Hyperphosphorylated tau is the major protein subunit of neurofibrillary tangles in Alzheimer's disease (AD) and related tauopathies. It is not understood, however, why the neurofibrillary tangle-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 in ␤-catenin phosphorylation and increases in nuclear translocation of ␤-catenin. Reduced levels of ␤-catenin antagonized the antiapoptotic effect of tau, whereas overexpressing ␤-catenin conferred resistance to apoptosis. These results reveal an antiapoptotic function of tau hyperphosphorylation, which likely inhibits competitively phosphorylation of ␤-catenin by GSK-3␤ and hence facilitates the function of ␤-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.Alzheimer's disease ͉ tau hyperphosphorylation ͉ glycogen synthase kinase-3 C hronic neurodegeneration characterized by accumulation of hyperphosphorylated tau and formation of neurofibrillary tangles (NFTs) is a hallmark lesion in Alzheimer's disease (AD) and related tauopathies (1-4). Although the mechanism underlying neurodegeneration remains elusive, the idea that neurons undergo apoptosis in the course of neurodegeneration is supported by studies showing that AD-related toxic stimuli, such as ␤-amyloid, cause cell death as manifested by up-regulation of apoptotic markers (5, 6). However, apoptosis accounts for only a minor proportion of neurons lost in AD brains (7); most NFT-bearing neurons undergo chronic degeneration (8-13) rather than apoptosis, even though they are constantly exposed to apoptotic stimuli, suggesting that mechanism(s) exist enabling neurons to escape apoptosis.Studies on postmortem AD brains have demonstrated that abnormally hyperphosphorylated tau is the major protein subunit of NFT (1-4), which suggests that hyperphosphorylation of tau may play a role in leading the neuronal cells to desert apoptosis. Tau is a microtubule-associated protein. The major function of tau is to promote microtubule assembly and maintain the stability of the microtubules. The roles of tau hyperphosphorylation and accumulation in the development of neurofibrillary degeneration seen in the AD brains (1-4) and related t...

10419approximation with two-body correlations has so far been tested only for the propagation of wave functions in model system-bath Hamiltonian~.~ These models used realistic potentials with barriers very similar to those encountered in H-exchange reactions, and the test calculations showed the mean field approximation with explicit system-bath correlation to be extremely accurate over times long enough for the wave packet to move away from the saddle point region. Nevertheless, numerical calculations of rate constants for problems with three or more degrees of freedom must be carried out to verify the applicability of the approach presented here to more challenging problems. Some such applications are in progress. Acknowledgment. This work has been supported by a JuniorFellowship from the Society of Fellows, Harvard University. The calculations reported were performed on a S U N 4/65 SPARC 1 station, funded by the Milton Fund Award of the Harvard Medical School.Ab initio electronic structure calculations using analytical energy derivative methods and automated potential energy surface walking techniques have been carried out on the tautomerization reaction path connecting formamide (F) H2N-CH0, through a transition state (TS), to formamidic acid (FA) HN-CHOH. The zero-point corrected F -FA, and F -TS energy differences are predicted to be 12.1 and 48.9 kcal/mol, respectively, when configuration interaction methods are used to treat electron correlation. An imaginary frequency of 23911' cm-' is obtained along the reaction coordinate at the TS. Isotopic substitution of F to generate H,N-CDO and subsequent calculation of the harmonic vibrational frequencies and eigenvectors allowed ambiguities in the assignment of the infrared spectrum of F to be resolved. The geometry of the F tautomer is found to be slightly nonplanar, but to have zero-point energy that permits the planar geometry to be dynamically accessed. Extensions to situations in which tautomerization is assisted by neighboring solvent molecule(s) are considered. In particular, the intimate involvement of a single H 2 0 solvent molecule reduces the zero-point-corrected F -FA and F -TS energy differences to 10.6 and 22.6 kcal/mol, respectively. Intimate solvent participation is thus found to much more strongly affect the activation energy than the overall thermodynamics in this case. The imaginary frequency corresponding to the reaction coordinate at the transition state changes to 20011' cm-I when a single H 2 0 is intimately involved.

SUMMARY Protein phosphatase 2A (PP2A) inhibition causes hy-perphosphorylation of tau and APP in Alzheimer’s disease (AD). However, the mechanisms underlying the downregulation of PP2A activity in AD brain remain unclear. We demonstrate that Cancerous Inhibitor of PP2A (CIP2A), an endogenous PP2A inhibitor, is overexpressed in AD brain. CIP2A-mediated PP2A inhibition drives tau/APP hyperphosphorylation and increases APP β-cleavage and Aβ production. Increase in CIP2A expression also leads to tau mislocalization to dendrites and spines and synaptic degeneration. In mice, injection of AAV-CIP2A to hippocampus induced AD-like cognitive deficits and impairments in long-term potentiation (LTP) and exacerbated AD pathologies in neurons. Indicative of disease exacerbating the feedback loop, we found that increased CIP2A expression and PP2A inhibition in AD brains result from increased Aβ production. In summary, we show that CIP2A overexpression causes PP2A inhibition and AD-related cellular pathology and cognitive deficits, pointing to CIP2A as a potential target for AD therapy.

Hyperphosphorylation of cytoskeletal proteins seen in Alzheimer's disease is most probably the result of an imbalanced regulation in protein kinases and protein phosphatases (PP) in the affected neurons. Previous studies have revealed that PP-2A and PP-1 play important roles in the pathogenesis. Employing human neuroblastoma cells, we found that 10 nM calyculin A (CA), a selective inhibitor of PP-2A and PP-1, significantly increased phosphorylation and accumulation of neurofilament (NF) in the cells. Levels of NF-M (middle chain) and NF-L (light chain) mRNA decreased after CA treatment. Additionally, CA led to a decreased cell viability determined by MTT and crystal violet assay. Melatonin efficiently protects the cell from CA-induced alterations in NF hyperphosphorylation and accumulation, suppressed NF gene expression as well as decreased cell viability. It is concluded that inhibition of PP-2A/PP-1 by CA induces abnormalities in NF metabolism and cell survival, and melatonin efficiently arrests the lesions.

Abnormally nitrated tau has been found recently in the neurofibrillary tangles of AlzheimerÕs disease (AD). However, whether and how nitration of tau is involved in AD pathology is not known. Herein, we found that in vitro incubation of peroxynitrite with recombinant tau resulted in nitration and oligomerization of tau in a dosage-dependent manner. Moreover, the nitrated tau showed a significantly decreased binding activity to taxol-stabilized microtubulesin in vitro. Further study demonstrated that peroxynitrite also induced tau nitration in neuroblastoma N2a cell line, and the nitrated tau was accumulated in the cells. We conclude that abnormal nitration of tau contributes to the impaired biological activity of tau in binding to the microtubules and the aggregation of tau, implying a novel mechanism responsible for the neurodegeneration seen in AD brain.