Self-assembly of beta-amyloid 42 is retarded by small molecular ligands at the stage of structural intermediates.

Assemblyof the amyloid-beta peptide (Abeta) into fibrils and its deposition in distinct brain areas is considered responsible for the pathogenesis of Alzheimer's disease (AD). Thus, inhibition of fibril assembly is a potential strategy for therapeutic intervention. Electron cryomicroscopy was used to monitor the initial, native assembly structure of Abeta42. In addition to the known fibrillar intermediates, a nonfibrillar, polymeric sheet-like structure was identified. A temporary sequence of supramolecular structures was revealed with (i) polymeric Abeta42 sheets during the onset of assembly, inversely related to the appearance of (ii) fibril intermediates, which again are time-dependently replaced by (iii) mature fibrils. A cell-based primary screening assay was used to identify compounds that decrease Abeta42-induced toxicity. Hit compounds were further assayed for binding to Abeta42, radical scavenger activity, and their influence on the assembly structure of Abeta42. One compound, Ro 90-7501, was found to efficiently retard mature fibril formation, while extended polymeric Abeta42 sheets and fibrillar intermediates are accumulated. Ro 90-7501 may serve as a prototypic inhibitor for Abeta42 fibril formation and as a tool for studying the molecular mechanism of fibril assembly.

[1]  E. Kellenberger,et al.  The wrapping phenomenon in air-dried and negatively stained preparations. , 1982, Ultramicroscopy.

[2]  Elena Orlova,et al.  Cryo‐electron microscopy structure of an SH3 amyloid fibril and model of the molecular packing , 1999, The EMBO journal.

[3]  P. Lansbury,et al.  The C‐Terminus of the β Protein is Critical in Amyloidogenesis a , 1993 .

[4]  Dominic M. Walsh,et al.  Protofibrillar Intermediates of Amyloid β-Protein Induce Acute Electrophysiological Changes and Progressive Neurotoxicity in Cortical Neurons , 1999, The Journal of Neuroscience.

[5]  D. Kirschner,et al.  Structural analysis of Alzheimer's beta(1-40) amyloid: protofilament assembly of tubular fibrils. , 1998, Biophysical journal.

[6]  H. Naiki,et al.  First-order kinetic model of Alzheimer's beta-amyloid fibril extension in vitro. , 1996, Laboratory investigation; a journal of technical methods and pathology.

[7]  S. Younkin,et al.  An increased percentage of long amyloid beta protein secreted by familial amyloid beta protein precursor (beta APP717) mutants. , 1994, Science.

[8]  Norman R. Farnsworth,et al.  Cancer Chemopreventive Activity of Resveratrol, a Natural Product Derived from Grapes , 1997, Science.

[9]  Peter T. Lansbury,et al.  Assembly of Aβ Amyloid Protofibrils: An in Vitro Model for a Possible Early Event in Alzheimer's Disease† , 1999 .

[10]  J. Rommens,et al.  Familial Alzheimer's disease in kindreds with missense mutations in a gene on chromosome 1 related to the Alzheimer's disease type 3 gene , 1995, Nature.

[11]  J. Dubochet,et al.  Cryo-electron microscopy of vitrified specimens , 1988, Quarterly Reviews of Biophysics.

[12]  U Aebi,et al.  Architecture and polymorphism of fibrillar supramolecular assemblies produced by in vitro aggregation of human calcitonin. , 1995, Journal of structural biology.

[13]  Synthetic peptide homologous to beta protein from Alzheimer disease forms amyloid-like fibrils in vitro. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[14]  D. Pollen,et al.  Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease , 1995, Nature.

[15]  A. Roher,et al.  Molecular modeling of the Abeta1-42 peptide from Alzheimer's disease. , 1998, Protein engineering.

[16]  C. Cotman,et al.  Assembly and aggregation properties of synthetic Alzheimer's A4/beta amyloid peptide analogs. , 1992, The Journal of biological chemistry.

[17]  P. Lansbury,et al.  Atomic force microscopic imaging of seeded fibril formation and fibril branching by the Alzheimer's disease amyloid-beta protein. , 1997, Chemistry & biology.

[18]  Carl W. Cotman,et al.  Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state , 1993, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[19]  M. Emmerling,et al.  Morphology and Toxicity of Aβ-(1-42) Dimer Derived from Neuritic and Vascular Amyloid Deposits of Alzheimer's Disease* , 1996, The Journal of Biological Chemistry.

[20]  R. Nicoll,et al.  Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[21]  J. Trojanowski,et al.  Full-length amyloid-beta (1-42(43)) and amino-terminally modified and truncated amyloid-beta 42(43) deposit in diffuse plaques. , 1996, The American journal of pathology.

[22]  C. Behl,et al.  Hydrogen peroxide mediates amyloid β protein toxicity , 1994, Cell.

[23]  T. Morgan,et al.  Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Carl W. Cotman,et al.  In vitro aging of ß-amyloid protein causes peptide aggregation and neurotoxicity , 1991, Brain Research.

[25]  J. Hofrichter,et al.  Kinetics of sickle hemoglobin polymerization. II. A double nucleation mechanism. , 1985, Journal of molecular biology.

[26]  B. Yankner,et al.  Beta-amyloid neurotoxicity requires fibril formation and is inhibited by congo red. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[27]  R. Wetzel,et al.  Physical, morphological and functional differences between ph 5.8 and 7.4 aggregates of the Alzheimer's amyloid peptide Abeta. , 1996, Journal of molecular biology.

[28]  L. Thal,et al.  Secretion of β-amyloid precursor protein cleaved at the amino terminus of the β-amyloid peptide , 1993, Nature.

[29]  M. Shearman,et al.  The Intracellular Component of Cellular 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) Reduction Is Specifically Inhibited by β‐Amyloid Peptides , 1995 .

[30]  P E Fraser,et al.  Examination of the structure of the transthyretin amyloid fibril by image reconstruction from electron micrographs. , 1995, Journal of molecular biology.

[31]  B. Seilheimer,et al.  A Biotechnological Method Provides Access to Aggregation Competent Monomeric Alzheimer's 1–42 Residue Amyloid Peptide , 1995, Bio/Technology.

[32]  B. Seilheimer,et al.  The toxicity of the Alzheimer's beta-amyloid peptide correlates with a distinct fiber morphology. , 1997, Journal of structural biology.

[33]  R. Karlsson,et al.  Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system. , 1991, Journal of immunological methods.

[34]  J. Hardy,et al.  Early-onset Alzheimer's disease caused by mutations at codon 717 of the β-amyloid precursor protein gene , 1991, Nature.

[35]  D. Kirschner,et al.  Neurotrophic and neurotoxic effects of amyloid beta protein: reversal by tachykinin neuropeptides. , 1990, Science.

[36]  S. Younkin,et al.  Amyloid β Protein (Aβ) in Alzheimeri's Disease Brain , 1995, The Journal of Biological Chemistry.

[37]  P. Lansbury.  Structural Neurology: Are Seeds at the Root of Neuronal Degeneration? , 1997, Neuron.

[38]  P. Fraser,et al.  Fibrillogenesis of Alzheimer Abeta peptides studied by fluorescence energy transfer. , 1997, Journal of molecular biology.

[39]  J. Dubochet,et al.  Cryo-negative staining. , 1998, Micron.

[40]  P. Cutler,et al.  Hemin and related porphyrins inhibit β‐amyloid aggregation , 1997 .

[41]  Veerle Baekelandt,et al.  Early Phenotypic Changes in Transgenic Mice That Overexpress Different Mutants of Amyloid Precursor Protein in Brain* , 1999, The Journal of Biological Chemistry.

[42]  S. Wagner,et al.  Amyloid production and deposition in mutant amyloid precursor protein and presenilin-1 yeast artificial chromosome transgenic mice , 1999, Nature Neuroscience.

[43]  Peter T. Lansbury,et al.  Observation of metastable Aβ amyloid protofibrils by atomic force microscopy , 1997 .

[44]  D. Kirschner,et al.  In vitro amyloid fibril formation by synthetic peptides corresponding to the amino terminus of apoSAA isoforms from amyloid-susceptible and amyloid-resistant mice. , 1998, Journal of structural biology.

[45]  R. Wickner,et al.  Prion domain initiation of amyloid formation in vitro from native Ure2p. , 1999, Science.

[46]  S. Younkin,et al.  Correlative Memory Deficits, Aβ Elevation, and Amyloid Plaques in Transgenic Mice , 1996, Science.

[47]  J. Hofrichter,et al.  Kinetics of sickle hemoglobin polymerization. I. Studies using temperature-jump and laser photolysis techniques. , 1985, Journal of molecular biology.

[48]  D. Selkoe,et al.  Amyloid beta-protein fibrillogenesis. Structure and biological activity of protofibrillar intermediates. , 1999, The Journal of biological chemistry.

[49]  J. Hardy,et al.  A locus for familial early–onset Alzhelmer's disease on the long arm of chromosome 14, proximal to the α1–antichymotrypsin gene , 1992, Nature Genetics.

[50]  Nybo,et al.  An Ultrastructural Study of Amyloid Intermediates in Aβ1–42 Fibrillogenesis , 1999, Scandinavian journal of immunology.

[51]  R. Wetzel,et al.  Aggregation state and neurotoxic properties of Alzheimer beta-amyloid peptide. , 1995, Neurodegeneration : a journal for neurodegenerative disorders, neuroprotection, and neuroregeneration.

[52]  S. Hirai,et al.  Electron micrograph of diffuse plaques. Initial stage of senile plaque formation in the Alzheimer brain. , 1989, The American journal of pathology.

[53]  P. Lansbury,et al.  Seeding “one-dimensional crystallization” of amyloid: A pathogenic mechanism in Alzheimer's disease and scrapie? , 1993, Cell.

[54]  D. Kirschner,et al.  On the nucleation and growth of amyloid beta-protein fibrils: detection of nuclei and quantitation of rate constants. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[55]  J. Swatton,et al.  Inhibition of fibril formation in beta-amyloid peptide by a novel series of benzofurans. , 1999, The Biochemical journal.

[56]  R. Henderson,et al.  Three-dimensional structure determination by electron microscopy of two-dimensional crystals. , 1982, Progress in biophysics and molecular biology.

[57]  P. Lansbury,et al.  Amyloid fibril formation requires a chemically discriminating nucleation event: studies of an amyloidogenic sequence from the bacterial protein OsmB. , 1992, Biochemistry.

[58]  P E Fraser,et al.  Structure of beta-crystallite assemblies formed by Alzheimer beta-amyloid protein analogues: analysis by x-ray diffraction. , 1993, Biophysical journal.

[59]  D. Selkoe,et al.  Translating cell biology into therapeutic advances in Alzheimer's disease , 1999, Nature.

[60]  Louise C. Serpell,et al.  Synchrotron X-ray studies suggest that the core of the transthyretin amyloid fibril is a continuous β-sheet helix , 1996 .

[61]  J. Kemp,et al.  β‐Amyloid‐Induced Cell Toxicity: Enhancement of 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide‐Dependent Cell Death , 1996, Journal of neurochemistry.

[62]  P. Fraser,et al.  pH-dependent structural transitions of Alzheimer amyloid peptides. , 1991, Biophysical journal.

[63]  P. Schuck.  Simultaneous radial and wavelength analysis with the Optima XL-A analytical ultracentrifuge , 1994 .

[64]  P. Greengard,et al.  Relative abundance of Alzheimer A beta amyloid peptide variants in Alzheimer disease and normal aging. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[65]  L. Tjernberg,et al.  Endogenous proteins controlling amyloid beta-peptide polymerization. Possible implications for beta-amyloid formation in the central nervous system and in peripheral tissues. , 1999, The Journal of biological chemistry.

[66]  D. Walsh,et al.  Amyloid beta-protein fibrillogenesis. Detection of a protofibrillar intermediate. , 1997, The Journal of biological chemistry.

[67]  J. Kemp,et al.  Controlling Polymerization of β-Amyloid and Prion-derived Peptides with Synthetic Small Molecule Ligands* , 2000, The Journal of Biological Chemistry.

[68]  U. Aebi,et al.  Watching amyloid fibrils grow by time-lapse atomic force microscopy. , 1999, Journal of molecular biology.