Amyloid Toxicity Is Independent of Polypeptide Sequence, Length and Chirality M. Teresa Pastor 1 ⁎, Nico Kümmerer 1 , Vanessa Schubert 2 Alexandra Esteras-Chopo 1 , Carlos G. Dotti 2 Manuela López de la Paz 1 and Luis Serrano 1 1 Structural and Computational Biology Unit, European Molecular Biology Laboratory , Meyerhofstrasse 1, D-69117 Heidelberg, Germany 2 Department of Clinical and Biological Science, Cavalieri Ottolenghi Scientific Institute, Universita Degli Studi di Torino, Azienda Ospedaliera San Luigi Gonzaga, Regione Gonzole 10, 10043 Orbassano (Torino), Italy Received 22 May 2007; received in revised form 7 August 2007; accepted 7 August 2007 Available online 21 August 2007 By using an amyloid sequence pattern, here we have identified putative six- residue amyloidogenic stretches in several relevant amyloid proteins. Hexapeptides synthesized on the bases of the sequence stretches matching the pattern have been shown to form amyloid fibrils in vitro. As larger pathological peptides such as Aβ 1–42 do, these short amyloid peptides form heterogeneous mixtures of small aggregates that induce cell death in PC12 cells and primary hippocampal neurons. Toxic mixtures of small aggregates from these hexapeptides bind to cell membranes and can be further internalized, as also observed for natural amyloid proteins. In neurons, toxic aggregates obtained from the full length Aβ 1–42 amyloid peptide or their amyloid stretch Aβ 16–21 peptide preferentially localize in synapses, leading to the re-organization of the underlying actin cytoskeleton. This process does not involve stereospecific interactions between membrane and toxic species as D-sequences are as toxic as L ones, suggesting that is not receptor mediated. Based on these results, we propose here that regardless of poly- peptide sequence, length and amino acid chirality, amyloid prefibrillar aggregates exert their cytotoxic effect through a common cell death mecha- nism related to a particular quaternary structure. The degree of toxicity of these species seems to depend, however, on cell membrane composition. © 2007 Elsevier Ltd. All rights reserved. Edited by J. Weissman Keywords: amyloid; amyloidoses; amyloid stretch; cytotoxicity; oligomers Introduction Aberrant aggregation of individual proteins into amyloid fibrils is related to numerous human diseases, including Alzheimer's and Parkinson's disease, type II diabetes and transmissible spongi- form encephalopathies among others. 1,2 Prior to amyloid fibril formation, amyloidogenic proteins partially unfold adopting a so-called amyloid prone state from which polymerization occurs. The pro- cess is favored by changes in local environment, protein concentration and by mutations that modify the stability or/and amyloid self-assembly propen- sity of the wild-type sequences. 3,4 Misfolded poly- peptides or proteins self-assemble through a com- mon cross-β structural motif resulting in highly stable fibrils that are deposited into plaques and that display common biophysical and tinctorial properties. 1,2 Amyloid formation is a complex process that takes place through several intermediates of various size and morphologies. 5,6 The role of the species formed in the amyloid pathway on the onset and the pro- gression of the disease is still a controversial issue. 7,8 Initially, fibrils were postulated as the species responsible for cell impairment and death. Cur- rently, there is accumulating evidence indicating that intermediate oligomers on the amyloid path- *Corresponding author. E-mail address: pastor@embl.de. Present addresses: C. G. Dotti, Department of Human Genetics, Catholic University of Leuven and Flanders Interuniversity Institute of Biotechnology (VIB4), Heerestraat 49, 3000 Leuven, Belgium; M. López de la Paz, Merz Pharmaceuticals GmbH, Altenhöferalle 3, D-60438 Frankfurt am Main, Germany; M. T. Pastor and L. Serrano, Department of Systems Byology, Centre for Genomic Regulation, Dr. Aiguader 88, E-08003 Barcelona, Spain. Abbreviations used: EM, electron microscopy; MTT, 3-[4,5-dimethylthiazole-zyl]-2,5-diphenyl tetrazolium bromide. doi:10.1016/j.jmb.2007.08.012 J. Mol. Biol. (2008) 375, 695–707 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.