylation level of tau protein treated with 5nM OA. OA-treated samples suggested tau protein was hyperphosphorylated at Ser-199/202,Ser-396 and Thr-231;2.When neurons was pretreated with HN(10μM,20μM)for 24h respectively,the hyperphosphorylation of tau induced by OA was suppressed,while the application of low HN(5μM)had no suppressive effects on OA-induced hyperphosphorylation;3.OA could selectively inhibit the activity of PP2A(P＜0.05);4.The activity of PP2A was up-regulated by 10μM and 20μM HN respectively(P＜0.05)but not 5μM HN;5.The expression level of GSK-3βwas not changed when neurons treated with OA(5nM 10nM 20nM)(P＞0.05);6.The expression level of GSK-3βhas not altered when neurons pretreated with different concentration of HN.Conclusions:1.OA induce the hyperphosphorylation of tau at Ser199/202,Ser396 and Thr231 by inhibit PP2A;2.HN suppressed OA—induced hyperphosphorylation of tau by up-regulated PP2A;3.Neither okadaic acid nor HN has effects on the expression of GSK-3β.
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