The transition of prion protein from a mainly a-structured isoform (PrPC) to a~sheet-containing protein (PrPSC) represents a major pathogenetic mechanism in prion diseases. To study the role of PrP structural conformation in prion-dependent neurodegeneration, we analysed the neurotoxicity of PrP in a and~conformations, using a recombinant protein encompassing amino acids 90-231 of the human PrP (hPrP90-231). Using controlled thermal denaturation (53°C, Ih) we converted hPrP90-231 in a structural isoform displaying Prpsc-related characteristics: high~sheet content, increased aggregability and a slight increase in the resistance to protease K. In virtue of these structural changes, hPrP90-231 powerfully affected the survival of SH-SY5Y cells, inducing a caspase-3 and p38-dependent apoptosis. Conversely, in the native a-helix-rich conformation, hPrP90-231 did not show significant cell toxicity. The relationship between the structural state of hPrP90-231 and its neurotoxicity was demonstrated, inducing the thermal denaturation of the peptide in the presence of Congo red that prevented both the transition of hPrP90-231 into a~-rich isoform and the acquisition of toxic properties. In conclusion, we report that the toxicity of hPrP90-231 is dependent on its three-dimensional structure, as is supposed to occur for the pathogen PrP during TSE.Transmissible spongiform encephalopathies (TSE) represent a group of fatal neurodegenerative disorders that affect both animals and humans, occurring through infective, genetic or sporadic mechanisms. TSE share a common histopathology with a pathognomonic triad: spongiform vacuolation of the gray matter, neuronal death and glial proliferation, with amyloid deposition less constantly observed. In contrast to other transmissible diseases in which the infectious particles are viruses or bacteria, the only agent currently associated with TSE is a structural isoform of the cellular prion protein (PrP C ) known as the scrapie conformation (PrPSC) (1).In Prpsc, large portions of the N-terminus of the protein adopt a~-sheet conformation, this feature being the main difference between the cellular and the pathogenic protein (2). Thus, the conversion of

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Because of high tendency of the prion protein (PrP) to aggregate, the exact PrP isoform responsible for prion diseases as well as the pathological mechanism that it activates remains still controversial. In this study, we show that a prefibrillar, monomeric or small oligomeric conformation of the human PrP fragment 90-231 (hPrP90-231), rather than soluble or fibrillar large aggregates, represents the neurotoxic species. In particular, we demonstrate that monomeric milddenatured hPrP90-231 (incubated for 1 h at 53°C) induces SH-SY5Y neuroblastoma cell death, while, when structured in large aggregates, it is ineffective. Using spectroscopic and cellular techniques we demonstrate that this toxic conformer is characterized by a high exposure of hydrophobic regions that favors the intracellular accumulation of the protein. Inside the cells hPrP90-231 is mainly compartmentalized into the lysosomes where it may trigger pro-apoptotic 'cell death' signals. The PrP toxic conformation, which we have obtained inducing a controlled in vitro conformational change of the protein, might mimic mild-unfolding events occurring in vivo, in the presence of specific mutations, oxidative reactions or proteolysis. Thus, in light of this model, we propose that novel therapeutic strategies, designed to inhibit the interaction of the toxic PrP with the plasmamembrane, could be beneficial to prevent the formation of intracellular neurotoxic aggregates and ultimately the neuronal death. Keywords: intracellular internalization, neurotoxicity, protein aggregation, PrP90-231. Transmissible spongiform encephalopathies (TSE), or prion diseases, are a group of neurodegenerative diseases that recognize as etiopathologic agent an aberrant isoform of the cellular prion protein (PrP C ), named PrP Sc (Prusiner 1998). PrP C is a ubiquitous membrane-bound protein, highly concentrated in the central nervous system in both neurons and glial cells, whose physiological function is still largely unknown. In PrP Sc large portions of the protein N-Terminus adopt a b-sheet configuration, being this feature the main difference between PrP c and PrP Sc (Prusiner 1991). In the past few years a large number of data has been published to better characterize both the molecular mechanisms involved in the conversion of PrP C into PrP Sc (Aguzzi 2006) and the intracellular signaling by which PrP Sc (Muller et al. 1993;Hetz et al. 2003), or some cytotoxic fragments derived from its sequence, may induce cell death (Forloni et al. 1993;Brown et al. 1996;Florio et al. 1998; Thellung et al. 2000a,b). To explain prion infectivity it was postulated that the pathogenic PrP Sc self-propagates in an autocatalytic manner using PrP C as a substrate (Prusiner 1982).A novel concept emerging in prion diseases research is represented by the dissociation between prion transmissibility/ These authors equally contributed and should be both considered as first author.2 Joint last and corresponding authors.Abbreviations used: CD, circular dichroism; hPrP90-231, human PrP fragment 9...

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