PrPSc deposition is detectable, suggesting that the protein could trigger the inflammatory and apoptotic cascades [6]. PrP fragments, corresponding to the protease-resistant portion of PrPSc, are widely used to study PrP pathogenic refolding and neurotoxicity. They are characterized by a flexible backbone that can undergo to a conformational rearrangement proposed as a model of PrPC-PrPSc conversion [7–10]. Although a definitive general agreement has not been obtained yet, it was proposed that these PrPSc-like fragments can transmit the disease after cerebral inoculation in rodents [11–14], supporting the ‘‘protein only’’ hypothesis for the pathogenesis of TSE [5]. We developed a model to study the neurotoxicity of PrPSc using a recombinant peptide encompassing residues 90–231 of human PrP (hPrP90–231), This peptide, in its native form, is a soluble monomer, mainly structured as a-helix, thus corresponding to a model of PrPC [15]. hPrP90–231 can be converted by mild thermal denaturation (1 hour at 53uC) in a b-sheet-rich conformation that renders the peptide insoluble, highly hydro- phobic and partially resistant to proteinase K [10,16]. All these biochemical features that resembles a PrPSc-like structure, allow hPrP90–231 to acquire biological activities in vitro, inducing microglial activation, astrocyte proliferation and apoptotic neuro- nal death [7,17–20]. Thus, also considering the recent demon- stration that different prion conformations comprise the infectious and neurotoxic entities [21], hPrP90–231 may represent a valuable model to study prion neurotoxicity and the potential development of novel therapeutic approaches [16,22]. Recently, we reported that Syrian hamster PrP27–30 was able to induce a ionic current through a lipid bilayer and that this effect was not mimicked by hPrP90–231 in its native a-helix-rich, monomeric conformation [23]. However, when incubated in high [Ca++] (10 mM), hPrP90–231 induced an effect super imposable to that of PrP27–30 [23]. However, neither the current/voltage relationship nor the relative increases of conductance in high external calcium concentration suggests that the divalent ion directly contributes to membrane permeability [23]. Thus, we hypothesized that the binding of Ca++ to the peptide may induce a conformational change in hPrP90–231 allowing a structural conformation similar to the Syrian hamster prion. Numerous studies have implicated alterations in Ca++ homeostasis in the neuronal dysfunction in several neurodegenerative diseases, including Alzheimer disease (AD) [24,25] and TSE [26,27]. Importantly, in AD, alterations of Ca++ signalling were bidirec- tionally related to the activation of the amyloidogenic pathway [24,28]. As far as TSE is concerned, most studies analyzed the effects of PrPC and PrPSc as regulator (or deregulator) of neuronal Ca++ homeostasis (for review see [27]), but a possible role of altered [Ca++] in PrPSc-mediated neurotoxicity is still underscored. Age-related deficits in neuronal [Ca++] regulatory systems are well characterized, involving higher intracellular [Ca++], enhanced Ca++ influx through voltage-sensitive calcium channels and reduced capacity of mitochondria to buffer Ca++ excess [29]. Recently, it was proposed that Ca++ deregulation occurring during the aging process may facilitate the formation of pathogenic Ab peptides, which in turn further alter neuronal Ca++ homeostasis, generating a vicious circle [30] that could occur also during TSE. The principal aim of this study has been to highlight both the structural features in PrPsc oligomers that push them in the toxic amyloidogenic cascade and the relationship between the acquire- ment of the toxic conformation with changes in the local environment. For this purpose we used the recombinant hPrP90–231 fragment, taking advantage on its conformational dependent toxicity, as detailed above [7]. We evaluated the role of high [Ca++], as determinant of the generation of PrP toxic Calcium Dependent Prion Protein Toxic Conformation molecules, analyzing the structural features of hPrP90–231 conformer generated in these experimental conditions that are able to induce the same biological effects of Syrian hamster purified PrPSc. We demonstrate that Ca++ binding to hPrP90–231 induces a conformational change in the peptide resembling that previously characterized as the neurotoxic conformer [7,10]. Importantly, in this PrPSc-like conformation, hPrP90–231 caused cytotoxic and membrane destabilizing effects identical to those induced by Syrian hamster PrP27–30, fully validating the proposed model. Finally, we provide a molecular model of the structural alterations induced by in the PrP molecule after Ca++ binding, in comparison with those induced by the disease-related pathogenic mutation E200K. Results Effect of Calcium on Ionic Currents Elicited by PrP Forms The Tip Dip technique allows single-channel recordings from artificial lipid bilayer by drastically reducing the surface area available for proteins to insert and, consequently, the background electrical noise. Figure 1 highlight the different ionic permeability induced in artificial membrane of PrP27–30 (left) compared to the recombinant hPrP90–231 (right) in agreement with previous data [23]. We also confirmed that hPrP90–231 conductance increases in elevated calcium ion concentration (10 mM) without apparently increasing the divalent ion flow through the membrane [23]. To separate the effect due to the transmembrane Ca++ ionic flow from any other possible interaction of divalent ions and PrP peptide or the influence of high [Ca++] on the lipid bilayer, we here recorded single channel events induced by the recombinant peptide in Tip Dip experiments using two procedures. In the first one, a classical dose/response curve was obtained changing [Ca++] in the recording pipette. The second procedure consisted in one hour pre-incubation of the PrP peptide in the different Ca++ solutions. After this period hPrP90–231 was resuspended (dilution 1:500) in the control trans solution (1.8 mM CaCl2) just before performing the electrophysiology measurements. The results in Figure 2 show that the effect of the different [Ca++] on membrane conductance is similar either using the divalent ion directly in contact with the bilayer or after one hour incubation. The current/voltage relationships (i/Vs) on the top of Figure 2 depict in both cases two distinct groups of data: current values obtained with the peptide exposed to 1.8 and 5 mM Ca++ and a second set of data concerning 10, 20 and 50 mM [Ca++]. The histograms in the bottom of Figure 2 highlight the difference between calcium concentration growth (vertical bars) and the increase of single channel conductance (chart boxes). At high [Ca++] (10, 20 and 50 mM) conductance values remain constant for both the experimental procedures. Clearly, this cannot be ascribed to a saturation of Ca++ permeability since current data obtained using pre-incubated hPrP90–231 (white bars) were recorded in physi- ological [Ca++]. Effect of Calcium on in Vitro Cell Toxicity of PrP Forms Data showed in Figure 2 suggest that membrane conductance increasing following the Ca++ treatment is mediated by changes in hPrP90–231 structural features rather than in the lipid bilayer permeability properties. We previously characterized the confor- mational changes of hPrP29–231 causing gain of toxicity: hPrP90– 231 can acquire toxic properties by mild thermal denaturation (53uC, 1 hour) [7,22], allowing hPrP90–231 to be highly internalized into SH-SY5Y in insoluble aggregates that caused lysosomal dysfunction and cell apoptosis [18]. In Figure 3 panel A the toxic properties acquired by hPrP90–231 after the thermal PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e38314