beta-amyloid (Abeta) has been proposed to play a role in the pathogenesis of Alzheimer's disease (AD). Deposits of insoluble Abeta are found in the brains of patients with AD and are one of the pathological hallmarks of the disease. It has been proposed that Abeta induces death by oxidative stress, possibly through the generation of peroxynitrite from superoxide and nitric oxide. In our current study, treatment with nitric oxide generators protected against Abeta-induced death, whereas inhibition of nitric oxide synthase afforded no protection, suggesting that formation of peroxynitrite is not critical for Abeta-mediated death. Previous studies have shown that aggregated Abeta can induce caspase-dependent apoptosis in cultured neurons. In all of the neuronal populations studied here (hippocampal neurons, sympathetic neurons, and PC12 cells), cell death was blocked by the broad spectrum caspase inhibitor N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone and more specifically by the downregulation of caspase-2 with antisense oligonucleotides. In contrast, downregulation of caspase-1 or caspase-3 did not block Abeta(1-42)-induced death. Neurons from caspase-2 null mice were totally resistant to Abeta(1-42) toxicity, confirming the importance of this caspase in Abeta-induced death. The results indicate that caspase-2 is necessary for Abeta(1-42)-induced apoptosis in vitro.

The stability of proteins that constitute the neurofibrillary tangles and senile plaques of Alzheimer disease suggests that they would be ideal substrates for nonenzymatic glycation, a process that occurs over long times, even at normal levels of glucose, ultimately resulting in the formation of advanced glycation end products (AGEs). AGE-modified proteins aggregate, and they generate reactive oxygen intermediates. Using monospecific antibody to AGEs, we have colocalized these AGEs with paired helical filament tau in neurofibrillary tangles in sporadic Alzheimer disease. Such neurons also exhibited evidence of oxidant stress: induction of malondialdehyde epitopes and heme oxygenase 1 antigen. AGErecombinant tau generated reactive oxygen intermediates and, when introduced into the cytoplasm of SH-SY5Y neuroblastoma cells, induced oxidant stress. We propose that in Alzheimer disease, AGEs in paired helical filament tau can induce oxidant stress, thereby promoting neuronal dysfunction.Proteins or lipids exposed to reducing sugars undergo nonenzymatic glycation and oxidation, initially with formation of Schiff bases and Amadori products on free amino groups, which ultimately undergo molecular rearrangement, to form irreversible advanced glycation end products (AGEs; refs. [1][2][3][4][5]. The AGEs are heterogeneous compounds of yellowbrown color and characteristic fluorescence (1-5). Accumulation of AGEs occurs on both intra-and extracellular structures, especially those whose turnover is prolonged. Although the formation of AGEs is accelerated in diabetes, it also occurs in normal aging. Proteins with many free amino groups (i.e., with high lysine content) are most readily glycated. AGE-modified proteins form crosslinks which result in aggregation and insolubility; they are also a continuing source ofpotentially damaging reactive oxygen intermediates (ROIs) and, when present extracellularly, interact with a distinct class of receptors (1-9). In cells, we have found that AGEs impart an oxidant stress manifested in endothelium by induction of heme oxygenase, activation of the transcription factor NF-KB, and formation of malondialdehyde epitopes of lipid peroxidation products (9). These perturbations, which result in changes in a spectrum of cellular properties (e.g., cell adherence, proliferation), were not accompanied by diminished cell viability (in short-term experiments), in keeping with a role for low levels of ROIs in signal transduction.The longstanding protein aggregates in Alzheimer disease (AD), such as paired helical filament (PHF) tau and amyloid .3protein (10)(11)(12) METHODS AGE ELISA, Immunoblotting, and Immunohistohemistry. AGE antigen was determined by using affinity-purified antibody to AGEs (9,13). This antibody selectively recognizes AGE forms of multiple proteins, but not the nonglycated counterparts (9) or formylated, maleylated, oxidized, or acetylated protein (9). To assay for AGE antigen (9), an ELISA was established by coating plates with brain homogenates/PHF tau (10-100 gg/ml) ove...

During mitosis in higher eukaryotic cells, the nuclear envelope membranes break down into distinct populations of vesicles and the proteins of the nuclear lamina and the nuclear pore complexes disperse in the cytoplasm. Since phosphorylation can alter protein-protein interactions and membrane traffic, we have examined the cell cycle-dependent phosphorylation of nuclear pore complex proteins. Nonmembrane nucleoporins Nup153, Nup214, and Nup358 that are modified by O-linked N-acetylglucosamine and recognized by a monoclonal antibody were phosphorylated throughout the cell cycle and hyperphosphorylated during M phase. Pore membrane glycoprotein gp210, that has a cytoplasmic, carboxyl-terminal domain facing the pore, was not phosphorylated in interphase but specifically phosphorylated in mitosis. Mutant and wild-type fusion proteins containing the cytoplasmic domain of gp210 were phosphorylated in vitro and their phosphopeptide maps compared to that of mitotic gp210. This analysis showed that Ser1880 of gp210 was phosphorylated in mitosis, possibly by cyclin B-p34cdc2 or a related kinase. Several nuclear pore complex proteins are therefore differentially phosphorylated during mitosis when pore complexes disassemble and reassemble.