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...

Paired helical filament (PHF) tau is the principal component of neurofibrillary tangles, a characteristic feature of the neurodegenerative pathology in Alzheimer's disease (AD). Post-translational modification of tau, especially phosphorylation, has been considered a major factor in aggregation and diminished microtubule interactions of PHF-tau. Recently, it has been recognized that PHF-tau is also subject to non-enzymatic glycation, with formation of advanced glycation end products (AGEs). We now show that as a consequence of glycation, PHF-tau from AD and AGE-tau generate oxygen free radicals, thereby activating transcription via nuclear factor-kappa B, increasing amyloid beta-protein precursor and release of approximately 4 kD amyloid beta-peptides. These data provide insight into how PHF-tau disturbs neuronal function, and add to a growing body of evidence that oxidant stress contributes to the pathogenesis of AD.

Endothelium serves as the cellular barrier retaining blood components within the intravascular space (1). This function is compromised in many pathophysiologic states . Most notably, the profound alterations in endothelial cell barrier function specifically induced by host mediators released in response to bacterial LPS contribute importantly to the pathogenesis of endotoxic/septic shock.The cytokine tumor necrosis factor/cachectin (TNF), one of several host mediators synthesized by macrophages after exposure to LPS, has been shown to be a central mediator of the shock state in Gram-negative sepsis (2, 3). Infusion ofTNF into animals produces many of abnormalities characteristic of Escherichia coli/LPS toxemia, including vascular leakage due to loss of barrier function (2, 4). Vascular endothelium has been shown to be a direct target for the action ofTNF, which causes modulation of many endothelial cell functions, such as coagulant activity and immunologic properties (2). Recent studies (5-7) suggest that the normal barrier function of the endothelium can also be altered directly by TNF. These considerations have led us to examine the effect of TNF on endothelial permeability. The results indicate that within 1-3 h after exposure to TNF, permeability of cultured endothelial monolayers to macromolecular and lower molecular weight solutes increased markedly ; this effect was temporally correlated with alterations in the cytoskeleton and cell shape, and the formation of intercellular gaps. Pertussis toxin blocked both the change in cell shape/cytoskelton and the increase in permeability, indicating that a pertussis toxin-sensitive G protein critically regulates this TNFinduced response. In contrast, other TNFinduced changes in endothelial cell function, such as perturbation of certain cell surface coagulant properties, were not affected by pertussis toxin. These data suggest the presence of more than one TNFdependent signal transduction pathway within endothelial cells: a pathway involving a pertussis toxin-sensitive G protein leads to increased vascular permeability, whereas, a separate mechanism underlies the modulation of other cellular properties by TNF.

In Alzheimer disease (AD), neurons are thought to be subjected to the deleterious cytotoxic effects of activated microglia. We demonstrate that binding of amyloidbeta peptide (A␤) to neuronal Receptor for Advanced Glycation Endproduct (RAGE), a cell surface receptor for A␤, induces macrophage-colony stimulating factor (M-CSF) by an oxidant sensitive, nuclear factor B-dependent pathway. AD brain shows increased neuronal expression of M-CSF in proximity to A␤ deposits, and in cerebrospinal fluid from AD patients there was Ϸ5-fold increased M-CSF antigen (P < 0.01), compared with age-matched controls. M-CSF released by A␤-stimulated neurons interacts with its cognate receptor, c-fms, on microglia, thereby triggering chemotaxis, cell proliferation, increased expression of the macrophage scavenger receptor and apolipoprotein E, and enhanced survival of microglia exposed to A␤, consistent with pathologic findings in AD. These data delineate an inflammatory pathway triggered by engagement of A␤ on neuronal RAGE. We suggest that M-CSF, thus generated, contributes to the pathogenesis of AD, and that M-CSF in cerebrospinal fluid might provide a means for monitoring neuronal perturbation at an early stage in AD.

HeLa, Vero, L, HEp2, and MDBK cells respond immediately to 0.2–0.5 µg/ml cytochalasin D (CD) with sustained contraction (contracture), loss of microvilli, expression of endoplasmic contents (zeiosis), nuclear protrusion, and extension of cytoplasmic processes. The development of these changes is depicted, and the dose-response patterns in these cell lines are described. MDBK is generally most resistant and HeLa most sensitive to these effects of CD. Cells in G1 are most sensitive to CD; responsiveness decreases progressively during early S and is least in mid S through G2. CD inhibits transport of [14C]deoxyglucose in HeLa by about 45% but has no significant effect on hexose uptake in Vero and MDBK; sugar transport is thus apparently unrelated to any morphologic effect of CD. Although spreading and attachment are impeded, CD does not decrease and may even enhance the adhesiveness of established monolayers. Contraction appears to be a primary early effect of CD, upon which other visible changes follow. It is prevented by some inhibitors of energy metabolism (deoxyglucose and dinitrophenol) and does not occur in glycerinated models without ATP. The possible bases of the contractile response to CD are discussed. Although direct or indirect action of CD on some microfilaments may occur, a generalized structural disruption of contractile filaments by CD is considered unlikely.