White, A. R., G. Multhaup, et al. (2002). "Contrasting, species-dependent modulation of copper-mediated neurotoxicity by the Alzheimer's disease amyloid precursor protein." J Neurosci 22(2): 365-76. The amyloid precursor protein (APP) of Alzheimer's disease (AD) has a copper binding domain (CuBD) located in the N-terminal cysteine-rich region that can strongly bind copper(II) and reduce it to Cu(I) in vitro. The CuBD sequence is similar among the APP family paralogs [amyloid precursor-like proteins (APLP1 and APLP2)] and its orthologs (including Drosophila melanogaster, Xenopus laevis, and Caenorhabditis elegans), suggesting an overall conservation in its function or activity. The APP CuBD is involved in modulating Cu homeostasis and amyloid beta peptide production. In this paper, we demonstrate for the first time that Cu-metallated full-length APP ectodomain induces neuronal cell death in vitro. APP Cu neurotoxicity can be induced directly or potentiated through Cu(I)-mediated oxidation of low-density lipoprotein, a finding that may have important implications for the role of lipoproteins and membrane cholesterol composition in AD. Cu toxicity induced by human APP, Xenopus APP, and APLP2 CuBDs is dependent on conservation of histidine residues at positions corresponding to 147 and 151 of human APP. Intriguingly, APP orthologs with different amino acid residues at these positions had dramatically altered Cu phenotypes. The corresponding C. elegans APL-1 CuBD, which has tyrosine and lysine residues at positions 147 and 151, respectively, strongly protected against Cu-mediated lipid peroxidation and neurotoxicity in vitro. Replacement of histidines 147 and 151 with tyrosine and lysine residues conferred this neuroprotective Cu phenotype to human APP, APLP2, and Xenopus APP CuBD peptides. Moreover, we show that the toxic and protective CuBD phenotypes are associated with differences in Cu binding and reduction. These studies identify a significant evolutionary change in the function of the CuBD in modulating Cu metabolism. Our findings also suggest that targeting of inhibitors to histidine residues at positions 147 and 151 of APP could significantly alter the oxidative potential of APP. Perini, G., V. Della-Bianca, et al. (2002). "Role of p75 neurotrophin receptor in the neurotoxicity by beta-amyloid peptides and synergistic effect of inflammatory cytokines." J Exp Med 195(7): 907-18. The neurodegenerative changes in Alzheimer's disease (AD) are elicited by the accumulation of beta-amyloid peptides (Abeta), which damage neurons either directly by interacting with components of the cell surface to trigger cell death signaling or indirectly by activating astrocytes and microglia to produce inflammatory mediators. It has been recently proposed that the p75 neurotrophin receptor (p75(NTR)) is responsible for neuronal damage by interacting with Abeta. By using neuroblastoma cell clones lacking the expression of all neurotrophin receptors or engineered to express full-length or various truncated forms of p75(NTR), we could show that p75(NTR) is involved in the direct signaling of cell death by Abeta via the function of its death domain. This signaling leads to the activation of caspases-8 and -3, the production of reactive oxygen intermediates and the induction of an oxidative stress. We also found that the direct and indirect (inflammatory) mechanisms of neuronal damage by Abeta could act synergistically. In fact, TNF-alpha and IL-1beta, cytokines produced by Abeta-activated microglia, could potentiate the neurotoxic action of Abeta mediated by p75(NTR) signaling. Together, our results indicate that neurons expressing p75(NTR), mostly if expressing also proinflammatory cytokine receptors, might be preferential targets of the cytotoxic action of Abeta in AD. Pappolla, M. A., M. J. Simovich, et al. (2002). "The neuroprotective activities of melatonin against the Alzheimer beta-protein are not mediated by melatonin membrane receptors." J Pineal Res 32(3): 135-42. Exposure of neuronal cells to the Alzheimer's amyloid beta protein (Abeta) results in extensive oxidative damage of bio-molecules that are profoundly harmful to neuronal homeostasis. It has been demonstrated that melatonin protects neurons against Abeta-mediated neurotoxicity, including cell death and a spectrum of oxidative lesions. We undertook the current study to determine whether melatonin membrane receptors are involved in the mechanism of neuroprotection against Abeta neurotoxicity. For this purpose, we characterized the free-radical scavenging potency of several compounds exhibiting various affinities for melatonin membrane receptors (MLT 1a and 1b). Abeta-mediated neurotoxicity was assessed in human neuroblastoma cells and in primary hippocampal neurons. In sharp contrast with melatonin, no neuroprotection against Abeta toxicity was observed when we used melatonin membrane receptor agonists that were devoid of antioxidant activity. In contrast, the cells were fully protected in parallel control experiments when either melatonin, or the structurally unrelated free-radical scavenger phenyl-N-t-butyl nitrone (PBN), were added to Abeta-containing culture media. This study demonstrates that the neuroprotective properties of melatonin against Abeta-mediated toxicity does not require binding of melatonin to a membrane receptor and is likely the result of the antioxidant and antiamyloidogenic features of the agent. Yip, C. M., E. A. Elton, et al. (2001). "Cholesterol, a modulator of membrane-associated Abeta-fibrillogenesis and neurotoxicity." J Mol Biol 311(4): 723-34. Recent studies have suggested that cholesterol, an important determinant of the physical state of biological membranes, plays a significant role in the development of Alzheimer's disease. We have employed in situ scanning probe microscopy, fluorescence anisotropy, and electron microscopy to investigate how cholesterol levels within total brain lipid bilayers effect amyloid beta-peptide (Abeta)-assembly. Fluorescence anisotropy measurements revealed that the relative fluidity of the total brain lipid membranes was influenced by the level of cholesterol and the addition of Abeta40 resulted in a decrease in the overall vesicle fluidity. In situ scanning probe microscopy performed on supported planar bilayers of total brain lipid revealed a correlation between membrane fluidity, as influenced by cholesterol level, and the extent of Abeta-insertion and subsequent fibrillogenesis. These observations were consistent with fluorescence microscopy studies of PC-12 and SH-SY5Y cell lines exposed to exogenous Abeta, which revealed an inverse correlation between membrane cholesterol level, and Abeta-cell surface binding and subsequent cell death. These results collectively suggest that Abeta-cell surface interactions are mediated by cellular cholesterol levels, the distribution of cholesterol throughout the cell, and membrane fluidity. Yan, S. D., A. M. Schmidt, et al. (2001). "Alzheimer's disease: inside, outside, upside down." Biochem Soc Symp(67): 15-22. Neurotoxicity of beta-amyloid peptide (A beta) in Alzheimer's disease (AD) is usually thought to arise from the nonspecific effects of high concentrations of A beta on vulnerable neurons, resulting in membrane destabilization and increasing intracellular calcium concentration. This review advances the hypothesis that at early stages of AD, when A beta is present in lower amounts, its ability to perturb the function of cellular targets is mediated by specific cofactors present on the cell surface and intracellularly. Receptor for advanced glycation endproducts (RAGE) is a cell-surface receptor which binds A beta and amplifies its effects on cells in the nanomolar range. The intracellular enzyme A beta-binding alcohol dehydrogenase (ABAD) is likely to engage nascent A beta formed in the endoplasmic reticulum, and to mediate cell stress from this site. The analysis of A beta interaction with RAGE and ABAD, as well as other cofactors, provides insight into new mechanisms and, potentially, identifies therapeutic targets relevant to neuronal dysfunction in AD. Yagyu, K., K. Kitagawa, et al. (2001). "Amyloid beta proteins inhibit Cl(-)-ATPase activity in cultured rat hippocampal neurons." J Neurochem 78(3): 569-76. Cl(-)-ATPase in the CNS is a candidate for an outwardly directed neuronal Cl(-) transporter requiring phosphatidylinositol-4-phosphate (PI4P) for its optimal activity. To test its pathophysiological changes in a phosphatidylinositol (PI) metabolism disorder, the effects of neurotoxic factors in Alzheimer's disease (AD), amyloid beta proteins (Abetas), on the Cl(-)-ATPase activity were examined using primary cultured rat hippocampal neurons. Amyloid beta proteins (1-40, 1-42 and 25-35) concentration-dependently (1-100 nM) and time-dependently (from 1 h to 6 day) decreased Cl(-)-ATPase activity and elevated intracellular Cl(-) concentrations ([Cl(-)]i), Abeta25-35 being the most potent. Addition of inositol or 8-Br-cyclic GMP completely reversed these Abeta-induced changes. The recoveries in enzyme activity were attenuated by an inhibitor of PI 4-kinase, 10 microM wortmannin or 20 microM quercetin, but not by a PI 3-kinase inhibitor, 50 nM wortmannin or 10 microM LY294002. The PI, PIP and PIP2 levels of the plasma membrane-rich fraction were lower in the Abeta-treated cells as compared with each control. In the Abeta-exposed culture, but not in control, stimulation by 10 microM glutamate for 10 min significantly increased fragmentation of DNA and decreased cell viability. Addition of inositol or 8-Br-cyclic GMP prevented the effect of Abeta-treatment on the neurotoxicity of glutamate. Thus, Abetas reduce neuronal Cl(-)-ATPase activity, resulting in an increase in [Cl(-)]i probably by lowering PI4P levels, and this may reflect a pre-apoptotic condition in early pathophysiological profiles of AD. Wang, S. S., D. L. Rymer, et al. (2001). "Reduction in cholesterol and sialic acid content protects cells from the toxic effects of beta-amyloid peptides." J Biol Chem 276(45): 42027-34. beta-Amyloid (Abeta) is the primary protein component of senile plaques associated with Alzheimer's disease and has been implicated in the neurotoxicity associated with the disease. A variety of evidence points to the importance of Abeta-membrane interactions in the mechanism of Abeta neurotoxicity and indicates that cholesterol and gangliosides are particularly important for Abeta aggregation and binding to membranes. We investigated the effects of cholesterol and sialic acid depletion on Abeta-induced GTPase activity in cells, a step implicated in the mechanism of Abeta toxicity, and Abeta-induced cell toxicity. Cholesterol reduction and depletion of membrane-associated sialic acid residues both significantly reduced the Abeta-induced GTPase activity. In addition, cholesterol and membrane-associated sialic acid residue depletion or inhibition of cholesterol and ganglioside synthesis protected PC12 cells from Abeta-induced toxicity. These results indicate the importance of Abeta-membrane interactions in the mechanism of Abeta toxicity. In addition, these results suggest that control of cellular cholesterol and/or ganglioside content may prove useful in the prevention or treatment of Alzheimer's disease. Kawahara, M. and Y. Kuroda (2001). "Intracellular calcium changes in neuronal cells induced by Alzheimer's beta-amyloid protein are blocked by estradiol and cholesterol." Cell Mol Neurobiol 21(1): 1-13. 1. The elevation of intracellular Ca2+ levels ([Ca2+]i) in immortalized hypothalamic neurons (GT1-7 cells) after exposure to Alzheimer's beta-amyloid protein (AbetaP[25-35]) was investigated using a multisite fluorometry system. 2. The marked rise in [Ca2+]i appeared after exposure to 5-20-microM AbetaP[25-35]. Analysis of the spatiotemporal patterns of [Ca2+]i changes revealed that the magnitude and the latency of the response to AbetaP in each cell were highly heterogeneous. 3. The preadministration of 17beta-estradiol, 17alpha-estradiol, phloretin and cholesterol, which influence the properties of membranes, such as membrane fluidity or membrane potential, significantly decreased the rise in [Ca2+]i. 4. These findings support the idea that disruption of calcium homeostasis by AbetaP channels may be the molecular basis of the neurotoxicity of AbetaP and of the pathogenesis of Alzheimer's disease. It is also suggested that membrane properties may play key roles in the expression of neurotoxicity. Kawahara, M., M. Kato, et al. (2001). "Effects of aluminum on the neurotoxicity of primary cultured neurons and on the aggregation of beta-amyloid protein." Brain Res Bull 55(2): 211-7. Recent epidemiological, neuropathological, and biochemical studies have suggested a possible link between the neurotoxicity of aluminum and the pathogenesis of Alzheimer's disease. However, this relationship remains controversial. To investigate detailed characteristics of neurotoxicity of aluminum, we used primary cultured neurons of rat cerebral cortex as an in vitro model system for the observation of morphological changes induced by chronic exposure to aluminum. Although the exposure to aluminum chloride (10-100 microM) for 1 week did not cause marked neuronal death, degeneration of neuritic processes and accumulation of tau protein and beta-amyloid protein appeared after chronic exposure to 50 microM aluminum chloride for more than 3 weeks. We also investigated the polymerization of beta-amyloid protein in vitro using the immunoblotting technique. We thus found that aluminum induced conformational changes in beta-amyloid protein and enhanced its aggregation in vitro. The aggregated beta-amyloid protein was dissolved by the addition of desferrioxamine, a chelator of aluminum. The aggregated beta-amyloid protein pre-incubated with aluminum formed fibrillar deposits on the surface of cultured neurons. Joseph, J., B. Shukitt-Hale, et al. (2001). "Copernicus revisited: amyloid beta in Alzheimer's disease." Neurobiol Aging 22(1): 131-46. The beta-amyloid hypothesis of Alzheimer's Disease (AD) has dominated the thinking and research in this area for over a decade and a half. While there has been a great deal of effort in attempting to prove its centrality in this devastating disease, and while an enormous amount has been learned about its properties (e.g., putative toxicity, processing and signaling), Abeta has not proven to be both necessary and sufficient for the development, neurotoxicity, and cognitive deficits associated with this disease. Instead, the few treatments that are available have emerged from aging research and are primarily directed toward modification of acetylcholine levels. Clearly, it is time to rethink this position and to propose instead that future approaches should focus upon altering the age-related sensitivity of the neuronal environment to insults involving such factors as inflammation and oxidative stress. In other words "solve the problems of aging and by extension those of AD will also be reduced." This review is being submitted as a rather Lutherian attempt to "nail an alternative thesis" to the gate of the Church of the Holy Amyloid to open its doors to the idea that aging is the most pervasive element in this disease and Abeta is merely one of the planets. Jhamandas, J. H., C. Cho, et al. (2001). "Cellular mechanisms for amyloid beta-protein activation of rat cholinergic basal forebrain neurons." J Neurophysiol 86(3): 1312-20. The deposition of amyloid beta-protein (Abeta) in the brain and the loss of cholinergic neurons in the basal forebrain are two pathological hallmarks of Alzheimer's disease (AD). Although the mechanism of Abeta neurotoxicity is unknown, these cholinergic neurons display a selective vulnerability when exposed to this peptide. In this study, application of Abeta(25-35) or Abeta(1-40) to acutely dissociated rat neurons from the basal forebrain nucleus diagonal band of Broca (DBB), caused a decrease in whole cell voltage-activated currents in a majority of cells. This reduction in whole cell currents occurs through a modulation of a suite of potassium conductances including calcium-activated potassium (I(C)), the delayed rectifier (I(K)), and transient outward potassium (I(A)) conductances, but not calcium or sodium currents. Under current-clamp conditions, Abeta evoked an increase in excitability and a loss of accommodation in cholinergic DBB neurons. Using single-cell RT-PCR technique, we determined that Abeta actions were specific to cholinergic, but not GABAergic DBB neurons. Abeta effects on whole cell currents were occluded in the presence of membrane-permeable protein tyrosine kinase inhibitors, genistein and tyrphostin B-44. Our data indicate that the Abeta actions on specific potassium conductances are modulated through a protein tyrosine kinase pathway and that these effects are selective to cholinergic but not GABAergic cells. These observations provide a cellular basis for the selectivity of Abeta neurotoxicity toward cholinergic basal forebrain neurons that are at the epicenter of AD pathology. Hashimoto, Y., T. Niikura, et al. (2001). "Detailed characterization of neuroprotection by a rescue factor humanin against various Alzheimer's disease-relevant insults." J Neurosci 21(23): 9235-45. A novel factor, termed Humanin (HN), antagonizes against neurotoxicity by various types of familial Alzheimer's disease (AD) genes [V642I and K595N/M596L (NL) mutants of amyloid precursor protein (APP), M146L-presenilin (PS) 1, and N141I-PS2] and by Abeta1-43 with clear action specificity ineffective on neurotoxicity by polyglutamine repeat Q79 or superoxide dismutase 1 mutants. Here we report that HN can also inhibit neurotoxicity by other AD-relevant insults: other familial AD genes (A617G-APP, L648P-APP, A246E-PS1, L286V-PS1, C410Y-PS1, and H163R-PS1), APP stimulation by anti-APP antibody, and other Abeta peptides (Abeta1-42 and Abeta25-35). The action specificity was further indicated by the finding that HN could not suppress neurotoxicity by glutamate or prion fragment. Against the AD-relevant insults, essential roles of Cys(8) and Ser(14) were commonly indicated, and the domain from Pro(3) to Pro(19) was responsible for the rescue action of HN, in which seven residues turned out to be essential. We also compared the neuroprotective action of S14G HN (HNG) with that of activity-dependent neurotrophic factor, IGF-I, or basic FGF for the antagonism against various AD-relevant insults (V642I-APP, NL-APP, M146L-PS1, N141I-PS2, and Abeta1-43). Although all of these factors could abolish neurotoxicity by Abeta1-43, only HNG could abolish cytotoxicities by all of them. HN and HN derivative peptides may provide a new insight into the study of AD pathophysiology and allow new avenues for the development of therapeutic interventions for various forms of AD. Drouet, B., A. Fifre, et al. (2001). "ApoE protects cortical neurones against neurotoxicity induced by the non-fibrillar C-terminal domain of the amyloid-beta peptide." J Neurochem 76(1): 117-27. Although the genetic link between the epsilon 4 allele of apolipoprotein E (apoE) and Alzheimer's disease (AD) is well established, the apoE isoform-specific activity underlying this correlation remains unclear. We have recently characterized the interaction of the soluble the amyloid-beta peptide (A beta) with model membrane and demonstrated that non-fibrillar A beta peptide, including N-terminal truncated forms of A beta, induced apoptotic cell death in primary rat cortical neurones in vitro. To further investigate the potential interaction between apoE and A beta in the pathogenesis of AD, we have determined the effect of apoE isoforms on the neurotoxicity of non-fibrillar A beta peptides. We demonstrate here that the apoE2 and E3 isoforms protect cortical neurones against apoptotic cell death induced by a non-fibrillar form of the A beta(1-40), A beta(12-42), A beta(29-40) and A beta(29-42) peptides, whereas apoE4 had no effect. This effect involves the formation of stable complexes between apoE and the C-terminal domain (e.g. amino acids 29-40) of A beta(1-40). Interestingly, apoE had no effect on the toxicity induced by aggregated A beta peptides, suggesting a lack of interaction between apoE and amyloid fibrils. Our results provide evidence that interaction with the C-terminal domain of A beta, apoE2 and E3, but not apoE4, inhibits the interactions of the non-fibrillar A beta peptide with the plasma membrane of neurones, A beta peptide aggregation and subsequent neurotoxicity. Cardoso, S. M., S. Santos, et al. (2001). "Functional mitochondria are required for amyloid beta-mediated neurotoxicity." Faseb J 15(8): 1439-41. Rymer, D. L. and T. A. Good (2000). "The role of prion peptide structure and aggregation in toxicity and membrane binding." J Neurochem 75(6): 2536-45. Prion diseases are neurodegenerative disorders associated with a conformational change in the normal cellular isoform of the prion protein, PrP(C), to an abnormal scrapie isoform, PrP(SC). Unlike the alpha-helical PrP(C), the protease-resistant core of PrP(SC) is predominantly beta-sheet and possesses a tendency to polymerize into amyloid fibrils. We performed experiments with two synthetic human prion peptides, PrP(106-126) and PrP(127-147), to determine how peptide structure affects neurotoxicity and protein-membrane interactions. Peptide solutions possessing beta-sheet and amyloid structures were neurotoxic to PC12 cells in vitro and bound with measurable affinities to cholesterol-rich phospholipid membranes at ambient conditions, but peptide solutions lacking stable beta-sheet structures and amyloid content were nontoxic and possessed less than one tenth of the binding affinities of the amyloid-containing peptides. Regardless of structure, the peptide binding affinities to cholesterol-depleted membranes were greatly reduced. These results suggest that the beta-sheet and amyloid structures of the prion peptides give rise to their toxicity and membrane binding affinities and that membrane binding affinity, especially in cholesterol-rich environments, may be related to toxicity. Our results may have significance in understanding the role of the fibrillogenic cerebral deposits associated with some of the prion diseases in neurodegeneration and may have implications for other amyloidoses. Pillot, T., B. Drouet, et al. (2000). "A nonfibrillar form of the fusogenic prion protein fragment [118-135] induces apoptotic cell death in rat cortical neurons." J Neurochem 75(6): 2298-308. Neuronal loss is a salient feature of prion diseases. However, its cause and mechanism, particularly its relationship with the accumulation and precipitation of the pathogenic, protease-resistant isoform PrP(Sc) of the cellular prion protein PrP(C), are still an enigma. Several studies suggest that neuronal loss could occur through a process of programmed cell death, which is consistent with the lack of inflammation in these conditions. By analogy with the pathological events occurring during the development of Alzheimer's disease, controversies still exist regarding the relationship between amyloidogenesis, prion aggregation, and neuronal loss. We recently demonstrated that a prion protein fragment (118-135) displayed membrane-destabilizing properties and was able to induce, in a nonfibrillar form, the fusion of unilamellar liposomes. To unravel the mechanism of prion protein neurotoxicity, we characterize the effects of the human Pr[118-135] peptide on rat cortical neurons. We demonstrate that low concentrations of the Pr[118-135] peptide, in a nonfibrillar form, induce a time- and dose- dependent apoptotic cell death, including caspase activation, DNA condensation, and fragmentation. This toxicity might involve oxidative stress, because antioxidant molecules, such as probucol and propyl gallate, protect neurons against prion peptide toxicity. By contrast, a nonfusogenic variant Pr[118-135, 0 degrees ] peptide, which displays the same amino acid composition but several amino acid permutations, is not toxic to cortical neurons, which emphasizes the critical role of the fusogenic properties of the prion peptide in its neurotoxicity. Taken together, our results suggest that the interaction between the Pr[118-135] peptide and the plasma membrane of neurons might represent an early event in a cascade leading to neurodegeneration. Nakagawa, T., H. Zhu, et al. (2000). "Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta." Nature 403(6765): 98-103. Apoptosis, or cellular suicide, is important for normal development and tissue homeostasis, but too much or too little apoptosis can also cause disease. The family of cysteine proteases, the so- called caspases, are critical mediators of programmed cell death, and thus far 14 family members have been identified. Some of these, such as caspase-8, mediate signal transduction downstream of death receptors located on the plasma membrane. Others, such as caspase-9, mediate apoptotic signals after mitochondrial damage. Stress in the endoplasmic reticulum (ER) can also result in apoptosis. Here we show that caspase-12 is localized to the ER and activated by ER stress, including disruption of ER calcium homeostasis and accumulation of excess proteins in ER, but not by membrane- or mitochondrial-targeted apoptotic signals. Mice that are deficient in caspase-12 are resistant to ER stress-induced apoptosis, but their cells undergo apoptosis in response to other death stimuli. Furthermore, we show that caspase-12-deficient cortical neurons are defective in apoptosis induced by amyloid-beta protein but not by staurosporine or trophic factor deprivation. Thus, caspase-12 mediates an ER-specific apoptosis pathway and may contribute to amyloid-beta neurotoxicity. Lorenzo, A., M. Yuan, et al. (2000). "Amyloid beta interacts with the amyloid precursor protein: a potential toxic mechanism in Alzheimer's disease." Nat Neurosci 3(5): 460-4. Amyloid beta protein (Abeta) deposition in the brain is a hallmark of Alzheimer's disease (AD). The fibrillar form of Abeta is neurotoxic, although the mechanism of its toxicity is unknown. We showed that conversion of Abeta to the fibrillar form markedly increased binding to specific neuronal membrane proteins, including amyloid precursor protein (APP). Nanomolar concentrations of fibrillar Abeta bound cell-surface holo-APP in cortical neurons. Reduced vulnerability of cultured APP-null neurons to Abeta neurotoxicity suggested that Abeta neurotoxicity involves APP. Thus Abeta toxicity may be mediated by the interaction of fibrillar Abeta with neuronal membrane proteins, notably APP. An Abeta-APP interaction reminiscent of the pathogenic mechanism of prions may thus contribute to neuronal degeneration in AD. Kawahara, M. and Y. Kuroda (2000). "Molecular mechanism of neurodegeneration induced by Alzheimer's beta-amyloid protein: channel formation and disruption of calcium homeostasis." Brain Res Bull 53(4): 389-97. The etiology of Alzheimer's disease has been suggested to be linked to the neurodegeneration induced by beta-amyloid protein (AbetaP), however, the mechanism underlying the latter remains unknown. We have previously shown the direct incorporation of AbetaP into neuronal membranes of immortalized hypothalamic neurons (GT1-7 cells) associated with the formation of calcium-permeable pores, and the elevation of the intracellular calcium concentrations in the GT1-7 cells. Taking together our results and those of numerous other studies, we hypothesize that the disruption of calcium homeostasis by AbetaP-channels may be the molecular basis of the neurotoxicity of AbetaP, and the development of Alzheimer's disease. It is also proposed that the constituents of membrane lipids may play important roles in the process of this channel formation. Our hypothesis may also explain the mechanism of development of other 'conformational diseases', such as prion disease or type 2 diabetes mellitus, which share some common features with Alzheimer's disease. Huang, X., M. P. Cuajungco, et al. (2000). "Alzheimer's disease, beta-amyloid protein and zinc." J Nutr 130(5S Suppl): 1488S-92S. Alzheimer's disease (AD) is characterized by amyloid deposits within the neocortical parenchyma and the cerebrovasculature. The main component of these predominantly extracellular collections, Abeta, which is normally a soluble component of all biological fluids, is cleaved out of a ubiquitously expressed parent protein, the amyloid protein precursor (APP), one of the type 1 integral membrane glycoproteins. Considerable evidence has indicated that there is zinc dyshomeostasis and abnormal cellular zinc mobilization in AD. We have characterized both APP and Abeta as copper/zinc metalloproteins. Zinc, copper and iron have recently been reported to be concentrated to 0.5 to 1 mmol/L in amyloid plaque. In vitro, rapid Abeta aggregation is mediated by Zn(II), promoted by the alpha-helical structure of Abeta, and is reversible with chelation. In addition, Abeta produces hydrogen peroxide in a Cu(II)/Fe(III)-dependent manner, and the hydrogen peroxide formation is quenched by Zn(II). Moreover, zinc preserves the nontoxic properties of Abeta. Although the zinc-binding proteins apolipoprotein E epsilon4 allele and alpha(2)-macroglobulin have been characterized as two genetic risk factors for AD, zinc exposure as a risk factor for AD has not been rigorously studied. Based on our findings, we envisage that zinc may serve twin roles by both initiating amyloid deposition and then being involved in mechanisms attempting to quench oxidative stress and neurotoxicity derived from the amyloid mass. Hence, it remains debatable whether zinc supplementation is beneficial or deleterious for AD until additional studies clarify the issue. Haik, S., J. M. Peyrin, et al. (2000). "Neurotoxicity of the putative transmembrane domain of the prion protein." Neurobiol Dis 7(6 Pt B): 644-56. It has been shown recently that the generation of an abnormal transmembrane form of the prion protein ((Ctm)PrP) is involved in the neurodegeneration process during inherited and infectious prion diseases but a causative relationship has never been established. We wanted to know if and how the proposed transmembrane domain of PrP could induce neuronal dysfunction. Thus, we investigated the neurotoxic properties of two peptides whose sequences are encompassed within this domain. We show that PrP peptides 118-135 and 105-132 as well as an amidated more soluble peptide 105-132 induce the death of pure cortical neurons originating from normal and PrP knockout mice. This can be correlated with the high propensity of these peptides to insert stably into and to destabilize cell membranes. Through this study, we have identified a novel mechanism of neurotoxicity for PrP, which directly involves membrane perturbation; this mechanism is independent of fibril formation and probably corresponds to the effect of the transmembrane insertion of (Ctm)PrP. Yatin, S. M., M. Aksenov, et al. (1999). "The antioxidant vitamin E modulates amyloid beta-peptide-induced creatine kinase activity inhibition and increased protein oxidation: implications for the free radical hypothesis of Alzheimer's disease." Neurochem Res 24(3): 427-35. Amyloid beta-peptide (Abeta), the main constituent of senile plaques in Alzheimer's disease (AD) brain, is hypothesized to be a key factor in the neurodegeneration seen in AD. Recently it has been shown by us and others that the neurotoxicity of Abeta occurs in conjunction with free radical oxidative stress associated with the peptide. Abeta(1-40) and several other fragments of the Abeta sequence are associated with free radicals in solution that are detectable using electron paramagnetic resonance spectroscopy. These free radicals were shown to attack brain cell membranes, initiate lipid peroxidation, increase Ca2+ influx and damage membrane and cytosolic proteins. In AD brain obtained under rapid autopsy protocol, the activity of the oxidatively-sensitive enzyme creatine kinase was shown to be significantly reduced. We reasoned that Abeta-associated free radical-induced modification of creatine kinase activity and other markers of cellular damage might be modulated by free radical scavengers. Accordingly, this study demonstrates that vitamin E can modulate Abeta(25-35)-induced oxidative damage to creatine kinase and cellular proteins in cultured embryonic hippocampal neurons. These results, consistent with the hypothesis of free radical-mediated Abeta toxicity in AD, are discussed with deference to potential free radical scavengers as therapeutic agents for slowing the progression of AD. Pillot, T., B. Drouet, et al. (1999). "The nonfibrillar amyloid beta-peptide induces apoptotic neuronal cell death: involvement of its C-terminal fusogenic domain." J Neurochem 73(4): 1626-34. The toxicity of the nonaggregated amyloid beta-peptide (1-40) [A beta(1-40)] on the viability of rat cortical neurons in primary culture was investigated. We demonstrated that low concentrations of A beta peptide, in a nonfibrillar form, induced a time- and dose-dependent apoptotic cell death, including DNA condensation and fragmentation. We compared the neurotoxicity of the A beta(1-40) peptide with those of several A beta-peptide domains, comprising the membrane-destabilizing C-terminal domain of A beta peptide (e.g., amino acids 29-40 and 29-42). These peptides reproduced the effects of the (1-40) peptide, whereas mutant nonfusogenic A beta peptides and the central region of the A beta peptide (e.g., amino acids 13-28) had no effect on cell viability. We further demonstrated that the neurotoxicity of the nonaggregated A beta peptide paralleled a rapid and stable interaction between the A beta peptide and the plasma membrane of neurons, preceding apoptosis and DNA fragmentation. By contrast, the peptide in a fibrillar form induced a rapid and dramatic neuronal death mainly through a necrotic pathway, under our conditions. Taken together, our results suggest that A beta induces neuronal cell death by either apoptosis and necrosis and that an interaction between the nonfibrillar C-terminal domain of the A beta peptide and the plasma membrane of cortical neurons might represent an early event in a cascade leading to neurodegeneration. Mason, R. P., R. F. Jacob, et al. (1999). "Distribution and fluidizing action of soluble and aggregated amyloid beta-peptide in rat synaptic plasma membranes." J Biol Chem 274(26): 18801-7. The effects of soluble and aggregated amyloid beta-peptide (Abeta) on cortical synaptic plasma membrane (SPM) structure were examined using small angle x-ray diffraction and fluorescence spectroscopy approaches. Electron density profiles generated from the x-ray diffraction data demonstrated that soluble and aggregated Abeta1-40 peptides associated with distinct regions of the SPM. The width of the SPM samples, including surface hydration, was 84 A at 10 degrees C. Following addition of soluble Abeta1-40, there was a broad increase in electron density in the SPM hydrocarbon core +/-0-15 A from the membrane center, and a reduction in hydrocarbon core width by 6 A. By contrast, aggregated Abeta1-40 contributed electron density to the phospholipid headgroup/hydrated surface of the SPM +/-24-37 A from the membrane center, concomitant with an increase in molecular volume in the hydrocarbon core. The SPM interactions observed for Abeta1-40 were reproduced in a brain lipid membrane system. In contrast to Abeta1-40, aggregated Abeta1-42 intercalated into the lipid bilayer hydrocarbon core +/-0-12 A from the membrane center. Fluorescence experiments showed that both soluble and aggregated Abeta1-40 significantly increased SPM bulk and protein annular fluidity. Physico-chemical interactions of Abeta with the neuronal membrane may contribute to mechanisms of neurotoxicity, independent of specific receptor binding. Malherbe, P., J. G. Richards, et al. (1999). "cDNA cloning of a novel secreted isoform of the human receptor for advanced glycation end products and characterization of cells co-expressing cell-surface scavenger receptors and Swedish mutant amyloid precursor protein." Brain Res Mol Brain Res 71(2): 159-70. The receptor for advanced glycation end products (RAGE) has been proposed as a cell surface receptor that binds amyloid-beta protein (Abeta), thereby triggering its cytotoxic effects [S.D. Yan, X. Chen, J. Fu, M. Chen, H. Zhu, A. Roher, T. Slattery, L. Zhao, M. Nagashima, J. Morser, A. Migheli, P. Nawroth, D. Stern, A.M. Schmidt, RAGE and amyloid-beta peptide neurotoxicity in Alzheimer's disease, Nature 382 (1996) 685-691.]. A cDNA library of human lung was screened for RAGE with an appropriate hybridization probe. In addition to cell surface RAGE, one clone was found which encodes a new version of RAGE, termed hRAGEsec, which lacks the 19 amino acids of the membrane-spanning region and is therefore secreted. Comparison with the genomic sequence revealed that the synthesis of the secreted isoform requires alternative splicing. The deduced protein sequence of the mature hRAGEsec consists of 321 amino acids with a predicted molecular mass of 35.66 kDa. The pattern of expression of hRAGEsec in human brain was analyzed by in situ hybridization histochemistry. The most intense expression of the gene in contrast to cell surface RAGE was detected in hippocampal CA3 pyramidal cells, dentate gyrus granule cells, cortical neurons as well as glial cells in white matter. To investigate the interaction between Abeta and RAGE and another scavenger receptor, SRA, under physiological conditions, they were co-expressed with human betaAPP(695)-SFAD in a human cell and the level of Abeta in the condition medium was assessed by immunoprecipitation and enzyme-linked immunosorbent assay (ELISA) analysis. A nearly 100% reduction of Abeta from the conditioned medium of hRAGE cells and approximately 40% reduction from the SRA-cells implied that hRAGE could be a prominent cell surface receptor interacting with Abeta. Kawahara, M. and Y. Kuroda (1999). "[Neurotoxicity of Alzheimer's beta-amyloid protein and membrane lipids]." Tanpakushitsu Kakusan Koso 44(13): 1982-7. Kanfer, J. N., G. Sorrentino, et al. (1999). "Amyloid beta peptide membrane perturbation is the basis for its biological effects." Neurochem Res 24(12): 1621-30. Experimental studies have indicated that the mechanisms offered for explaining the neurotoxicity of amyloid beta peptide (AbetaP) are diverse, and include altered enzyme activities, disrupted calcium homeostasis, and increased free radical formation. AbetaP appears to interact at the cell membrane with a multitude of receptor sites and also inserts physically into the membrane matrix. This membrane insertion affects the membrane fluidity and potentially influences the function of resident membrane proteins. We propose a unifying hypothesis to explain the experimental observations of the diverse cellular responses to AbetaP. The indiscriminate physical insertion of AbetaP into the cell membrane unspecifically activates a host of membrane processes by perturbation of the membrane proteins. This recurrent activation of membrane processes eventually culminates in neuronal cell death. We recommend that successful therapeutic interventions should be directed at reducing or preventing the interaction of AbetaP with neuronal cell membranes. Hartley, D. M., D. M. Walsh, et al. (1999). "Protofibrillar intermediates of amyloid beta-protein induce acute electrophysiological changes and progressive neurotoxicity in cortical neurons." J Neurosci 19(20): 8876-84. Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is thought to be caused in part by the age-related accumulation of amyloid beta-protein (Abeta). The presence of neuritic plaques containing abundant Abeta-derived amyloid fibrils in AD brain tissue supports the concept that fibril accumulation per se underlies neuronal dysfunction in AD. Recent observations have begun to challenge this assumption by suggesting that earlier Abeta assemblies formed during the process of fibrillogenesis may also play a role in AD pathogenesis. Here, we present the novel finding that protofibrils (PF), metastable intermediates in amyloid fibril formation, can alter the electrical activity of neurons and cause neuronal loss. Both low molecular weight Abeta (LMW Abeta) and PF reproducibly induced toxicity in mixed brain cultures in a time- and concentration-dependent manner. No increase in fibril formation during the course of the experiments was observed by either Congo red binding or electron microscopy, suggesting that the neurotoxicity of LMW Abeta and PF cannot be explained by conversion to fibrils. Importantly, protofibrils, but not LMW Abeta, produced a rapid increase in EPSPs, action potentials, and membrane depolarizations. These data suggest that PF have inherent biological activity similar to that of mature fibrils. Our results raise the possibility that the preclinical and early clinical progression of AD is driven in part by the accumulation of specific Abeta assembly intermediates formed during the process of fibrillogenesis. Harkany, T., T. Hortobagyi, et al. (1999). "Neuroprotective approaches in experimental models of beta-amyloid neurotoxicity: relevance to Alzheimer's disease." Prog Neuropsychopharmacol Biol Psychiatry 23(6): 963-1008. 1. beta-Amyloid peptides (A beta s) accumulate abundantly in the Alzheimer's disease (AD) brain in areas subserving information acquisition and processing, and memory formation. A beta fragments are produced in a process of abnormal proteolytic cleavage of their precursor, the amyloid precursor protein (APP). While conflicting data exist in the literature on the roles of A beta s in the brain, and particularly in AD, recent studies have provided firm experimental evidence for the direct neurotoxic properties of A beta. 2. Sequence analysis of A beta s revealed a high degree of evolutionary conservation and inter-species homology of the A beta amino acid sequence. In contrast, synthetic A beta fragments, even if modified fluorescent or isotope-labeled derivatives, are pharmacological candidates for in vitro and in vivo modeling of their cellular actions. During the past decade, acute injection, prolonged mini-osmotic brain perfusion approaches or A beta infusions into the blood circulation were developed in order to investigate the effects of synthetic A beta s, whereas transgenic models provided insight into the distinct molecular steps of pathological APP cleavage. 3. The hippocampus, caudate putamen, amygdala and neocortex all formed primary targets of acute neurotoxicity screening, but functional consequences of A beta infusions were primarily demonstrated following either intracerebroventricular or basal forebrain (medial septum or magnocellular basal nucleus (MBN)) infusions of A beta fragments. 4. In vivo investigations confirmed that, while the active core of A beta is located within the beta(25-35) sequence, the flanking peptide regions influence not only the folding properties of the A beta fragments, but also their in vivo neurotoxic potentials. 5. It has recently been established that A beta administration deranges neuron-glia signaling, affects the glial glutamate uptake and thereby induces noxious glutamatergic stimulation of nerve cells. In fact, a critical role for N-methyl-D-aspartate (NMDA) receptors was postulated in the neurotoxic processes. Additionally, A beta s might become internalized, either after their selective binding to cell-surface receptors or after membrane association in consequence of their highly lipophilic nature, and induce free radical generation and subsequent oxidative injury. Ca(2+)-mediated neurotoxic events and generation of oxygen free radicals may indeed potentiate each other, or even converge to the same neurotoxic events, leading to cell death. 6. Neuroprotection against A beta toxicity was achieved by both pre- and post-treatment with NMDA receptor channel antagonists. Moreover, direct radical-scavengers, such as vitamin E or vitamin C, attenuated A beta toxicity with high efficacy. Interestingly, combined drug treatments did not necessarily result in additive enhanced neuroprotection. 7. Similarly to the blockade of NMDA receptors, the neurotoxic action of A beta s could be markedly decreased by pharmacological manipulation of voltage-dependent Ca(2+)-channels, serotonergic IA or adenosine A1 receptors, and by drugs eliciting membrane hyperpolarization or indirect blockade of Ca(2+)-mediated intracellular consequences of intracerebral A beta infusions. 8. A beta neurotoxicity might be dose-dependently modulated by trace metals. In spite of the fact that zinc (Zn) may act as a potent inhibitor of the NMDA receptor channel, high Zn doses accelerate A beta fibril formation, stabilize the beta-sheet conformation and thereby potentiate A beta neurotoxicity. Combined trace element supplementation with Se, Mn, or Mg, which prevails over the expression of detoxifying enzymes or counteracts intracellular elevations of Ca2+, may reduce the neurotoxic impact of A beta s. 9. Alterations in the regulatory functions of the hypothalamo-pituitary-adrenal axis may contribute significantly to neurodegenerative changes in the brain. Furthermore, AD patients exhibit substantially increased circadia Ekinci, F. J., K. U. Malik, et al. (1999). "Activation of the L voltage-sensitive calcium channel by mitogen-activated protein (MAP) kinase following exposure of neuronal cells to beta-amyloid. MAP kinase mediates beta-amyloid-induced neurodegeneration." J Biol Chem 274(42): 30322-7. Neuronal degeneration in Alzheimer's disease (AD) has been variously attributed to increases in cytosolic calcium, reactive oxygen species, and phosphorylated forms of the microtubule-associated protein tau. beta-Amyloid (betaA), which accumulates extracellularly in AD brain, induces calcium influx in culture via the L voltage-sensitive calcium channel. Since this channel is normally activated by protein kinase A-mediated phosphorylation, we examined kinase activities recruited following betaA treatment of cortical neurons and SH-SY-5Y neuroblastoma. betaA increased channel phosphorylation; this increase was unaffected by the protein kinase A inhibitor H89 but was reduced by the mitogen-activated protein (MAP) kinase inhibitor PD98059. Pharmacological and antisense oligonucleotide-mediated reduction of MAP kinase activity also reduced betaA-induced accumulation of calcium, reactive oxygen species, phospho-tau immunoreactivity, and apoptosis. These findings indicate that MAP kinase mediates multiple aspects of betaA-induced neurotoxicity and indicates that calcium influx initiates neurodegeneration in AD. betaA increased MAP kinase-mediated phosphorylation of membrane-associated proteins and reduced phosphorylation of cytosolic proteins without increasing overall MAP kinase activity. Increasing MAP kinase activity with epidermal growth factor did not increase channel phosphorylation. These findings indicate that redirection, rather than increased activation, of MAP kinase activity mediates betaA-induced neurotoxicity. Drouet, B., M. Pincon-Raymond, et al. (1999). "Laminin 1 attenuates beta-amyloid peptide Abeta(1-40) neurotoxicity of cultured fetal rat cortical neurons." J Neurochem 73(2): 742-9. A growing amount of evidence indicates the involvement of extracellular matrix components, especially laminins, in the development of Alzheimer's disease, although their role remains unclear. In this study, we clearly demonstrate that laminin 1 inhibits beta-amyloid peptide (Abeta)-induced neuronal cell death by preventing the fibril formation and interaction of the Abeta peptide with cell membranes. The presence of laminin at a laminin/Abeta peptide molar ratio of 1:800 significantly inhibits the Abeta-induced apoptotic events, together with inhibition of amyloid fibril formation. The inhibitory effects of laminin 1 were time- and dose-dependent, whereas laminin 2 had less effect on Abeta neurotoxicity. A preincubation of laminin and Abeta was not required to observe the protective effect of laminin, suggesting a direct interaction between laminin 1 and Abeta. Moreover, laminin had no effect on the toxicity of the fibrillar Abeta peptide, suggesting an interaction of laminin with nonfibrillar species of the Abeta peptide, sequestering the peptide in a soluble form. These data extend our understanding of laminin-dependent binding of Abeta and highlight the possible modulation role of laminin regarding Abeta aggregation and neurotoxicity in vivo. Butterfield, D. A., S. M. Yatin, et al. (1999). "Amyloid beta-peptide-associated free radical oxidative stress, neurotoxicity, and Alzheimer's disease." Methods Enzymol 309: 746-68. Given the increasing evidence of oxidative stress in AD brain and studies from different perspectives that appear to show a converging, central role for A beta in the pathogenesis and etiology of AD, insight into A beta-associated free radical oxidative stress will likely lead to a greater understanding of AD and, potentially, to better therapeutic strategies in this disorder. This article outlined methods to investigate markers of oxidative stress induced by A beta in brain membrane systems. Especially important are markers for protein oxidation, lipid peroxidation, and ROS generation by A beta. Oxidative stress and its sequelae are likely related to both necrotic and apoptotic mechanisms of neurotoxicity, and A beta-associated free radical oxidative stress may be of fundamental importance in Alzheimer's disease etiology and pathogenesis. The methods described here provide some means for investigating this possibility. Butterfield, D. A., T. Koppal, et al. (1999). "Vitamin E as an antioxidant/free radical scavenger against amyloid beta-peptide-induced oxidative stress in neocortical synaptosomal membranes and hippocampal neurons in culture: insights into Alzheimer's disease." Rev Neurosci 10(2): 141-9. Amyloid beta-peptide (Abeta), the major constituent in senile plaques in Alzheimer's disease (AD) brain, is thought by many researchers to be central to neurotoxicity in AD brain. Increasing evidence from many laboratories indicates that AD brain is under oxidative stress, with strong evidence of protein oxidation, lipid peroxidation, and peroxynitrite damage. A link between the central role of Abeta and oxidative stress in AD brain may be Abeta-associated free radical oxidative stress. If so, antioxidants such as vitamin E should modulate Abeta-induced oxidative damage and neurotoxicity in brain cells. This review summarizes studies of Abeta-associated free radical oxidative stress and its inhibition by vitamin E in cortical synaptosomal membranes and hippocampal neuronal cells in culture. Taken together with the recent report that vitamin E slows the progression of AD, this review strongly supports a central role of Abeta-associated free radical oxidative stress in neurotoxicity in AD brain. Bianca, V. D., S. Dusi, et al. (1999). "beta-amyloid activates the O-2 forming NADPH oxidase in microglia, monocytes, and neutrophils. A possible inflammatory mechanism of neuronal damage in Alzheimer's disease." J Biol Chem 274(22): 15493-9. The deposition of beta-amyloid in the brain is the key pathogenetic event in Alzheimer's disease. Among the various mechanisms proposed to explain the neurotoxicity of beta-amyloid deposits, a new one, recently identified in our and other laboratories, suggests that beta-amyloid is indirectly neurotoxic by activating microglia to produce toxic inflammatory mediators such as cytokines, nitric oxide, and oxygen free radicals. Three findings presented here support this mechanism, showing that beta-amyloid peptides (25-35), (1-39), and (1-42) activated the classical NADPH oxidase in rat primary culture of microglial cells and human phagocytes: 1) The exposure of the cells to beta-amyloid peptides stimulates the production of reactive oxygen intermediates; 2) the stimulation is associated with the assembly of the cytosolic components of NADPH oxidase on the plasma membrane, the process that corresponds to the activation of the enzyme; 3) neutrophils and monocytes of chronic granulomatous disease patients do not respond to beta-amyloid peptides with the stimulation of reactive oxygen intermediate production. Data are also presented that the activation of NADPH oxidase requires that beta-amyloid peptides be in fibrillary state, is inhibited by inhibitors of tyrosine kinases or phosphatidylinositol 3-kinase and by dibutyryl cyclic AMP, and is potentiated by interferon-gamma or tumor necrosis factor-alpha. Bergamaschini, L., S. Canziani, et al. (1999). "Alzheimer's beta-amyloid peptides can activate the early components of complement classical pathway in a C1q-independent manner." Clin Exp Immunol 115(3): 526-33. beta-Amyloid (beta-A) accumulates in the brain of patients with Alzheimer's disease (AD) and is presumably involved in the pathogenesis of this disease, on account of its neurotoxicity and complement-activating ability. Although assembly of beta-A in particular aggregates seems to be crucial, soluble non-fibrillar beta-A may also be involved. Non-fibrillar beta-A does not bind C1q, so we investigated alternative mechanisms of beta-A-dependent complement activation in vitro. On incubation with normal human plasma, non-fibrillar beta-A 1-42, and truncated peptide 1-28, induced dose-dependent activation of C1s and C4, sparing C3, as assessed by densitometric analysis of immunostained membrane after SDS-PAGE and Western blotting. The mechanism of C4 activation was not dependent on C1q, because non-fibrillar beta-A can still activate C1s and C4 in plasma genetically deficient in C1q (C1qd). In Factor XII-deficient plasma (F.XIId) the amount of cleaved C4 was about 5-10% less that in C1qd and in normal EDTA plasma; the reconstitution of F.XIId plasma with physiologic concentrations of F.XII resulted in an increased (8-15%) beta-A-dependent cleavage of C4. Thus our results indicate that the C1q-independent activation of C1 and C4 can be partially mediated by the activation products of contact system. Since the activation of contact system and of C4 leads to generation of several humoral inflammatory peptides, non-fibrillar beta-A might play a role in initiating the early inflammatory reactions leading to a multistep cascade contributing to neuronal and clinical dysfunction of AD brain. Subramaniam, R., T. Koppal, et al. (1998). "The free radical antioxidant vitamin E protects cortical synaptosomal membranes from amyloid beta-peptide(25-35) toxicity but not from hydroxynonenal toxicity: relevance to the free radical hypothesis of Alzheimer's disease." Neurochem Res 23(11): 1403-10. Amyloid beta-peptide (Abeta) is a key factor in the neurotoxicity of Alzheimer's disease (AD). Recent research has shown that Abeta-mediated neurotoxicity involves free radicals and that Abeta peptides can initiate multiple membrane alterations, including protein oxidation and lipid peroxidation, eventually leading to neuronal cell death. Research also has emphasized the role of 4-hydroxynonenal (HNE), a downstream product of lipid peroxidation, in being able to mimic some of the effects of Abeta peptides. In the current investigation, electron paramagnetic resonance (EPR) studies of spin labeled cortical synaptosomal membrane proteins has been employed to study conformational changes in proteins, spectrophotometric methods have been used to measure protein carbonyl content, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for mitochondrial function has been used to study the effect of vitamin E on samples that were treated with Abeta or HNE. The free radical dependence of beta-amyloid-associated toxicity was confirmed by the ability of the free radical scavenger vitamin E to prevent the toxic effects of Abeta. In contrast, HNE was still toxic in the presence of vitamin E. These results support our Abeta-associated free radical model for neurotoxicity in AD brain and are discussed with reference to potential therapeutic strategies for AD. Schulz, J. G., D. Megow, et al. (1998). "Evidence that glypican is a receptor mediating beta-amyloid neurotoxicity in PC12 cells." Eur J Neurosci 10(6): 2085-93. Docking of beta-amyloid fibrils to neuronal or glial cell membranes may be an early, necessary and intervenable step during the progression of Alzheimer's disease. Formation of neurofibrillary tangles and amyloid plaques as well as neurotoxicity and inflammation may be direct or indirect consequences. In an attempt to find a receptor that mediates those effects, we assessed rat pheochromocytoma PC12 cell 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction after addition of beta-amyloid to the culture medium. Presence of competitive substances in the medium, cell-surface treatment and specific block of cellular synthesis pathways helped to identify the heparan sulphate moiety of a glycosylphosphatidylinositol-anchored protein likely to represent glypican as a possible receptor mediating beta-amyloid neurotoxicity. Nishimoto, I. (1998). "A new paradigm for neurotoxicity by FAD mutants of betaAPP: a signaling abnormality." Neurobiol Aging 19(1 Suppl): S33-8. We have demonstrated that normal betaAPP695 behave as a signaling receptor and indicated that point mutations at V642 create autoactive betaAPP in signal transduction. Cellular expression of those familial Alzheimer's disease-associated mutants causes neuronal cells to undergo apoptotic death; and procedures inhibiting the signal of normal betaAPP block the mutant-induced apoptosis. We have also shown that the mutant-induced death is mediated by intracellular G protein activity but not by secretion of Abeta peptides. Accordingly, the mutant-induced death requires a cytoplasmic domain but not the 41st and 42nd residues of the Abeta region. These studies provide a novel insight that betaAPP may play a normal role as a death receptor and that Alzheimer's disease-relevant abnormality occurred in this function may lead neurons to suicidal degeneration. Muller, W. E., G. P. Eckert, et al. (1998). "Effects of beta-amyloid peptides on the fluidity of membranes from frontal and parietal lobes of human brain. High potencies of A beta 1-42 and A beta 1-43." Amyloid 5(1): 10-5. beta-amyloid peptide (A beta) and several A beta-fragments decrease the fluidity of human cortex membranes in a concentration dependent fashion. The effect of A beta on membrane fluidity increases with peptide length, is most pronounced for A beta 1-43 and can be seen at concentrations as low as 100 nmol/l. While the fragment A beta 25-35 is active, scrambled peptide (A beta 35-25) when investigated under similar conditions shows no effects on membrane fluidity. The effect of A beta peptides on fluidity of the phospholipid bilayer is more pronounced in the hydrocarbon core (labeled with the fluorescence probe 1,6-diphenylhexa-1,3,5-triene) than in the region of the hydrophilic heads (labeled with the fluorescence probe 1-[4'-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene). It is suggested that the effect of A beta on neuronal membranes is probably a major initial mechanism in a cascade of events finally leading to neurotoxicity and cell death in Alzheimer's disease. McLaurin, J., T. Franklin, et al. (1998). "Phosphatidylinositol and inositol involvement in Alzheimer amyloid-beta fibril growth and arrest." J Mol Biol 278(1): 183-94. A key pathological feature of Alzheimer's disease is the formation and accumulation of amyloid fibres. The major component is the 39 to 42 residue amyloid-beta peptide (Abeta) which is an internal proteolytic fragment of the integral membrane amyloid precursor protein. Aggregation of Abeta into insoluble amyloid fibres is a nucleation-dependent event that may be modulated by the presence of amyloid-associated molecules. Fibril formation is also associated with neurotoxicity which may be the result of specific Abeta interactions with membrane proteins and/or lipids. Using circular dichroism spectroscopy, tyrosine fluorescence spectroscopy and electron microscopy, we have examined the binding of Abeta peptides 1-40 (Abeta40) and 1-42 (Abeta42) to the glycolipid, phosphatidylinositol (PI), and different inositol headgroups. At pH 6.0 and in the presence of PI vesicles, both Abeta40 and Abeta42 adopted an amyloidogenic beta-structure. In contrast, at neutral pH only Abeta42 folded into a beta-structure in the presence of PI vesicles. To determine whether the induction of beta-structure stemmed from interactions with the headgroup of PI, the effects of inositol derivatives on Abeta were also examined. At pH 7.0, myo-inositol was sufficient to induce beta-structure in Abeta42 but had no effect on the conformation of Abeta40. Myo-inositol may promote beta-structure as a result of its ability to be both a hydrogen-bond donor and acceptor. Mono-, di- and triphosphorylated forms of inositol had reduced ability to induce beta-structure in both peptides. The results from this study indicate that interaction of Abeta40 and Abeta42 with PI acts as a seed for fibril formation while myo-inositol stabilizes a soluble Abeta42 micelle. Lambert, M. P., A. K. Barlow, et al. (1998). "Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins." Proc Natl Acad Sci U S A 95(11): 6448-53. Abeta1-42 is a self-associating peptide whose neurotoxic derivatives are thought to play a role in Alzheimer's pathogenesis. Neurotoxicity of amyloid beta protein (Abeta) has been attributed to its fibrillar forms, but experiments presented here characterize neurotoxins that assemble when fibril formation is inhibited. These neurotoxins comprise small diffusible Abeta oligomers (referred to as ADDLs, for Abeta-derived diffusible ligands), which were found to kill mature neurons in organotypic central nervous system cultures at nanomolar concentrations. At cell surfaces, ADDLs bound to trypsin-sensitive sites and surface-derived tryptic peptides blocked binding and afforded neuroprotection. Germ-line knockout of Fyn, a protein tyrosine kinase linked to apoptosis and elevated in Alzheimer's disease, also was neuroprotective. Remarkably, neurological dysfunction evoked by ADDLs occurred well in advance of cellular degeneration. Without lag, and despite retention of evoked action potentials, ADDLs inhibited hippocampal long-term potentiation, indicating an immediate impact on signal transduction. We hypothesize that impaired synaptic plasticity and associated memory dysfunction during early stage Alzheimer's disease and severe cellular degeneration and dementia during end stage could be caused by the biphasic impact of Abeta-derived diffusible ligands acting upon particular neural signal transduction pathways. Kanfer, J. N., G. Sorrentino, et al. (1998). "Phospholipases as mediators of amyloid beta peptide neurotoxicity: an early event contributing to neurodegeneration characteristic of Alzheimer's disease." Neurosci Lett 257(2): 93-6. There is a consensus that by some still to be defined mechanism amyloid beta peptide, which accumulates in Alzheimer's disease brain tissue, contributes to the characteristic neurodegeneration. We suggest that one of these mechanisms for amyloid beta peptide is the ability to activate cellular phospholipases. Excessive phospholipid hydrolysis would produce a variety of lipidic second messengers. These catabolites would then evoke unnecessary stereotypic responses. This indiscriminate activation of the phospholipases could be responsible for the increased amounts of phospholipid catabolites found in Alzheimer's disease brain tissue. Failure to maintain regeneration of the membrane components would result in a loss of essential cellular neuronal processes. Hirakura, Y., Y. Satoh, et al. (1998). "Membrane perturbation by the neurotoxic Alzheimer amyloid fragment beta 25-35 requires aggregation and beta-sheet formation." Biochem Mol Biol Int 46(4): 787-94. The beta-amyloid peptide (beta AP) is a major proteinaceous component of senile plaques and cerebrovascular amyloid deposits found in the brain of patients with Alzheimer's disease. beta AP is reported to be neurotoxic only when it forms beta-sheet structure and aggregates. In the present study, we report that the neurotoxic core of beta AP, beta AP-25-35 (beta 25-35), perturbs liposome membranes, induces membrane current, and exhibits hemolytic activity only in a buffer condition where the peptide forms beta-sheet structure and spontaneously aggregates. In contrast, beta 25-35 in its monomeric random coil structure does not perturb lipid membranes significantly, and exhibits no hemolytic activity. Also, the membrane current was inhibited by Congo Red. The ability of beta 25-35 to interact with membranes highly correlates with its neurotoxicity reported previously. These results suggest that membrane perturbation by aggregated beta 25-35 constitutes the molecular basis of the peptide's neurotoxicity. Giulian, D., L. J. Haverkamp, et al. (1998). "The HHQK domain of beta-amyloid provides a structural basis for the immunopathology of Alzheimer's disease." J Biol Chem 273(45): 29719-26. The beta-amyloid peptide 1-42 (Abeta1-42), a major component of neuritic and core plaques found in Alzheimer's disease, activates microglia to kill neurons. Selective modifications of amino acids near the N terminus of Abeta showed that residues 13-16, the HHQK domain, bind to microglial cells. This same cluster of basic amino acids is also known as a domain with high binding affinity for heparan sulfate. Both Abeta binding to microglia and Abeta induction of microglial killing of neurons were sensitive to heparitinase cleavage and to competition with heparan sulfate, suggesting membrane-associated heparan sulfate mediated plaque-microglia interactions through the HHQK domain. Importantly, small peptides containing HHQK inhibited Abeta1-42 cell binding as well as plaque induction of neurotoxicity in human microglia. In vivo experiments confirmed that the HHQK peptide reduces rat brain inflammation elicited after infusion of Abeta peptides or implantation of native plaque fragments. Strategies which exploit HHQK-like agents may offer a specific therapy to block plaque-induced microgliosis and, in this way, slow the neuronal loss and dementia of Alzheimer's disease. Fu, W., H. Luo, et al. (1998). "Catecholamines potentiate amyloid beta-peptide neurotoxicity: involvement of oxidative stress, mitochondrial dysfunction, and perturbed calcium homeostasis." Neurobiol Dis 5(4): 229-43. Oxidative stress and mitochondrial dysfunction are implicated in the neuronal cell death that occurs in physiological settings and in neurodegenerative disorders. In Alzheimer's disease (AD) degenerating neurons are associated with deposits of amyloid beta-peptide (A beta), and there is evidence for increased membrane lipid peroxidation and protein oxidation in the degenerating neurons. Cell culture studies have shown that A beta can disrupt calcium homeostasis and induce apoptosis in neurons by a mechanism involving oxidative stress. We now report that catecholamines (norepinephrine, epinephrine, and dopamine) increase the vulnerability of cultured hippocampal neurons to A beta toxicity. The catecholamines were effective in potentiating A beta toxicity at concentrations of 10-200 microM, with the higher concentrations (100-200 microM) themselves inducing cell death. Serotonin and acetylcholine were not neurotoxic and did not modify A beta toxicity. Levels of membrane lipid peroxidation, and cytoplasmic and mitochondrial reactive oxygen species, were increased following exposure to neurons to A beta, and catecholamines exacerbated the oxidative stress. Subtoxic concentrations of catecholamines exacerbated decreases in mitochondrial energy charge and transmembrane potential caused by A beta, and higher concentrations of catecholamines alone induced mitochondrial dysfunction. Antioxidants (vitamin E, glutathione, and propyl gallate) protected neurons against the damaging effects of A beta and catecholamines, whereas the beta-adrenergic receptor antagonist propanolol and the dopamine (D1) receptor antagonist SCH23390 were ineffective. Measurements of intracellular free Ca2+ ([Ca2+]i) showed that A beta induced a slow elevation of [Ca2+]i which was greatly enhanced in cultures cotreated with catecholamines. Collectively, these data indicate a role for catecholamines in exacerbating A beta-mediated neuronal degeneration in AD and, when taken together with previous findings, suggest roles for oxidative stress induced by catecholamines in several different neurodegenerative conditions. Butterfield, D. A., L. Martin, et al. (1996). "A beta (25-35) peptide displays H2O2-like reactivity towards aqueous Fe2+, nitroxide spin probes, and synaptosomal membrane proteins." Life Sci 58(3): 217-28. Amyloid beta peptides (A beta s) are found in abnormally high accumulations in brains of persons with Alzheimer's disease, and are believed to contribute to cognitive decline in this disorder. Synthetic A beta and its peptide fragment 25-35 [A beta (25-35)] are toxic to cells in culture; however, the exact mechanism of amyloid peptide toxicity is not known. An emerging hypothesis contends that A beta toxicity results from peptide-mediated free radical reactions and generation of reactive oxygen species. Recently, we reported that reactivity of A beta toward the oxidation-sensitive enzyme glutamine synthetase is related to the peptide's reactivity toward the spin trap phenyl-tert-butyl nitrone (PBN). Neuronal damage may be due, in part, to oxidative processes initiated by amyloid-derived free radicals species. This work presents evidence from electron paramagnetic resonance (EPR) spin labeling techniques and spectrophotometric assays that a portion of synthetic A beta (25-35) demonstrates hydrogen peroxide-like reactivity toward Fe2+, nitroxide spin probes, and neocortical synaptasomal membrane proteins. These results are discussed with reference to free radical membrane damage and neurotoxicity in Alzheimer's disease.