A Reciprocal Interaction Of Nuclear Tau Hyperphosphorylation And Nuclear Calcium Signaling Activation

Background: Alzheimer's disease(AD),the most common neurodegenerative disease.Intracellular neurofibrillary tangles(NFT)which are composed of intracellular hyperphosphorylated microtubular-associated proteins Tau,are the most important symbolic pathological changes.Calcium ions(Ca2+)impact nearly every aspect of cellular life.Calcium dysregulation contribute to pathology of AD.Nuclear calcium signaling is the most important mechanism underlying gene transcription and synaptic activity.Does nuclear calcium signaling involve in the pathology of AD? Whether phosphorylated Tau activates nuclear calcium signaling pathway and whether nuclear calcium signal facilitates nuclear Tau hyperphosphorylation remain uncovered.We transfected human Tau(h Tau)in human embryonic kidney 293 cells(HEK293)and detected the subcellular distribution of Tau phosphorylated in different phosphorylation site.We further investigated the interaction of nuclear phosphorylated Tau and nuclear calcium signaling activation.Objective: To explore the subcellular distribution of Tau phosphorylated in different phosphorylation site and the interaction of nuclear phosphorylated Tau and nuclear calcium signaling activation.Methods: Calcium fluorescence indicator Fluo-3AM was employed to monitor the calcium concentration.Nuclear protein extraction was used to get the nuclear protein.Co-immunofluorescence was used to confirm the location of the phosphorylated T205 and the phosphorylated S214 in nuclei.OA treatment was used to induce Tau phosphorylation.Immunoblot was used to evaluate the quantity of protein such as nuclear phosphorylated Tau,nuclear phosphorylated Ca MKIV and nuclear phosphorylated CREB.Site-directed mutagenesis was employed to construct the Tau T205 A,T205E,S214 A,S214E plasmids to explore the effect of site-specific phosphorylation Tau on nuclear calcium signal.Results: We found that expression of human wild-type Tau in HEK293 cells caused elevation of cytoplasmic and nuclear calcium concentration with Tau hyperphosphorylation and phosphorylated Tau at Thr205 and Ser214 were almost exclusively detected in nuclei.Phosphorylation of Tau at Thr205 triggered the elevation of nuclear calcium concentration and modulated the nuclear Ca2+-Ca MKIV-CREB pathway.Phosphorylation of Tau at Ser214 only activated nuclear phosphorylated Ser129 of CREB.OA treatment further increased nuclear Tau phosphorylation with elevation of nuclear calcium concentration and activation of Ca MKIV-CREB pathway in nuclei,indicating that nuclear phosphorylated Tau triggers nuclear calcium dyshomeostasis and nuclear calcium signaling.Transfection of CAMBP4,a calmodulin binding peptide that can inhibit nuclear calcium signal,efficiently decreased the phosphorylated Thr196 of Ca MKIV,decreased nuclear Tau phosphorylation,indicating that nuclear activation of Ca MKIV is required for nuclear Tau phosphorylation.Overexpression of CAMBP4 decreased GSK-3? phosphorylation at Ser9 in nuclear fraction,which indicated Ca MKIV is required for phosphorylated Ser9 of GSK-3?.We further observed that OA treatment increased the inhibitory phosphorylation of GSK-3? at Ser9 in nuclei,which indicated activation of GSK-3? may not contribute to the OA-induced Tau hyperphosphorylation in the nuclei.Conclusion: These data together indicated that phosphorylated Thr205 and phosphorylated Ser214 of Tau localize in nuclear and nuclear phosphorylated Tau triggers nuclear calcium dysregulation and the activation of nuclear Ca2+-Ca MKIV-CREB pathway.In turn activation of nuclear calcium signaling pathway is required for nuclear Tau phosphorylation.