10 mM) and it is unlikely to find such a high concentration of the divalent ions or such a high temperature (53uC in the thermal denaturation model) in a living organism. However, two consid- erations should be taken in account. As shown in Figure 5, the conversion of the peptide in the toxic form can take place even in ‘‘physiological’’ conditions, such as prolonged incubation at 1.8 mM Ca++ and 37uC. The experiments reported in Fig. 5 contain indications that the toxic conformation of hPrP90–231 is mainly due to small oligomers. Higher toxicity level and membrane conductance occur early, after 5, 15 and 24 hours and declines later, presumably due to the formation of bigger aggregates. Thus, it is realistic to think that high Ca++ level and/or high temperature work as catalyst to speed up in vitro a spontaneous reaction. In a healthy system the rate of spontaneous toxic PrP species formation is compatible with the ability to efficiently eliminate the misfolded proteins. It is also possible that the slow production of reactive peptides would favor the direct aggregation of monomeric PrP in amorphous big aggregates without the formation of harmful intermediated oligomeric form [64]. However, it should be also considered a different scenario. If the rate of PrP misfolded proteins formation drastically increases, all the system devoted to annihilation of aberrant proteins would be plugged and this will favored a harmful unbalanced situation. Misfolded proteins will increase in concentration allowing the formation of reactive oligomers before the peptides would reach the amorphous state of big aggregates. Small oligomers in their short lifespan will be able to react with the biological membrane causing unregulated ionic flows, most of the time resulting in a depolarization of the cell membrane potential. Depolarized neuronal cells could be considered a first stage, even if reversible, of the disease progression. In addition to that, the formation of membrane discontinuity could also be the pathway allowing extracellularly accumulated PrP peptides to leak in the cell to form aggregates in the cytosol. Cytotoxicity could be the result of both these factors. Pathogenic PrP, by changing the membrane voltage, is able to increase intracellular Ca++ concentration through the activation of both ion channels and ionotropic receptors. Thus, it is likely that elevation of intracellular Ca++ may interfere with internalized and neosynthesized PrP molecules causing a structural alteration within the cell. In this model we have the starting of a vicious circle in which altered PrP molecules affect intracellular membrane permeability to Ca++ that reaching high intracellular levels contribute to the generation of new toxic species that altogether with the altered Ca++ homeostasis induce synapse dysfunction and neuronal death. In conclusion, we suggest that increased membrane permeabil- ity due to the structural rearrangement of PrP peptide is a spontaneous long lasting reaction. Increase of membrane conduc- tance could be linked to pathological conditions once there is an overproduction of PrP misfolded protein. In this case the system is not capable to eliminate the dangerous peptide that, both for environmental conditions and for its concentration, is allowed to form highly reactive oligomers able to strongly interact with the cell membrane. The possibility to form high conductance ionic pathways could be also the vehicle to allow PrP protein inside the cell. Here the peptide could aggregate and form deposits [18]. Alternatively misfolded PrP could form oligomers and cause damage to internal membrane such as mitochondria, lysosomes and nuclear envelope, determine a slowly but inexorable collapse of the cell organization. Materials and Methods Ethics Statement: N/A. Calcium Dependent Prion Protein Toxic Conformation Prion Samples The protease-resistant core (PrP27–30) of PrPTSE extracted from brains of TSE-affected hamsters PrP27–30 was kindly provided by the Pocchiari’s group [31]. Recombinant prion peptide PrP 90–231 (hPrP90–231) was synthesized in E. coli and purified as previously reported [15]. The same procedure was used to purify the E200K and D202N hPrP90–231 mutants [17]. GST was purified from the empty pGEX-4T-2 vector following the same procedure used to purify hPrP90–231 [7]. Cell Cultures and Treatments SH-SY5Y human neuroblastoma cells (ICLC-Biological Bank and Cell Factory, IRCCS IST Genova, code HTL95013) were cultured in MEM/F12 (Euroclone, Milano, Italy) supplemented with 15% fetal bovine serum (Gibco-Invitrogen, Milano, Italy), 2 mM glutamine (Euroclone, Milano, Italy), pen/strep 100 mg/ ml (Euroclone) and grown in 5% CO2 atmosphere at 37uC. Twenty-four hours after plating, unless otherwise stated, culture medium was replaced with fresh medium containing a reduced (2%) FBS content, to induce growth arrest and minimize spontaneous apoptosis that serum withdrawal causes in this cell model [32]. Cells were treated for 2 days with a single administration of hPrP90–231 directly to the culture medium to mimic the interaction of PrPSc deposits with the neurons as occurred in vivo [33]. Survival Assay Mitochondrial function, as index of cell viability, was evaluated by measuring the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, Milano, Italy). The cleavage of MTT to a purple formazan byproduct by mitochondrial dehydrogenases was spectrophotometrically quan- tified, as reported [34]. Briefly, cells were incubated for 1 h with 0.25 mg/mL MTT in serum-free Dulbecco’s modified Eagle’s medium at 37uC; medium, was removed and formazan crystals dissolved in dimethyl-sulfoxide (DMSO). Absorbance values were measured at wave length of 570 nm. Experiments were performed in quadruplicate and repeated at least three times. Data are reported as mean values 6 standard error (S.E.) Statistical analysis was performed by means of one-way ANOVA followed by Newman and Keuls test. A p value less than or equal to 0.05 was considered statistically significant. Electrophysiological Assay Single-channel recordings from lipid bilayer were obtained using the Tip-Dip method. Patch clamp pipettes (Garner Glass 7052) were made using a P97 Sutter Instruments puller (Novato, CA), coated with Sylgard (Dow Corning, Midland, MI) and fire- polished to a tip diameter of 1–1.5 mm and 5–7 MOhm resistance. 1,2-diphytanoyl-sn-glycero-3-phosphocholine (Diph-PC, Avanti Polar Lipids, Inc., Birmingham, AL) was used to arise the lipid monolayer. An Axopatch 200 B amplifier and pClamp 8 (Molecular Device, Novato, CA) were used to record and analyze, digitized at 5 kHz and filtered at 1000 Hz the detected current. Membrane voltages (Vm) and ion currents were expressed considering relative to the pipette solution. An inward current is defined as a cation moving from the electrode to the bath chamber. Data are presented as mean 6 SEM. Values obtained from different experiments were tested for statistical differences using independent two population t test or ANOVA routine (Origin software; Origin Lab, Northampton, MA). Different buffers were employed in the two compartments. The ion compositions of the trans solutions were defined in order to PLoS ONE | www.plosone.org 12 July 2012 | Volume 7 | Issue 7 | e38314