Neurons Re-enter The Cell Cycle During Tau Hyperphosphorylation

Alzheimer’s disease (AD) is the most common neurodegenerative disease. The major pathological changes of AD are intracellular neurofibrillary tangles (NFTs) and extracellular senile plaques (SP). Clinical research found that amount of NFTs correlates positively with the severity of cognitive impairment in AD. Hyperphosphorylated microtubule associated protein tau is the major protein subunit of NFTs.Tau protein is a microtubule associated protein. The major function of tau is to promote microtubule assembly and maintain the stability of microtubules. Hyperphosphorylated tau loses the function and aggregates into paired helical filaments and NFTs, leading to neuronal microtubule disruption, body-axonal transport of nutrients and information transmission impairment and neuronal degenerative death. The major biochemical mechanism of the abnormal tau hyperphosphorylation is the imbalance between protein kinases and protein phosphatases in the involved neurons. It has been found that cAMP-dependent protein kinase (PKA) play an important role in tau phosphorylation. Forskolin is a specific activator of PKA. Activating PKA in vivo can directly induce tau hyperphosphorylation. Meanwhile, prephosphorylation of tau by PKA make it more susceptible to be phosphorylated by GSK-3and CDK5.Neuronal cell cycle re-enter is one of the main neuropathological features in AD. Central neurons are usually considered in the terminally differentiated state, meaning that they no longer enter the cell cycle. However, more and more studies show that there is cell cycle re-entry in the neurons of AD brains. Cell cycle re-entry will result in neuronal death. Cell cycle regulators, such as cyclin B, PCNA and Cdc2, were abnormally expressed and activated in the neurons containing NFTs in the AD brain. Cyclin D1is the key factor to regulate neurons to leave Go phase and enter G1phase, and the level of cyclin D1is elevated in the AD brain. There is cell cycle re-entry in neurons containing NFTs in the AD brain, which suggests a positive correlation between hyperphosphorylation of tau and neuronal cell cycle re-retey.Part1. Forskolin induces tau hyperphosphorylation in cultured primary hippocampal neurons(1) Objective To investigate tau hyperphosphorylation induced by Forskolin in cultured primary hippocampal neurons.(2) Methods In this study, cultured primary hippocampal neurons from newborn (1day after birth) SD rats were treated with4μmol/L Forskolin, a specific activator of PKA, for0h,3h,6h,12h and24h. Levels of phosphorylated tau at Ser214, Ser396and Ser202/Thr205sites were measured by Western blot and immunofluorescence. Image J software was used to quantitatively analyze the protein bands. SPSS (13.0) software was used to analyze the data (presented as mean±SD) by One-way ANOVA followed by LSD. Each experiment was repeated3times for statistic analysis.(3) Results Primary hippocampal neurons from newborn (1day after birth) SD rats were cultured for48h and then were treated with4μmol/L Forskolin for0h,3h,6h,12h and24h. Levels of phosphorylated tau was measured by Western blot and immunofluorescence. The level of tau phosphorylation at Ser214site was increased at 6h (F=10.059, P<0.005) and12h and it was restored to the level of0h at24h; the level of tau phosphorylation at Ser396site was elevated at6h (F=4.813, P<0.05), peaked at12h (F=4.813, P<0.005), and then back to the level of0h at24h; the level of tau phosphorylation at Ser202/Thr205site was elevated at3h and6h, peaked at12h (F=2.720, P<0.005), and then back to the level of3h at24h; the level of total tau did not change (F=0.720, P>0.05). The highest level of tau phosphorylation at Ser214, Ser396and Ser202/Thr205sites was seen at6h,12h and12h respectively after treatment with4μmol/L Forskolin. Hyperphosphorylated tau at Ser214, Ser396and Ser202/Thr205sites and total tau were mainly distributed in the cytoplasm.(4) Conclusions4μmol/L Forskolin induced tau hyperphosphorylation at Ser214, Ser396and Ser202/Thr205sites in cultured primary hippocampal neurons, and the highest level of tau phosphorylation was seen at6h,12h and12h, respectively.Part2. Neurons re-enter the cell cycle during tau hyperphosphorylation(1) Objective To investigate the correlation between hyperphosphorylation of tau and neuronal cell cycle re-entey.(2) Methods In this study, cultured primary hippocampal neurons from newborn (1day after birth) SD rats were treated with4μmol/L Forskolin, a specific activator of PKA, to induce hyperphosphorylation of tau. Levels of cyclin D1and cyclin B1were measured by Western blot. The co-localization of hyperphosphorylated tau (at Ser214, Ser396or Ser202/Thr205sites) and cyclin D1or cyclin B1was measured by double-labeling immunofluorescence. Image J software was used to quantitatively analyze the protein bands. SPSS (13.0) software was used to analyze the data (presented as mean±SD) by One-way ANOVA followed by LSD. Each experiment was repeated3times for statistic analysis. (3) Results Primary hippocampal neurons from newborn (1day after birth) SD rats were cultured for48h and then were treated with4μmol/L Forskolin for6h and12h to induce hyperphosphorylation of tau, and DMSO was used as a solvent control. Levels of cyclin D1and cyclin B1were measured by Western blot. Levels of cyclin D1and cyclin B1were both increased at6h and12h (cyclin D1:F=433.106, P<0.005; cyclin B1:F=39.135, P<0.005). Primary hippocampal neurons from newborn (1day after birth) SD rats were cultured for48h and then were treated with4μmol/L Forskolin for6h. The co-localization of hyperphosphorylated tau at Ser214site and cyclin D1or cyclin B1was measured by double-labeling immunofluorescence. Levels of phosphorylated tau at Ser214site, cyclin D1and cyclin B1were elevated at6h, and hyperphosphorylated tau at Ser214site and cyclin D1or cyclin B1were co-localized in the cytoplasm. Primary hippocampal neurons from newborn (1day after birth) SD rats were cultured for48h and then were treated with4μmol/L Forskolin for12h. The co-localization of hyperphosphorylated tau (at Ser396or Ser202/Thr205sites) and cyclin Dl or cyclin B1were measured by double-labeling immunofluorescence. Levels of phosphorylation of tau (at Ser396or Ser202/Thr205sites) and cyclin D1and cyclin B1were elevated, and hyperphosphorylated tau (at Ser396or Ser202/Thr205sites) and cyclin D1or cyclin B1were co-localized in the cytoplasm.(4) Conclusions4μmol/L Forskolin induced tau hyperphosphorylation and concomitant increases in the level of cyclin D1and cyclin B1in hippocampal neurons, and hyperphosphorylated tau and cyclin D1or cyclin B1were co-localized in the cytoplasm.