Recent evidence has suggested that truncation of tau protein at the caspase cleavage site D421 precedes hyperphosphorylation and may be necessary for the assembly of tau into filaments in Alzheimer's disease and other tauopathies. Here we have investigated the time course of the appearance of phosphorylated and truncated tau in the brain and spinal cord of mice transgenic for mutant human P301S tau protein. This mouse line recapitulates the essential molecular and cellular features of the human tauopathies, including tau hyperphosphorylation, tau filament formation, and neurodegeneration. Soluble tau was strongly phosphorylated at 1 to 6 months of age. Low levels of phosphorylated, sarkosyl-insoluble tau were detected at 2 months, with a steady increase up to 6 months of age. Tau truncated at D421 was detected at low levels in Tris-soluble and detergent-soluble tau at 3 to 6 months of age. By immunoblotting, it was not detected in sarkosyl-insoluble tau. However, by immunoelectron microscopy, a small percentage of tau in filaments from brain and spinal cord of transgenic mice was truncated at D421. Similar findings were obtained using dispersed filaments from Alzheimer's disease and FTDP-17 brains. The late appearance and low abundance of tau ending at D421 indicate that it is unlikely that truncation at this site is necessary for the assembly of tau into filaments. (Am J Pathol 2008,

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.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 phosphorylatio...

In Alzheimer's disease, neurofibrillary degeneration results from the aggregation of abnormally phosphorylated Tau proteins into filaments and it may be related to the reactivation of mitotic mechanisms. In order to investigate the link between Tau phosphorylation and mitosis, Xenopus laevis oocytes in which most of the M-phase regulators have been discovered were used as a cell model. The human Tau isoform htau412 (2+3-10+) was microinjected into prophase I oocytes that were then stimulated by progesterone that activate cyclindependent kinase pathways. Hyperphosphorylation of the Tau isoform, which is characterized by a decrease of its electrophoretic mobility and its labelling by a number of phosphorylation-dependent antibodies, was observed at the time of germinal vesicle breakdown. Surprisingly, Tau immunoreactivity, considered as typical of Alzheimer's pathology (AT100 and phospho-Ser422), was observed in meiosis II. Because meiosis II is considered as a mitosis-like phase, we investigated if our observation was also relevant to a neuronelike model. Abnormal Tau phosphorylation was detected in mitotic human neuroblastoma SY5Y cells overexpressing Tau. Regarding AT100-immunoreactivity and phosphoSer422, we suggest that phosphatase 2A inhibition and a phosphorylation combination of mitotic kinases may lead to this Alzheimer-type phosphorylation. Our results not only demonstrate the involvement of mitotic kinases in Alzheimertype Tau phosphorylation but also indicate that Xenopus oocyte could be a useful model to identify the kinases involved in this process. Keywords: Alzheimer's disease, microtubule-associated Tau proteins, mitosis, oocyte maturation, phosphorylation. The mechanisms leading to neurodegenerative disorders referred to as tauopathies, such as Alzheimer's disease (AD) are still unknown but recent evidences had shown that neurones affected in these diseases and undergoing neurofibrillary degeneration may re-express some cell cycle genes notably implicated in the G 2 /M transition (Vincent et al. 1998;Husseman et al. 2000).Neurofibrillary degeneration is one of the neuropathological hallmarks of AD. It results from the aggregation of abnormally phosphorylated Tau proteins into paired helical filaments (PHFs). Tau proteins are mainly found in neurones. They play important roles in the polymerization and stability of microtubules (MTs). Moreover, phosphorylation is a key post-translational modification involved in their regulation. At least 30 phosphorylation sites have been described in Tau proteins, most of which occur on Ser/Pro and Thr/Pro motifs.Despite the fact that many phosphorylation sites are common between Tau aggregated into filaments in AD and normal Tau from biopsy-derived material, some differences exist and support the idea of an abnormal phosphorylation of Tau proteins in AD (for review see Buée et al. 2000). This latter is also a common feature among tauopathies. Despite the lack of evidence, a possible link may exist between abnormal Tau

Increasing evidence suggests that an inhibition of the proteasome, as demonstrated in ParkinsonÕs disease, might be involved in AlzheimerÕs disease. In this disease and other Tauopathies, Tau proteins are hyperphosphorylated and aggregated within degenerating neurons. In this state, Tau is also ubiquitinated, suggesting that the proteasome might be involved in Tau proteolysis. Thus, to investigate if proteasome inhibition leads to accumulation, hyperphosphorylation and aggregation of Tau, we used neuroblastoma cells overexpressing Tau proteins. Surprisingly, we showed that the inhibition of the proteasome led to a bidirectional degradation of Tau. Following this result, the cellular mechanisms that may degrade Tau were investigated.

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tau gene mutations cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Here we have used Xenopus oocyte maturation as an indicator of microtubule function. We show that wildtype four-repeat Tau protein inhibits maturation in a concentration-dependent manner, whereas three-repeat Tau has no effect. Of the seven four-repeat Tau proteins with FTDP-17 mutations tested, five (G272V, ⌬K280, P301L, P301S, and V337M) failed to interfere significantly with oocyte maturation, demonstrating a greatly reduced ability to interact with microtubules. One mutant protein (R406W) almost behaved like wildtype Tau, and one (S305N) inhibited maturation more strongly than wild-type Tau. With the exception of R406W, wild-type Tau and all the mutants studied were similarly phosphorylated during the Xenopus oocyte maturation, and this was independent of their effects on this process. Data obtained with R406W and S305N may be related to charge changes (phosphorylation and basic amino acids). Our results demonstrate variable effects of FTDP-17 mutations on microtubules in an intact cell situation. Those findings establish Xenopus oocyte maturation as a system allowing the study of the functional effects of tau gene mutations in a quantitative manner.

Filamentous inclusions made of the microtubule-associated protein tau in a hyperphosphorylated state are a defining feature of a large number of human neurodegenerative diseases. Here we show that (trans,trans)-1-fluoro-2,5-bis(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (FSB), a fluorescent Congo red derivative, labels tau inclusions in tissue sections from a mouse line transgenic for human P301S tau and in cases of familial frontotemporal dementia and sporadic Pick’s disease. Labelling by FSB required the presence of tau filaments. More importantly, tau inclusions in the spinal cord of human P301S tau transgenic mice were labelled following a single intravenous injection of FSB. These findings indicate that FSB can be used to detect filamentous tau in vivo.

In Alzheimer's disease, the peptidyl prolyl cis/trans isomerase Pin1 binds to phospho-Thr231 on Tau proteins and, hence, is found within degenerating neurons, where it is associated to the large amounts of abnormally phosphorylated Tau proteins. Conversely, Pin1 may restore the tubulin polymerization function of these hyperphosphorylated Tau. In the present work, we investigated, both at the cellular and molecular levels, the role of Pin1 in Alzheimer's disease through the study of its interactions with phosphorylated Tau proteins. We also showed that in neuronal cells, Pin1 upregulates the expression of cyclin D1. This, in turn, could facilitate the transition from quiescence to the G1 phase (re-entry in cell cycle) in a neuron and, subsequently, neuronal dedifferentiation and apoptosis. The involvement of Pin1 in the G0/G1 transition in neurons points to its function as a good target for the development of new therapeutic strategies in neurodegenerative disorders.