Prion Strain Discrimination Based on Rapid In Vivo Amplification and Analysis by the Cell Panel Assay

Prion strain identification has been hitherto achieved using time-consuming incubation time determinations in one or more mouse lines and elaborate neuropathological assessment. In the present work, we make a detailed study of the properties of PrP-overproducing Tga20 mice. We show that in these mice the four prion strains examined are rapidly and faithfully amplified and can subsequently be discriminated by a cell-based procedure, the Cell Panel Assay

In Vitro and In Vivo Neurotoxicity of Prion Protein Oligomers

The mechanisms underlying prion-linked neurodegeneration remain to be elucidated, despite several recent advances in this field. Herein, we show that soluble, low molecular weight oligomers of the full-length prion protein (PrP), which possess characteristics of PrP to PrPsc conversion intermediates such as partial protease resistance, are neurotoxic in vitro on primary cultures of neurons and in vivo after subcortical stereotaxic injection. Monomeric PrP was not toxic. Insoluble, fibrillar forms of PrP exhibited no toxicity in vitro and were less toxic than their oligomeric counterparts in vivo. The toxicity was independent of PrP expression in the neurons both in vitro and in vivo for the PrP oligomers and in vivo for the PrP fibrils. Rescue experiments with antibodies showed that the exposure of the hydrophobic stretch of PrP at the oligomeric surface was necessary for toxicity. This study identifies toxic PrP species in vivo. It shows that PrP-induced neurodegeneration shares common mechanisms with other brain amyloidoses like Alzheimer disease and opens new avenues for neuroprotective intervention strategies of prion diseases targeting PrP oligomers

Atypical BSE (BASE) Transmitted from Asymptomatic Aging Cattle to a Primate

BACKGROUND: Human variant Creutzfeldt-Jakob Disease (vCJD) results from foodborne transmission of prions from slaughtered cattle with classical Bovine Spongiform Encephalopathy (cBSE). Atypical forms of BSE, which remain mostly asymptomatic in aging cattle, were recently identified at slaughterhouses throughout Europe and North America, raising a question about human susceptibility to these new prion strains. METHODOLOGY/PRINCIPAL FINDINGS: Brain homogenates from cattle with classical BSE and atypical (BASE) infections were inoculated intracerebrally into cynomolgus monkeys (Macacca fascicularis), a non-human primate model previously demonstrated to be susceptible to the original strain of cBSE. The resulting diseases were compared in terms of clinical signs, histology and biochemistry of the abnormal prion protein (PrPres). The single monkey infected with BASE had a shorter survival, and a different clinical evolution, histopathology, and prion protein (PrPres) pattern than was observed for either classical BSE or vCJD-inoculated animals. Also, the biochemical signature of PrPres in the BASE-inoculated animal was found to have a higher proteinase K sensitivity of the octa-repeat region. We found the same biochemical signature in three of four human patients with sporadic CJD and an MM type 2 PrP genotype who lived in the same country as the infected bovine. CONCLUSION/SIGNIFICANCE: Our results point to a possibly higher degree of pathogenicity of BASE than classical BSE in primates and also raise a question about a possible link to one uncommon subset of cases of apparently sporadic CJD. Thus, despite the waning epidemic of classical BSE, the occurrence of atypical strains should temper the urge to relax measures currently in place to protect public health from accidental contamination by BSE-contaminated products

Prion Protein Is a Key Determinant of Alcohol Sensitivity through the Modulation of N-Methyl-D-Aspartate Receptor (NMDAR) Activity

The prion protein (PrP) is absolutely required for the development of prion diseases; nevertheless, its physiological functions in the central nervous system remain elusive. Using a combination of behavioral, electrophysiological and biochemical approaches in transgenic mouse models, we provide strong evidence for a crucial role of PrP in alcohol sensitivity. Indeed, PrP knock out (PrP−/−) mice presented a greater sensitivity to the sedative effects of EtOH compared to wild-type (wt) control mice. Conversely, compared to wt mice, those over-expressing mouse, human or hamster PrP genes presented a relative insensitivity to ethanol-induced sedation. An acute tolerance (i.e. reversion) to ethanol inhibition of N-methyl-D-aspartate (NMDA) receptor-mediated excitatory post-synaptic potentials in hippocampal slices developed slower in PrP−/− mice than in wt mice. We show that PrP is required to induce acute tolerance to ethanol by activating a Src-protein tyrosine kinase-dependent intracellular signaling pathway. In an attempt to decipher the molecular mechanisms underlying PrP-dependent ethanol effect, we looked for changes in lipid raft features in hippocampus of ethanol-treated wt mice compared to PrP−/− mice. Ethanol induced rapid and transient changes of buoyancy of lipid raft-associated proteins in hippocampus of wt but not PrP−/− mice suggesting a possible mechanistic link for PrP-dependent signal transduction. Together, our results reveal a hitherto unknown physiological role of PrP on the regulation of NMDAR activity and highlight its crucial role in synaptic functions

Toxic Effects of Intracerebral PrP Antibody Administration During the Course of BSE Infection in Mice

The absence of specific immune response is a hallmark of prion diseases. However, in vitro and in vivo experiments have provided evidence that an anti-PrP humoral response could have beneficial effects. Prophylactic passive immunization performed at the time of infection delayed or prevented disease. Nonetheless, the potential therapeutic effect of PrP antibodies administered shortly before the clinical signs has never been tested in vivo. Moreover, a recent study showed the potential toxicity of PrP antibodies administered intracerebrally. We aimed at evaluating the effect of a prolonged intracerebral anti-PrP antibody administration at the time of neuroinvasion in BSE infected Tg20 mice

In Vivo Neurotoxicity of Ovine PrP Oligomers and Fibrils

<div><p>(A) Schematic representation of a coronal section of a mouse brain with arrows indicating the injection sites of the PrP preparations.</p><p>(B–I) Nissl-like staining (gallocyanine) of the hippocampal region CA₂-CA₃ from the brains of mice injected with the PrP preparations or control buffer. Top panels represent the low magnification image (scale bar = 100 μm) and bottom panels the high magnification image (scale bar = 20 μm) of the lesioned regions (and anatomically corresponding region for the buffer and nontoxic PrP monomers). (B–E) Wild-type C57BL/6 mice, (F–I) PrP0/0 C57BL/6 mice.</p><p>(J and K) Higher toxicity of PrP oligomers versus PrP fibrils (wild-type C57BL/6 mice shown). Apoptotic pyramidal neurons in the lesioned hippocampal region have been labeled with the ApopTag BrdU kit and appear in green (scale bar = 50 μm).</p></div

Neurotoxicity of Oligomeric PrP

<div><p>(A–C) E15 cortical neurons from PrP^+/+ (A) or PrP^0/0 (B) were exposed to various concentrations of murine PrP oligomers (red bars) for 72 h, and then cell viability was measured using MTT. Untreated neurons (black bars) and neurons treated with either equivalent volumes of vehicle solution (light blue bars) or a peptide mimicking the linker region of the mouse tandem PrP (dark blue bars) were chosen as controls (see [C] for molecular details of the peptide).</p><p>(D) Cell viability measurements of neurons treated with either ovine PrP oligomers (dark red bars) or ovine PrP monomers (light red bars). The black and light blue bars correspond to untreated and treated with vehicle solution controls, respectively.</p><p>(E) To compare the toxicity of the PrP preparations with a known toxic PrP peptide, PrP^+/+ neurons were incubated with the PrP peptide 105–132 (dark green bars) and its scrambled version (light green bars) at two concentrations reported to induce neuronal death [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030125#ppat-0030125-b018" target="_blank">18</a>]. The cell viability results are expressed as a percentage relative to the untreated control (black bar) + SEM. The results are representative of at least two independent experiments performed with triplicate samples. The significance of the results was evaluated using a two-tailed unpaired student <i>t</i>-test with Welch corrections when needed (significant = *, 0.01 < <i>p</i> ≤ 0.05; very significant = **, <i>p</i> ≤ 0.01).</p><p>(F) Hoechst 33342 nuclear staining of embryonic cortical neurons incubated with toxic recombinant proteins. Left: No protein control, showing normal fully rounded cell nuclei. Middle: incubated with mouse PrP oligomers. Right: incubated with ovine PrP oligomers. Condensed nuclei characteristic of apoptotic cells are seen in treated cells (white arrows).</p></div