title: Native metastable prefibrillar oligomers are the most neurotoxic species among amyloid aggregates.
abstract: Many proteins belonging to the amyloid family share the tendency to misfold and aggregate following common steps, and display similar neurotoxicity. In the aggregation pathway different kinds of species are formed, including several types of oligomers and eventually mature fibers. It is now suggested that the pathogenic aggregates are not the mature fibrils, but the intermediate, soluble oligomers. Many kinds of aggregates have been described to exist in a metastable state and in equilibrium with monomers. Up to now it is not clear whether a specific structure is at the basis of the neurotoxicity. Here we characterized, starting from the early aggregation stages, the oligomer populations formed by an amyloid protein, salmon calcitonin (sCT), chosen due to its very slow aggregation rate. To prepare different oligomer populations and characterize them by means of photoinduced cross-linking SDS-PAGE, Energy Filtered-Transmission Electron Microscopy (EF-TEM) and Circular Dichroism (CD) spectroscopy, we used Size Exclusion Chromatography (SEC), a technique that does not influence the aggregation process leaving the protein in the native state. Taking advantage of sCT low aggregation rate, we characterized the neurotoxic potential of the SEC-separated, non-crosslinked fractions in cultured primary hippocampal neurons, analyzing intracellular Ca(2+) influx and apoptotic trend. We provide evidence that native, globular, metastable, prefibrillar oligomers (dimers, trimers and tetramers) were the toxic species and that low concentrations of these aggregates in the population was sufficient to render the sample neurotoxic. Monomers and other kind of aggregates, such as annular or linear protofibers and mature fibers, were totally biologically inactive.
authors.affiliation: Department of Technology and Health, Istituto Superiore di Sanità, 00161 Roma, Italy. Electronic address: marco.diociaiuti@iss.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Diociaiuti
authors.affiliation: Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, 00161 Roma, Italy.
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Macchia
authors.affiliation: Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, 00161 Roma, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Paradisi
authors.affiliation: National Center for Rare Diseases, Istituto Superiore di Sanità, 00161 Roma, Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Frank
authors.affiliation: Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, 00161 Roma, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Camerini
authors.affiliation: Department of Technology and Health, Istituto Superiore di Sanità, 00161 Roma, Italy.
authors.forename: Pietro
authors.initials: P
authors.lastname: Chistolini
authors.affiliation: Department of Therapeutic Research and Medicine Evaluation, Istituto Superiore di Sanità, 00161 Roma, Italy.
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Gaudiano
authors.affiliation: Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, 00161 Roma, Italy.
authors.forename: Tamara Corinna
authors.initials: TC
authors.lastname: Petrucci
authors.affiliation: Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, 00161 Roma, Italy.
authors.forename: Fiorella
authors.initials: F
authors.lastname: Malchiodi-Albedi
mesh_terms: D000682:Amyloid / Q000737:chemistry* / Q000633:toxicity*; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000473:pathology*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004594:Electrophysiology; D006624:Hippocampus / Q000187:drug effects / Q000473:pathology*; D046529:Microscopy, Electron, Transmission; D010777:Photochemistry; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000682:Amyloid; D003432:Cross-Linking Reagents; D002118:Calcium
keywords: Amyloid oligomers; Neurodegenerative diseases; Neurotoxicity; Structural biophysics