Prion 2016 poster abstracts

Until now, the 3-dimensional structure of infectious mammalian prions and how this differs from non-infectious amyloid fibrils remained unknown. Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity. One of the major challenges has been the production of highly homogeneous material of demonstrable high specific infectivity to allow direct correlation of particle structure with infectivity. We have recently developed novel methods to obtain exceptionally pure preparations of prions from prion-infected murine brain and have shown that pathogenic PrP in these high-titer preparations is assembled into rod-like assemblies (Wenborn et al. 2015. Sci. Rep. 10062). Our preparations contain very high titres of infectious prions which faithfully transmit prion strain-specific phenotypes when inoculated into mice making them eminently suitable for detailed structural analysis. We are now undertaking structural characterization of prion assemblies and comparing these to the structure of non-infectious PrP fibrils generated from recombinant Pr

Detection and characterization of proteinase K-sensitive disease-related prion protein with thermolysin

Disease-related PrPSc [pathogenic PrP (prion protein)] is classically distinguished from its normal cellular precursor, PrPC(cellular PrP) by its detergent insolubility and partial resistance to proteolysis. Although molecular diagnosis of prion disease has historically relied upon detection of protease-resistant fragments of PrPSc using PK (proteinase K), it is now apparent that a substantial fraction of disease-related PrP is destroyed by this protease. Recently, thermolysin has been identified as a complementary tool to PK, permitting isolation of PrPSc in its full-length form. In the present study, we show that thermolysin can degrade PrPC while preserving both PK-sensitive and PK-resistant isoforms of disease-related PrP in both rodent and human prion strains. For mouse RML (Rocky Mountain Laboratory) prions, the majority of PK-sensitive disease-related PrP isoforms do not appear to contribute significantly to infectivity. In vCJD (variant Creutzfeldt–Jakob disease), the human counterpart of BSE (bovine spongiform encephalopathy), up to 90% of total PrP present in the brain resists degradation with thermolysin, whereas only ∼15% of this material resists digestion by PK. Detection of PK-sensitive isoforms of disease-related PrP using thermolysin should be useful for improving diagnostic sensitivity in human prion diseases

Superoxide Dismutase 1 and tgSOD1G93A Mouse Spinal Cord Seed Fibrils, Suggesting a Propagative Cell Death Mechanism in Amyotrophic Lateral Sclerosis

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that specifically affects motor neurons and leads to a progressive and ultimately fatal loss of function, resulting in death typically within 3 to 5 years of diagnosis. The disease starts with a focal centre of weakness, such as one limb, and appears to spread to other parts of the body. Mutations in superoxide dismutase 1 (SOD1) are known to cause disease and it is generally accepted they lead to pathology not by loss of enzymatic activity but by gain of some unknown toxic function(s). Although different mutations lead to varying tendencies of SOD1 to aggregate, we suggest abnormal proteins share a common misfolding pathway that leads to the formation of amyloid fibrils.Methodology/Principal Findings: Here we demonstrate that misfolding of superoxide dismutase 1 leads to the formation of amyloid fibrils associated with seeding activity, which can accelerate the formation of new fibrils in an autocatalytic cascade. The time limiting event is nucleation to form a stable protein "seed" before a rapid linear polymerisation results in amyloid fibrils analogous to other protein misfolding disorders. This phenomenon was not confined to fibrils of recombinant protein as here we show, for the first time, that spinal cord homogenates obtained from a transgenic mouse model that overexpresses mutant human superoxide dismutase 1 (the TgSOD1(G93A) mouse) also contain amyloid seeds that accelerate the formation of new fibrils in both wildtype and mutant SOD1 protein in vitro.Conclusions/Significance: These findings provide new insights into ALS disease mechanism and in particular a mechanism that could account for the spread of pathology throughout the nervous system. This model of disease spread, which has analogies to other protein misfolding disorders such as prion disease, also suggests it may be possible to design assays for therapeutics that can inhibit fibril propagation and hence, possibly, disease progression

Plasmacytoid Dendritic Cells Sequester High Prion Titres at Early Stages of Prion Infection

In most transmissible spongiform encephalopathies prions accumulate in the lymphoreticular system (LRS) long before they are detectable in the central nervous system. While a considerable body of evidence showed that B lymphocytes and follicular dendritic cells play a major role in prion colonization of lymphoid organs, the contribution of various other cell types, including antigen-presenting cells, to the accumulation and the spread of prions in the LRS are not well understood. A comprehensive study to compare prion titers of candidate cell types has not been performed to date, mainly due to limitations in the scope of animal bioassays where prohibitively large numbers of mice would be required to obtain sufficiently accurate data. By taking advantage of quantitative in vitro prion determination and magnetic-activated cell sorting, we studied the kinetics of prion accumulation in various splenic cell types at early stages of prion infection. Robust estimates for infectious titers were obtained by statistical modelling using a generalized linear model. Whilst prions were detectable in B and T lymphocytes and in antigen-presenting cells like dendritic cells and macrophages, highest infectious titers were determined in two cell types that have previously not been associated with prion pathogenesis, plasmacytoid dendritic (pDC) and natural killer (NK) cells. At 30 days after infection, NK cells were more than twice, and pDCs about seven-fold, as infectious as lymphocytes respectively. This result was unexpected since, in accordance to previous reports prion protein, an obligate requirement for prion replication, was undetectable in pDCs. This underscores the importance of prion sequestration and dissemination by antigen-presenting cells which are among the first cells of the immune system to encounter pathogens. We furthermore report the first evidence for a release of prions from lymphocytes and DCs of scrapie-infected mice ex vivo, a process that is associated with a release of exosome-like membrane vesicles

Exosomes are released from scrapie-infected B cells <i>ex vivo</i>.

<p>Spleens were dissected from 129/Sv×C57BL/6 mice 30 days after i.p. inoculation with 1% (w/v) RML I6200. MACS-isolated B lymphocytes were cultured under passive leakage (A) and basal (B) conditions essentially as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#ppat-1002538-t004" target="_blank">Table 4</a> and tissue culture supernatants were isolated by sequential centrifugation (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#s4" target="_blank">Materials and Methods</a>). After centrifugation at 100,000× g for 2 h pellets were resuspended in PBS, absorbed onto carbon-coated grids and negatively stained with 1% uranyl acetate. Cup-shaped exosome-like membrane particles of different sizes (see arrows) are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#ppat-1002538-g001" target="_blank">Figure 1B</a>. Twenty randomly recorded images (surface area: 2.82 µm²) from each condition were counted and the number of exosome-like particles (1.7±1.2 (A) and 22.8±6.5 (B) per surface area, p≪0.001) determined in a blinded manner. Scale bar: 0.2 µm.</p

Isolation of splenic cell types by magnetic-activated cell sorting.

<p><b>A</b>: Schematic representation for the isolation of specific splenic cell types from mice. Splenocytes were released by repeated collagenase digestion from freshly dissected spleens, followed by removal of erythrocytes and purification of splenocytes on Lympholyte M gradients. Splenic cell types are isolated by positive selection with magnetic beads coated with cell type-specific mAbs as specified. <b>B</b>: The purities of MACS-isolated cells were analysed by FACS using cell-type specific mAbs and isotype controls as specified in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#s4" target="_blank">Materials and Methods</a>. One representative out of three experiments is shown. (Bv) CD11^low B220⁺ pDCs, isolated with murine plasmacytoid dendritic antigen-1 (mPDCA-1) showed a purity of about 90% in three independent experiments. (Bvi) The macrophage population, isolated with CD11b microbeads after depletion of CD11c⁺ cells was contaminated with CD11c⁺ CD11b⁺ mDCs. Macrophages were therefore isolated by FACS instead (<b>C</b>). <b>C</b>: Splenocytes labelled with mAbs against anti-CD11b (M1/70) and anti-CD11c (HL3) were isolated by FACS using a DAKO cell sorter.</p

Infectious titers of MACS-isolated cells after homogenization by sonication and ribolyzation.

‡<p>Inputs of infectious cell homogenates are expressed as cell number equivalents. Aliquots of 30 µl were inoculated i.c. into groups of six Tga20 mice for mouse bioassay and 300 µl aliquots were layered onto prion-susceptible cells per well for SCEPA, respectively.</p>†<p>Infectious titers were calculated according to the Spearman-Karber method <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#ppat.1002538-Dougherty1" target="_blank">[55]</a> and are expressed as log LD₅₀/g ± SE for bioassay and log TCIU/g ± SE for SCEPA.</p>$<p>Infectious titers were calculated using a GLM with binomial family complementary log-log link and expressed as mean log TCIU/g ± SE of two independent experiments with six technical repeats each.</p>#<p>Controls represent MACS-isolated pDCs and B lymphocytes from spleens 129/Sv×C57BL/6 mice inoculated with 1% (w/v) uninfected CD1 brain homogenates and sacrificed at 30 dpi.</p>*<p>Level of significance for maximum likelihood estimates (GLM) between infectious titers of ribolyzed versus sonicated pDCs as determined by SCEPA.</p><p>Four 129/Sv×C57BL/6 mice were inoculated i.p. with 100 µl of 1% (w/v) RML and 1% (w/v) uninfected CD1 brain homogenate (control), respectively. At 30 d.p.i spleens were dissected and pDCs and B cells isolated by MACS according to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#ppat-1002538-g001" target="_blank">Figure 1</a>. Aliquots of 1×10⁷ cells/ml OFCS, supplemented with protease inhibitors were homogenized by sonication <i>(A)</i> or ribolyzation <i>(B)</i> according to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#s4" target="_blank">Materials and Methods</a>. To determine infectious titers the cell homogenates were serially diluted 1∶10 and inoculated intracerebrally into Tga20 mice or transferred onto layers of susceptible PK1-2 cells in parallel experiments. Infectious titers were determined by non-parametric statistical analysis for bioassay (Spearman and Karber) and GLM for SCEPA and expressed as log LD50 units/10⁶ cells and log TCIU/10⁶ cells, respectively. A 10⁻² dilution of cell homogenates corresponds to 2×10⁵ cell equivalents/ml or 6×10³ cell equivalents per 30 µl inoculum for mouse bioassay and 6×10⁴ cell equivalents per 300 µl per well for SCEPA, respectively. Infectious titers represent log mean values ± SE of six independent experiments for SCEPA and log mean values ± SE of a single experiment for mouse bioassay.</p