Histone Deacetylase-2Induces Tau Hyperphosphorylation Via Activation Of GSK-3β

Background: AD (Alzheimer’s disease, AD) is an increasingly severe neurodegenerativedisease which can finally lead to death. Typically, it is famous for its learning and memorydecline, living ability abolish, abnormal psychiatric symptoms (like depression, apprehension,hallucinations and insomnia) as well as behavior disorder et al. NFT (neurofibrillary tangles)within cells, SP (senile plaques) outside of the cells, together with the progressively neuronalloss are the main pathological symptoms of AD patients. Hyperphosphorylatedmicrotubule-associated protein tau is the main component of NFT. Studies have shown thatHDAC2(Histone deacetyltransferase-2, HDAC2) is abnormally increased in AD patients,transgenic mice models of AD and AD related neurotoxic insult in vitro, which is incorrelation with the down regulation of genes implicated in learning and memory as well assynaptic plasticity. Knockout of HDAC2can reverse the above neurodegeneration in ADtransgenic mouse models. But the relationship between highly increased HDAC2andhyperphosphorylated tau is still unknown.Objects: To try to explore the relationship between abnormally increased HDAC2andhyperphosphorylated tau in in vitro models and the underlying pathological mechanism.Methods: Transient transfection of pEGFP-HDAC2into HEK293/tau cells, then western blotwas conducted to measure the protein levels of pT231, pS396, pS404, R134d, and severalkinases plus phosphatase implicated in tau hyperphosphorylation. Co-immunoprecipitationwas used to detect if HDAC2could have an direct impact on GSK-3β andimmunofluorescence technique was followed to clarify the subcellular distribution ofHDAC2and GSK-3β.Results: 1. HDAC2induces tau highly hyperphosphorylated in HEK293/tau cells. Overexpression of HDAC2induces tau highly hyperphosphorylated in its pT231, pS404sites, with the percentage is140.2%(p=0.017) and150.7%(p=0.0058), respectively. Meanwhile, total tau doesn’t change at all in this process.2. Two other members of class I HDACs can’t induce tau to be highly phosphorylated. HDAC1and HDAC3are all belong to the class I HDACs and their overexpression doesn’t have any influence on tau phosphorylation states in either sites of pT231, pS396, pS404or the total tau R134d (p>0.05).3. HDAC2induces tau hyperphosphorylated via activation of GSK-3β. Protein levels were detected after the introduction of HDAC2and results showing that only inhibitory site pS9-GSK-3p were decreased, while total GSK-3p, pY216-GSK-3p, pS21-GSK-3a and total GSK-3a remains unchanged, which implies us that overexpression of HDAC2could activate GSK-3β, not GSK-3a. Meanwhile all of the other kinases like PKA, PKC, ERK or phosphate PP2Ac related to tau hyperphosphorylation were all tested and none of the above showed any changes during that process. All of the above results indicate that HDAC2overexpression induces tau hyperphosphorylation most likely by activating GSK-3β, other than other kinases or phosphates. In addition, pEGFP-N1-dnGSK and GSK-3β inhibitor SB216763abolished that phosphotylation states (p>0.05), further indicating that GSK-3β activation mediated HDAC2induced tau hyperphosphorylation.4. HDAC2and GSK-3p doesn’t bind directly to have that effect. pEGFP-HDAC2and HA-GSK-3β were co-transfected into293/tau cells and co-immunoprecipitation results showing that they didn’t bind directly to each other, which imply us that the activation of GSK-3β is indirectly mediated by HDAC2. Furthermore, co-staining of HDAC2and GSK-3β shows that they don’t co-localization.Conclusion:HDAC2induces tau hyperphosphorylation in its pT231and pS404sites via the activation of GSK-3β, and this effect can’t be mimicked by his class I family member HDAC1and HDAC3.