The pathological prion protein forms ionic conductance in lipid bilayer

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative pathologies characterized by the accumulation of amyloid fibrils mainly composed of the pathological isoform of the prion protein (PrP(TSE)). PrP(TSE) pre-amyloid fibrils are supposed to induce neurodegenerative lesions possibly through the alteration of membrane permeability. The effect of PrP(TSE) on cellular membranes has been modeled in vitro by synthetic peptides that are, however, only partially representative of PrP(TSE) isoforms found in vivo. In the present work we show that a synthetic membrane exposed to PrP27-30 extracted from TSE-infected hamster brains changes its permeability because of the formation of molecular pores that alter the conductance of the synthetic lipid bilayer. Synthetic membrane challenged with the recombinant prion peptide PrP90-231 shows a much lower conductance. Elevation of calcium ion concentration not only increases the current amplitude due to the action of both PrP27-30 and PrP90-231 on the membrane, but also amplifies the interaction of PrP90-231 with the lipid bilayer. (C) 2011 Elsevier B.V. All rights reserved

Mutant PrPCJD prevails over wild-type PrPCJD in the brain of V210I and R208H genetic Creutzfeldt-Jakob disease patients.

Creutzfeldt–Jakob disease (CJD) is a neurodegenerative disorder characterized by the deposition of the pathological conformer (PrPCJD) of the host encoded cellular prion protein (PrPC). In genetic CJD associated with V210I or R208H PrP substitutions, the pathogenic role of mutant residues is still poorly understood. To understand how V210I or R208H PrP mutations facilitate the development of the disease, we determined by mass spectrometry the quantitative ratio of mutant/wild-type PrPCJD allotypes in brains from affected subjects. We found that the mutant PrPCJD allotypes moderately exceeds of 2- or 3-fold the amount of the wild-type counterpart suggesting that these mutations mainly exert their pathogenic effect on the onset of the pathogenic cascade. Different mechanisms can be hypothesized to explain the pathogenic role of mutant residues: V210I and R208H substitutions can increase the concentration of PrPC and the probability to form insoluble aggregates, or they may facilitate the formation of pathological intermediates, or, alternatively, they may increase the affinity for ligands that are involved in the initial phases of PrPCJD formation and aggregation. Whatever the mechanism, the enrichment found for the mutated PrPCJD species indicates that these altered structures are more prone, with respect to the non-mutated ones, to be captured in the polymer- ization process either at the onset or during the development of the disease

Influenza viruses circulation in a tertiary care children hospital in Rome: a comparison between 2022 and the previous 5 years

Abstract Background Influenza surveillance aims to determine onset, duration and intensity of the seasonal Influence-like Illness (ILI); data collection begins in the week 42 of a year and ends in the week 17 of the following year. In this observational study, we report the experience of a tertiary care children hospital in Rome about Influenza viruses circulation during the calendar year 2022 (January-December) in comparison with the previous five years (2017–2021), with a special focus on the weeks 18–41, usually not under surveillance. Methods This retrospective study involved 36782 respiratory samples referred to 21354 patients (pts), median age 2.63 years, admitted with respiratory symptoms at Bambino Gesù Children’s Hospital in the years 2017–2022. Respiratory viruses were detected by molecular Allplex™ Respiratory Panel Assays (Seegene, Korea). Results Regarding the pre pandemic years, 2017–2019, distribution of Flu positive patients focused in the first weeks of the year (weeks 1–17). During the pandemic period, Flu was not detected. In 2022, 239 Flu viruses were identified: 37 FluA (weeks 1–17), 29 FluA (weeks 18–41) and 168 FluA and 5 FluB (weeks 42–52). For the year 2022, during the non-epidemic period, the number of Flu viruses detected corresponded to 12.1% of total Flu detected, respect to 0-1.7% for the previous five years (p < 0.001). Conclusions When compared with pre SARS-CoV-2 pandemic years, our data show a significant increase in Influenza cases during weeks 18–41/2022 and reveal an unexpected summer circulation of these viruses: just weeks 26–30 showed to be influenza virus free. A national year-round Flu surveillance could be useful to understand if changing in influenza epidemiology is transitional or likely to persist in the following years

Mutant PrPCJD prevails over wild-type PrPCJD in the brain of V210I and R208H genetic Creutzfeldt-Jakob disease patients

Creutzfeldt\u2013Jakob disease (CJD) is a neurodegenerative disorder characterized by the deposition of the pathological conformer (PrPCJD) of the host encoded cellular prion protein (PrPC). In genetic CJD associated with V210I or R208H PrP substitutions, the pathogenic role of mutant residues is still poorly understood. To understand how V210I or R208H PrP mutations facilitate the development of the disease, we determined by mass spectrometry the quantitative ratio of mutant/wild-type PrPCJD allotypes in brains from affected subjects. We found that the mutant PrPCJD allotypes moderately exceeds of 2- or 3-fold the amount of the wild-type counterpart suggesting that these mutations mainly exert their pathogenic effect on the onset of the pathogenic cascade. Different mechanisms can be hypothesized to explain the pathogenic role of mutant residues: V210I and R208H substitutions can increase the concentration of PrPC and the probability to form insoluble aggregates, or they may facilitate the formation of pathological intermediates, or, alternatively, they may increase the affinity for ligands that are involved in the initial phases of PrPCJD formation and aggregation. Whatever the mechanism, the enrichment found for the mutated PrPCJD species indicates that these altered structures are more prone, with respect to the non-mutated ones, to be captured in the polymerization process either at the onset or during the development of the disease

Calcium Binding Promotes Prion Protein Fragment 90–231 Conformational Change toward a Membrane Destabilizing and Cytotoxic Structure

The pathological form of prion protein (PrP(Sc)), as other amyloidogenic proteins, causes a marked increase of membrane permeability. PrP(Sc) extracted from infected Syrian hamster brains induces a considerable change in membrane ionic conductance, although the contribution of this interaction to the molecular mechanism of neurodegeneration process is still controversial. We previously showed that the human PrP fragment 90–231 (hPrP(90–231)) increases ionic conductance across artificial lipid bilayer, in a calcium-dependent manner, producing an alteration similar to that observed for PrP(Sc). In the present study we demonstrate that hPrP(90–231), pre-incubated with 10 mM Ca(++) and then re-suspended in physiological external solution increases not only membrane conductance but neurotoxicity as well. Furthermore we show the existence of a direct link between these two effects as demonstrated by a highly statistically significant correlation in several experimental conditions. A similar correlation between increased membrane conductance and cell degeneration has been observed assaying hPrP(90–231) bearing pathogenic mutations (D202N and E200K). We also report that Ca(++) binding to hPrP(90–231) induces a conformational change based on an alteration of secondary structure characterized by loss of alpha-helix content causing hydrophobic amino acid exposure and proteinase K resistance. These features, either acquired after controlled thermal denaturation or induced by D202N and E200K mutations were previously identified as responsible for hPrP(90–231) cytotoxicity. Finally, by in silico structural analysis, we propose that Ca(++) binding to hPrP(90–231) modifies amino acid orientation, in the same way induced by E200K mutation, thus suggesting a pathway for the structural alterations responsible of PrP neurotoxicity

Structure of the proposed Ca⁺⁺ binding site in the wild type PrP90–231 protein.

<p>On the left the site with the residues still involved in an H-bond pattern in model I (light cyan); on the right the resulting complex after Arg156 shift and Ca⁺⁺ binding in model III (orange). Hydrogen bonds (dotted red) and coordinative bonds (green) are explicitly indicated.</p

Increased proteinase K (PK) resistance of hPrP90–231 <i>w.t.</i> in the presence of increasing Ca⁺⁺ concentrations.

<p>hPrP90–231 w.t. was thermally denatured or preincubated with increasing concentration of Ca⁺⁺ before being subjected to PK digestion with different enzyme concentrations. Partial resistance was evaluated in Western blot experiments using the anti-PrP antibody 3F4. Left panel shows representative immunoblots, while the graph on the right depicts the densitometry analysis of three independent experiments. Data are expressed as percentage of the respective recombinant peptide input. * = p<0.05 and ** = p<0.01 <i>vs</i>. control values. In native conditions or in the presence of low Ca⁺⁺ concentrations, the peptide showed no resistance to PK digestion. After incubation with 5 or 10 mM Ca⁺⁺ a partial resistance was observed for a peptide/PK ratio of 500∶1. Thermal denaturation induced a higher resistance as compared to Ca⁺⁺ binding, being the immunolabeled peptide band detectable also for a peptide/PK ratio of 200∶1.</p

Thioflavin T binding to hPrP90–231 w.t. HPrP90–231 in native, conformation, after mild thermal denaturation (53°C, for 1 hr), after incubation for 1 hr in the presence of 10 mM Ca⁺⁺, or after a combination of both treatments was tested for Th T binding in a fluorimetric assay, as index of β-sheet content.

<p>As previously reported, thermal denaturation increased the Th T binding as compared to the native peptide, but this effect was slightly higher after incubation with Ca⁺⁺. No additivity between thermal denaturation and incubation in the presence of 10 mM Ca⁺⁺ was observed.</p

Calcium concentration modulates the ionic conductance induced by hPrP90–231 recombinant peptide.

<p>A: current/voltage relationship obtained from Tip Dip experiment using the peptide in 5 different calcium concentrations. B: the current/voltage relationships were obtained from experiments performed in 1.8 mM calcium concentration but the peptide was prior incubated for 1 hour in 4 different calcium concentrations. C: the histogram shows the conductance values versus calcium concentrations either present in the external solution during the experiment or only used during hPrP90–231 incubation. The conductance in the two different conditions show similar values and reaches its maximum at 10 mM calcium. Black and white bars visualize the calcium increase (n = 5).</p