Abstract
Among tau phosphorylation sites, some phosphoepitopes referred to as abnormal ones are exclusively found on tau aggregated into filaments in Alzheimer's disease. Recent data suggested that molecular mechanisms similar to those encountered during mitosis may play a role in abnormal tau phosphorylation. In particular, TG-3 phosphoepitope is associated with early stages of neurofibrillary tangles (NFTs). In this study, we reported a suitable cell model consisting of SH-SY5Y cells stably transfected with an inducible p25 expression vector. It allows investigation of tau phosphorylation by p25-Cdk5 kinase complex in a neuronal context and avoiding p25-induced cytotoxicity. Immunoblotting analyses showed that p25-Cdk5 strongly phosphorylates tau protein not only at the AT8 epitope but also at the AT180 epitope and at the Alzheimer's mitotic epitope TG-3. Further biochemical analyses showed that abnormal phosphorylated tau accumulated in cytosol as a microtubule-free form, suggesting its impact on tau biological activity. Since tau abnormal phosphorylation occurred in dividing cells, TG-3 immunoreactivity was also investigated in differentiated neuronal ones, and both TG-3-immunoreactive tau and nucleolin, another early marker for NFT, were also generated. These data suggest that p25-Cdk5 is responsible for the mitotic-like phosphoepitopes present in NFT and argue for a critical role of Cdk5 in neurodegenerative mechanisms.
Highlights
Play a role in abnormal tau phosphorylation
Recent data suggested that molecular mecha- abnormal tau phosphorylation in neurofibrillary tangles (NFTs) formation requires idennisms similar to those encountered during mitosis may tification of kinases leading to these specific phosphoepitopes
Abnormal tau phosphorylation is likely to be a critical mechanism involved in tau aggregation and neurofibrillary degeneration in Alzheimer’s disease (1, 2)
Summary
SH-SY5Y human neuroblastoma cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1 mM nonessential amino acids, and 50 units/ml penicillin/ streptomycin (Invitrogen) in a 5% CO2 humidified incubator at 37 °C. Tau-SY5Y cells were previously described (4, 25) P25 cDNA was cloned into pcDNA4/TO vector (Invitrogen). Stable cell lines that constitutively express tetracycline repressor were generated by transfection of pcDNA6/TR vector (Invitrogen) using ExGen500 (Euromedex, France) according to the manufacturer’s instructions. Isolated clones, maintained in medium with 5 g/ml blasticidin, were transfected with inducible expression vector alone (mock) or with p25 cDNA. Cells were differentiated for 7 days in Dulbecco’s modified Eagle’s medium/F-12 medium supplemented with 2 mM L-glutamine, 50 units/ml penicillin/streptomycin, 7 g/ml progesterone, 1% insulin/ transferrin/selenium (Invitrogen), and 10 ng/ml NGF 2.5 S (Sigma).
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