Phosphorylation Of Tau Antagonizes Apoptosis And The Underlying Mechanisms

Part 1 Phosphorylation of tau antagonizes apoptosis and the underlying mechanismsHyperphosphorylated tau is the major protein subunit of neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) and related tauopathies. It is not understood, however, why the NFTs-containing neurons seen in the AD brains do not die of apoptosis but rather degeneration even though they are constantly awash in a proapoptotic environment. Here, we show that cells overexpressing tau exhibit marked resistance to apoptosis induced by various apoptotic stimuli, which also causes correlated tau hyperphosphorylation and glycogen synthase kinase-3 (GSK-3) activation. GSK-3 overexpression did not potentiate apoptotic stimulus-induced cell apoptosis in the presence of high levels of tau. The resistance of neuronal cells bearing hyperphosphorylated tau to apoptosis was also evident by the inverse staining pattern of PHF-1-positive tau and activated caspase-3 or fragmented nuclei in cells and the brains of rats or tau-transgenic mice. Tau hyperphosphorylation was accompanied by decreases inβ-catenin phosphorylation and increases in nuclear translocation ofβ-catenin. Reduced levels ofβ-catenin antagonized tau's anti-apoptotic effect, while overexpressingβ-catenin conferred resistance to apoptosis. These results reveal a novel anti-apoptotic function of tau hyperphosphorylation, which likely inhibits competitively phosphorylation ofβ-catenin by GSK-3βand hence facilitates the function ofβ-catenin. Our findings suggest that tau phosphorylation may lead the neurons to escape from an acute apoptotic death, implying the essence of neurodegeneration seen in the AD brains and related tauopathies. Part 2 Overexpression of dishevelled-1 attenuates wortmannin-induced hyperphosphorylation of cytoskeletal proteins in N2a cellNeurofibrillary tangles (NFTs) are one of the hallmark lesions of Alzheimer's disease (AD) and NFTs are made up of paired helical filaments (PHF), whose main component is the abnormally hyperphosphorylated microtubule-associated protein tau. In addition to tau, abnormally hyperphosphorylated neurofilament is also the protein component of NFTs. Studies have shown that Wnt signaling pathways play important roles in AD and overexpression of dishevelled-1 (DVL-1) mimics the Wnt signal. There is no report about the role of DVL-1 in neurofilament phosphorylation, and the role of DVL-1 in tau phosphorylation in neuronal cells is also not known. To investigate the effect of DVL-1 on Alzheimer-like hyperphosphorylation of cytoskeletal proteins, we used wortmannin to produce a cell model with hyperphosphorylation of cytoskeletal proteins in mouse Neuroblastoma 2a (N2a) cell. Then, cultured N2a cells were transiently transfected with DVL-1 expression plasmid using LipofectamineTM 2000 and were treated with wortmannin. Western blot and immunofluorescence microscopy were used to measure the phosphorylation of neurofilament and tau. Level of phosphorylated neurofilament at SMI31 epitope and phosphorylated tau determined by PHF-1 was increased at 1 h and 3 h and back to normal at 6 h after wortmannin 1μM treatment. The highest level of phosphorylated neurofilament and phosphorylated tau was seen at 1 h and 3 h after wortmannin treatment, respectively. When DVL-1 protein was overexpressed, the hyperphosphorylation of neurofilament at SMI31 and SMI32 epitopes and tau at PHF-1 (Ser-396/404), M4 (Thr-231/Ser-235), and Tau-1 (Ser-198/199/202) epitopes was attenuated. Taken together, our findings suggests that overexpression of mouse DVL-1 protein inhibits wortmannin-induced hyperphosphorylation of neurofilament and tau in N2a cells.Part 3 Transient Expression of Mouse DVL-1 cDNA Recombinant Plasmid in Cultured Mouse Neuroblastoma 2a CellObjective To establish a transient expression system of mouse DVL-1 cDNA recombinant plasmid in cultured wild-type mouse Neuroblastoma 2a (N2a) cell for the further use in studying the role of Wnt signaling in Alzheimer's disease (AD). Methods After being amplified and purified, the recombinant plasmid was transfected into cultured N2a cell by LipofectamineTM 2000. The transfection and expression were examined by Western blot and immunofluorescence microscopy. Results A successful amplification and purification of the recombinant plasmid PCS2+MT-MDVL1 was evaluated by Cla I digestion, and the successful transfection and expression of the fusion protein DVL-1-c-Myc in N2a cells was determined by Western blot and immunofluorescence microscopy using anti-c-Myc tag antibody. The transfection efficiency was 57.6% evaluated by immunofluorescence. Conclusion A transient expression system of the fusion protein DVL-1-c-Myc was established in the present study, which can serve as a tool in studying the role of Wnt signaling in AD.