Amyloid beta-protein fibrillogenesis - Structure and biological activity of protofibrillar intermediates
JOURNAL OF BIOLOGICAL CHEMISTRY 274: (36) 25945-25952 SEP 3 1999
Walsh DM, Hartley DM, Kusumoto Y, Fezoui Y, Condron MM, Lomakin A, Benedek GB, Selkoe DJ, Teplow DB

Alzheimer's disease is characterized by extensive cerebral amyloid deposition. Amyloid deposits associated with damaged neuropil and blood vessels contain abundant fibrils formed by the amyloid beta-protein (A beta). Fibrils, both in vitro and in vivo, are neurotoxic. For this reason, substantial effort has been expended to develop therapeutic approaches to control A beta production and amyloidogenesis. Achievement of the latter goal is facilitated by a rigorous mechanistic understanding of the fibrillogenesis process. Recently, we discovered a novel intermediate in the pathway of A beta fibril formation, the amyloid protofibril (Walsh, D, RI,, Lomakin, A., Benedek, G. B., Condron, RI. Ri,, and Teplow, D. B. (1997) J. Biol. Chem. 272-, 22364-22372). We report here results of studies of the assembly, structure, and biological activity of these polymers. We find that protofibrils: 1) are in equilibrium with low molecular weight A beta (monomeric or dimeric); 2) have a secondary structure characteristic of amyloid fibrils; 3) appear as beaded chains in rotary shadowed preparations examined electron microscopically; 4) give rise to mature amyloid-like fibrils; and 5) affect the normal metabolism of cultured neurons. The implications of these results for the development of therapies for Alzheimer's disease and for our understanding of fibril assembly are discussed.

Chelation and intercalation: Complementary properties in a compound for the treatment of Alzheimer's disease
Cherny RA, Barnham KJ, Lynch T, Volitakis I, Li QX, McLean CA, Multhaup G, Beyreuher K, Tanzi RE, Masters CL, Bush AI

Selective application of metal chelators to homogenates of human Alzheimer's disease (AD) brain has led us to propose that the architecture of aggregated beta-amyloid peptide, whether in the form of plaques or soluble oligomers, is determined at least in part by high-affinity binding of transition metals, especially copper and zinc. Of the two metals, copper is implicated in reactive oxygen species generating reactions, while zinc appears to be associated with conformational and antioxidant activity. We tested the copper chelators trientine, penicillamine, and bathophenanthroline for their ability to mobilize brain A beta as measured against our benchmark compound bathocuproine (BC). All of these agents were effective in solubilizing brain A beta, although BC was the most consistent across the range of AD brain tissue samples tested. Similarly, all of the copper chelators depleted copper in the high-speed supernatants. BC alone had no significant effect upon zinc levels in the soluble fraction. BC extraction of brain tissue from C100 transgenic mice (which express human A beta but do not develop amyloid) revealed SDS-resistant dimers as A beta was mobilized from the sediment;able to the soluble fraction. NMR analysis showed that, in addition to its copper chelating properties, BC interacts with A beta to form a complex independent of the presence of copper. Such hybrid copper chelating and "chain breaking" properties may form the basis of a rational design for a therapy for Alzheimer's disease.

Characterization of copper interactions with Alzheimer amyloid beta peptides: Identification of an attomolar-affinity copper binding site on amyloid beta 1-42
JOURNAL OF NEUROCHEMISTRY 75: (3) 1219-1233 SEP 2000
Atwood CS, Scarpa RC, Huang XD, Moir RD, Jones WD, Fairlie DP, Tanzi RE, Bush AI

Cu and Zn have been shown to accumulate in the brains of Alzheimer's disease patients. We have previously reported that Cu2+ and Zn2+ bind amyloid beta (A beta), explaining their enrichment in plaque pathology. Here we detail the stoichiometries and binding affinities of multiple cooperative Cu2+-binding sites on synthetic A beta 1-40 and A beta 1-42. We have developed a ligand displacement technique (competitive metal capture analysis) that uses metal-chelator complexes to evaluate metal ion binding to A beta, a notoriously self-aggregating peptide. This analysis indicated that there is a very-high-affinity Cu2+-binding site on A beta 1-42 (log K-app = 17.2) that mediates peptide precipitation and that the tendency of this peptide to self-aggregate in aqueous solutions is due to the presence of trace Cu2+ contamination (customarily similar to 0.1 mu M). In contrast, A beta 1-40 has much lower affinity for Cu2+ at this site (estimated log K-app = 10.3), explaining why this peptide is less self-aggregating. The greater Cu2+-binding affinity of A beta 1-42 compared with A beta 1-40 is associated with significantly diminished negative cooperativity, The role of trace metal contamination in inducing A beta precipitation was confirmed by the demonstration that A beta peptide (10 micro-M) remained soluble for 5 days only in the presence of high-affinity Cu2+-selective chelators.

Oxidative stress in Alzheimer's disease
Smith MA, Rottkamp CA, Nunomura A, Raina AK, Perry G

Oxidative balance is emerging as an important issue in understanding the pathogenesis of Alzheimer's disease. Examination of Alzheimer's disease brain has demonstrated a great deal of oxidative damage, associated with both hallmark pathologies (senile plaques and neurofibrillary tangles) as well as in normal appearing pyramidal neurons. While this suggests that oxidative stress is a proximal event in Alzheimer's disease pathogenesis, the mechanisms by which redox balance is altered in the disease remains elusive. Determining which of the proposed sources of free radicals, which include mitochondrial dysfunction, amyloid-beta-mediated processes, transition metal accumulation and genetic factors like apolipoprotein E and presenilins, is responsible for redox imbalance will. lead to a better understanding of Alzheimer's disease pathogenesis and novel therapeutic approaches.

Evidence that the beta-amyloid plaques of Alzheimer's disease represent the redox-silencing and entombment of A beta by zinc
JOURNAL OF BIOLOGICAL CHEMISTRY 275: (26) 19439-19442 JUN 30 2000
Cuajungco MP, Goldstein LE, Nunomura A, Smith MA, Lim JT, Atwood CS, Huang XD, Farrag YW, Perry G, Bush AI

A beta binds Zn2+, Cu2+ and Fe3+ in vitro, and these metals are markedly elevated in the neocortex and especially enriched in amyloid plaque deposits of individuals with Alzheimer's disease (AD). Zn2+ precipitates A beta in vitro, and Cu2+ interaction with A beta promotes its neurotoxicity, correlating with metal reduction and the cell-free generation of H2O2 (A beta 1-42 > A beta 1-40 > ratA beta 1-40). Because Zn2+ is redox-inert, we studied the possibility that it may play an inhibitory role in H2O2 –mediated A beta toxicity. In competition to the cytotoxic potentiation caused by coincubation with Cu2+, Zn2+ rescued primary cortical and human embryonic kidney 293 cells that were exposed to A beta 1-42, correlating with the effect of Zn2+ in suppressing Cu2+-dependent H2O2 formation from A beta 1-42. Since plaques contain exceptionally high concentrations of Zn2+, we examined the relationship between oxidation (8-OH guanosine) levels in AD-affected tissue and histological amyloid burden and found a significant negative correlation. These data suggest a protective role for Zn2+ in AD, where plaques form as the result of a more robust Zn2+ antioxidant response to the underlying oxidative attack.

Metal binding modes of Alzheimer's amyloid beta-peptide in insoluble aggregates and soluble complexes
BIOCHEMISTRY 39: (23) 7024-7031 JUN 13 2000
Miura T, Suzuki K, Kohata N, Takeuchi H

Aggregation of the amyloid beta-peptide (A beta) into insoluble fibrils is a key pathological event in Alzheimer's disease. Zn(II) induces the A beta aggregation at acidic-to-neutral pH, while Cu(TI) is an effective inducer only at mildly acidic pH. We have examined Zn(II) and Cu(II) binding modes of A beta and their pH dependence by Raman spectroscopy. The Raman spectra dearly demonstrate that three histidine residues in the N-terminal hydrophilic region provide primary metal binding sites and the solubility of the metal-A beta complex is correlated with the metal binding mode. Zn(II) binds to the N-tau atom of the histidine imidazole ring and the peptide aggregates through intermolecular His(N-tau)-Zn(II)-His(N-tau) bridges. The N-tau-metal ligation also occurs in Cu(II)-induced A beta aggregation at mildly acidic pH. At neutral pH, however, Cu(II) binds to N-pi, the other nitrogen of the histidine imidazole ring, and to deprotonated amide nitrogens of the peptide main chain. The chelation of Cu(II) by histidine and main-chain amide groups results in soluble Cu(II)-A beta complexes. Under normal physiological conditions, Cu(II) is expected to protect A beta against Zn(II)-induced aggregation by competing with Zn(II) for histidine residues of A beta.


Copper catalyzed oxidation of alzheimer A beta
Atwood CS, Huang XD, Khatri A, Scarpa RC, Kim YS, Moir RD, Tanzi RE, Roher AE, Bush AI

A beta derived from amyloid plaques of Alzheimer's disease-affected brain contain several oxidative posttranslational modifications. In this study we have characterized the amino acid content of human amyloid-derived A beta and compared it with that of human synthetic A beta subjected to metal-catalyzed oxidation. Human amyloid derived A beta has an increased content of arginine (46%) and glutamate/glutamine residues (28%), but a decreased content of histidine residues (-32%) as compared to the expected amino acid content. Incubation of synthetic human A beta with Cu(II), but not Fe(III), in the presence of H2O2 similarly induced a decrease in histidine residues (-79%), but also a decrease in tyrosine residues (-28%). Our results suggest that histidine and tyrosine are most vulnerable to metal mediated oxidative attack, consistent with our earlier findings that Cu coordinated via histidine residues is redox competent. Our results suggest that the loss of histidine residues in human amyloid-derived A beta may be a result of Cu oxidation, and that unidentified post-translational mechanisms operate to modify other amino acids of A beta in vivo.