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

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... 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. Tau aggregation is a common feature among neurodegenerative disorders referred to as tauopathies. Mechanisms leading to tau aggregation and neurofibrillary degeneration are poorly understood. However, abnormal phosphorylation seems to be involved in tau conformational changes and aggregation (1, 2). Phosphorylation is a key post-translational modification involved in the regulation of tau function regarding microtubule polymerization and stability. Whereas many phosphorylation sites are common between tau aggregated into filaments (tau-PHF) in AD 1 and normal tau from biopsy-derived material, some phosphoepitopes referred to as abnormal ones are exclusively found on tau-PHF (for a review, see Ref. 3). Our recent data suggest that molecular mechanisms similar to those encountered during mitosis may play a role in the formation of abnormal tau phosphoepitopes (4). Understanding the role of abnormal tau phosphorylation in NFT formation requires identification of kinases leading to these specific phosphoepitopes. A large number of kinases can phosphorylate tau on specific Thr or Ser residues (for a review, see Ref. 3). It was found that many kinases, belonging to the Pro-directed protein kinase family, have an enzymatic activity for one of the 17 Ser/Thr-Pro motifs in full-length tau. Other kinases, however, such as protein kinase A were found to phosphorylate Ser or Thr residues that are not followed by a Pro, whereas GSK-3␤ can modify both Ser/Thr residues that are or not followed by a Pro (for a review, see Ref. 5). A number of phosphorylation-dependent antibodies were used to monitor tau phosphorylation. For instance, AD2 recognizes phosphorylated residues Ser 396 and Ser 404 (6), whereas AT8 and AT180 recognize phospho-Ser 202 / Ser 205 and phospho-Thr 231 , respectively (7, 8). Among abnormal tau phosphoepitopes, TG-3 recognizes phospho-Thr 231 in a conformation-dependent manner. This phosphoepitope is exclusively found in mitotic cells (9) with only the exception of degenerating neurons of AD and seems to be associated with early stages of the disease (10, 11). The Cdc2 kinase probably generates TG-3 epitope in mitotic cells, but the kinase(s) responsible for this phosphorylation in neurons remain unknown (9, 12, 13). For instance, it was reported that Thr 231 could be phosphorylated in vitro by four kinases (GSK3␤, p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase 2) (14). Nevertheless, one of the most evident candidates is Cdk5, the neuronal Cdc2-like kinase. Several data support the idea of Cdk5 involvement in AD (15-17). Cdk5 activity is regulated in neuronal cells by its binding with its activating partner p35, p39, and the p35 proteolytic fragment, p25 protein (for a review, see Ref. 18). p35 proteolysis into p25 occurs in response to diverse insults that probably trigger calpain activation (19 -21). p25-Cdk5 complex leads to a deregulated kinase activity that has been linked to tau hyperphosphorylation and neurotoxicity (16, 22, 23) . Tau phosphorylation has been well characterized in vitro (24), but data on Cdk5-induced tau phosphorylation in vivo and in cellular models are lacking. In the present study, biochemical studies have been undertaken to investigate tau phosphorylation by p25-Cdk5 complex. The toxicity linked with p25 expression was avoided by using an appropriate cell model that expresses inducible p25-Cdk5 kinase activity. EXPERIMENTAL PROCEDURES Cell Culture and Transfections 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% CO 2 humidified incubator at 37°C. Tau-SY5Y cells were previously described (4, 25). They constitutively