Essential Components of Synthetic Infectious Prion Formation De Novo

Prion diseases are a class of neurodegenerative diseases that are uniquely infectious. Whilst their general replication mechanism is well understood, the components required for the formation and propagation of highly infectious prions are poorly characterized. The protein-only hypothesis posits that the prion protein (PrP) is the only component of the prion; however, additional co-factors are required for its assembly into infectious prions. These can be provided by brain homogenate, but synthetic lipids and non-coding RNA have also been used in vitro. Here, we review a range of experimental approaches, which generate PrP amyloid assemblies de novo. These synthetic PrP assemblies share some, but not necessarily all, properties of genuine infectious prions. We will discuss the different experimental approaches, how a prion is defined, the non-protein requirements of a prion, and provide an overview of the current state of prion amplification and generation in vitro

Amyloid-β(1-42) aggregation initiates its cellular uptake and cytotoxicity

The accumulation of amyloid beta peptide(1-42) (Abeta(1-42)) in extracellular plaques is one of the pathological hallmarks of Alzheimer disease (AD). Several studies have suggested that cellular reuptake of Abeta(1-42) may be a crucial step in its cytotoxicity, but the uptake mechanism is not yet understood. Abeta may be present in an aggregated form prior to cellular uptake. Alternatively, monomeric peptide may enter the endocytic pathway and conditions in the endocytic compartments may induce the aggregation process. Our study aims to answer the question whether aggregate formation is a prerequisite or a consequence of Abeta endocytosis. We visualized aggregate formation of fluorescently labeled Abeta(1-42) and tracked its internalization by human neuroblastoma cells and neurons. beta-Sheet-rich Abeta(1-42) aggregates entered the cells at low nanomolar concentration of Abeta(1-42). In contrast, monomer uptake faced a concentration threshold and occurred only at concentrations and time scales that allowed Abeta(1-42) aggregates to form. By uncoupling membrane binding from internalization, we found that Abeta(1-42) monomers bound rapidly to the plasma membrane and formed aggregates there. These structures were subsequently taken up and accumulated in endocytic vesicles. This process correlated with metabolic inhibition. Our data therefore imply that the formation of beta-sheet-rich aggregates is a prerequisite for Abeta(1-42) uptake and cytotoxicity

Aggregation of full length immunoglobulin light chains from AL amyloidosis patients is remodeled by epigallocatechin-3-gallate

Intervention into amyloid deposition with anti-amyloid agents like the polyphenol Epigallocatechin-3-gallate (EGCG) is emerging as an experimental secondary treatment strategy in systemic light chain amyloidosis (AL). In both AL and Multiple Myeloma (MM), soluble immunoglobulin light chains (LC) are produced by clonal plasma cells, but only in AL they form amyloid deposits in vivo. We investigated the amyloid formation of patient-derived LC and their susceptibility to EGCG in vitro to probe commonalities and systematic differences in their assembly mechanisms. We isolated nine LC from urine of AL and MM patients. We quantified their thermodynamic stabilities, and monitored their aggregation under physiological conditions by ThT fluorescence, light scattering, SDS-stability and atomic force microscopy. LC from all patients formed amyloid-like aggregates, albeit with individually different kinetics. LC existed as dimers, ~50% of which were linked by disulfide bridges. Our results suggest that cleavage into LC monomers is required for efficient amyloid formation. The kinetics of AL LC displayed a transition point in concentration dependence, which MM LC lacked. The lack of concentration dependence of MM LC aggregation kinetics suggests that conformational change of the light chain is rate-limiting for these proteins. Aggregation kinetics displayed two distinct phases, which corresponded to the formation of oligomers and amyloid fibrils, respectively. EGCG specifically inhibited the second aggregation phase and induced the formation of SDS-stable, non-amyloid LC aggregates. Our data suggest that EGCG intervention does not depend on the individual LC sequence and is similar to the mechanism observed for amyloid-{beta} and {alpha}-synuclein

Amyloid-β(1-42) Aggregation Initiates Its Cellular Uptake and Cytotoxicity

The accumulation of amyloid β peptide(1-42) (Aβ(1-42)) in extracellular plaques is one of the pathological hallmarks of Alzheimer disease (AD). Several studies have suggested that cellular reuptake of Aβ(1-42) may be a crucial step in its cytotoxicity, but the uptake mechanism is not yet understood. Aβ may be present in an aggregated form prior to cellular uptake. Alternatively, monomeric peptide may enter the endocytic pathway and conditions in the endocytic compartments may induce the aggregation process. Our study aims to answer the question whether aggregate formation is a prerequisite or a consequence of Aβ endocytosis. We visualized aggregate formation of fluorescently labeled Aβ(1-42) and tracked its internalization by human neuroblastoma cells and neurons. β-Sheet-rich Aβ(1-42) aggregates entered the cells at low nanomolar concentration of Aβ(1-42). In contrast, monomer uptake faced a concentration threshold and occurred only at concentrations and time scales that allowed Aβ(1-42) aggregates to form. By uncoupling membrane binding from internalization, we found that Aβ(1-42) monomers bound rapidly to the plasma membrane and formed aggregates there. These structures were subsequently taken up and accumulated in endocytic vesicles. This process correlated with metabolic inhibition. Our data therefore imply that the formation of β-sheet-rich aggregates is a prerequisite for Aβ(1-42) uptake and cytotoxicity

Structural effects of the highly protective V127 polymorphism on human prion protein

Prion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). A single point mutation (G127V) in human PrP prevents prion disease, however the structural basis for its protective effect remains unknown. Here we show that the mutation alters and constrains the PrP backbone conformation preceding the PrP β-sheet, stabilising PrP dimer interactions by increasing intermolecular hydrogen bonding. It also markedly changes the solution dynamics of the β2-α2 loop, a region of PrP structure implicated in prion transmission and cross-species susceptibility. Both of these structural changes may affect access to protein conformers susceptible to prion formation and explain its profound effect on prion disease

Amyloid-β(1-42) Aggregation Initiates Its Cellular Uptake and Cytotoxicity

The accumulation of amyloid β peptide(1-42) (Aβ(1-42)) in extracellular plaques is one of the pathological hallmarks of Alzheimer disease (AD). Several studies have suggested that cellular reuptake of Aβ(1-42) may be a crucial step in its cytotoxicity, but the uptake mechanism is not yet understood. Aβ may be present in an aggregated form prior to cellular uptake. Alternatively, monomeric peptide may enter the endocytic pathway and conditions in the endocytic compartments may induce the aggregation process. Our study aims to answer the question whether aggregate formation is a prerequisite or a consequence of Aβ endocytosis. We visualized aggregate formation of fluorescently labeled Aβ(1-42) and tracked its internalization by human neuroblastoma cells and neurons. β-Sheet-rich Aβ(1-42) aggregates entered the cells at low nanomolar concentration of Aβ(1-42). In contrast, monomer uptake faced a concentration threshold and occurred only at concentrations and time scales that allowed Aβ(1-42) aggregates to form. By uncoupling membrane binding from internalization, we found that Aβ(1-42) monomers bound rapidly to the plasma membrane and formed aggregates there. These structures were subsequently taken up and accumulated in endocytic vesicles. This process correlated with metabolic inhibition. Our data therefore imply that the formation of β-sheet-rich aggregates is a prerequisite for Aβ(1-42) uptake and cytotoxicity

Aggregation of Full-length Immunoglobulin Light Chains from Systemic Light Chain Amyloidosis (AL) Patients Is Remodeled by Epigallocatechin-3-gallate

Intervention into amyloid deposition with anti-amyloid agents like the polyphenol epigallocatechin-3-gallate (EGCG) is emerging as an experimental secondary treatment strategy in systemic light chain amyloidosis (AL). In both AL and multiple myeloma (MM), soluble immunoglobulin light chains (LC) are produced by clonal plasma cells, but only in AL do they form amyloid deposits in vivo We investigated the amyloid formation of patient-derived LC and their susceptibility to EGCG in vitro to probe commonalities and systematic differences in their assembly mechanisms. We isolated nine LC from the urine of AL and MM patients. We quantified their thermodynamic stabilities and monitored their aggregation under physiological conditions by thioflavin T fluorescence, light scattering, SDS stability, and atomic force microscopy. LC from all patients formed amyloid-like aggregates, albeit with individually different kinetics. LC existed as dimers, ∼50% of which were linked by disulfide bridges. Our results suggest that cleavage into LC monomers is required for efficient amyloid formation. The kinetics of AL LC displayed a transition point in concentration dependence, which MM LC lacked. The lack of concentration dependence of MM LC aggregation kinetics suggests that conformational change of the light chain is rate-limiting for these proteins. Aggregation kinetics displayed two distinct phases, which corresponded to the formation of oligomers and amyloid fibrils, respectively. EGCG specifically inhibited the second aggregation phase and induced the formation of SDS-stable, non-amyloid LC aggregates. Our data suggest that EGCG intervention does not depend on the individual LC sequence and is similar to the mechanism observed for amyloid-β and α-synuclein

Small-molecule conversion of toxic oligomers to nontoxic β-sheet-rich amyloid fibrils

Several lines of evidence indicate that prefibrillar assemblies of amyloid-{beta} (A{beta}) polypeptides, such as soluble oligomers or protofibrils, rather than mature, end-stage amyloid fibrils cause neuronal dysfunction and memory impairment in Alzheimer's disease. These findings suggest that reducing the prevalence of transient intermediates by small molecule-mediated stimulation of amyloid polymerization might decrease toxicity. Here we demonstrate the acceleration of A{beta} fibrillogenesis through the action of the orcein-related small molecule O4, which directly binds to hydrophobic amino acid residues in A{beta} peptides and stabilizes the self-assembly of seeding-competent, {beta}-sheet-rich protofibrils and fibrils. Notably, the O4-mediated acceleration of amyloid fibril formation efficiently decreases the concentration of small, toxic A{beta} oligomers in complex, heterogeneous aggregation reactions. In addition, O4 treatment suppresses inhibition of long-term potentiation by A{beta} oligomers in hippocampal brain slices. These results support the hypothesis that small, diffusible prefibrillar amyloid species rather than mature fibrillar aggregates are toxic for mammalian cells

The SHiP experiment at the proposed CERN SPS Beam Dump Facility

The Search for Hidden Particles (SHiP) Collaboration has proposed a general-purpose experimental facility operating in beam-dump mode at the CERN SPS accelerator to search for light, feebly interacting particles. In the baseline configuration, the SHiP experiment incorporates two complementary detectors. The upstream detector is designed for recoil signatures of light dark matter (LDM) scattering and for neutrino physics, in particular with tau neutrinos. It consists of a spectrometer magnet housing a layered detector system with high-density LDM/neutrino target plates, emulsion-film technology and electronic high-precision tracking. The total detector target mass amounts to about eight tonnes. The downstream detector system aims at measuring visible decays of feebly interacting particles to both fully reconstructed final states and to partially reconstructed final states with neutrinos, in a nearly background-free environment. The detector consists of a 50$\mathrm { \,m}$ long decay volume under vacuum followed by a spectrometer and particle identification system with a rectangular acceptance of 5 m in width and 10 m in height. Using the high-intensity beam of 400$\,\mathrm {GeV}$ protons, the experiment aims at profiting from the $4\times 10^{19}$ protons per year that are currently unexploited at the SPS, over a period of 5–10 years. This allows probing dark photons, dark scalars and pseudo-scalars, and heavy neutral leptons with GeV-scale masses in the direct searches at sensitivities that largely exceed those of existing and projected experiments. The sensitivity to light dark matter through scattering reaches well below the dark matter relic density limits in the range from a few ${\mathrm {\,MeV\!/}c^2}$ up to 100 MeV-scale masses, and it will be possible to study tau neutrino interactions with unprecedented statistics. This paper describes the SHiP experiment baseline setup and the detector systems, together with performance results from prototypes in test beams, as it was prepared for the 2020 Update of the European Strategy for Particle Physics. The expected detector performance from simulation is summarised at the end

Track reconstruction and matching between emulsion and silicon pixel detectors for the SHiP-charm experiment

In July 2018 an optimization run for the proposed charm cross section measurement for SHiP was performed at the CERN SPS. A heavy, moving target instrumented with nuclear emulsion films followed by a silicon pixel tracker was installed in front of the Goliath magnet at the H4 proton beam-line. Behind the magnet, scintillating-fibre, drift-tube and RPC detectors were placed. The purpose of this run was to validate the measurement's feasibility, to develop the required analysis tools and fine-tune the detector layout. In this paper, we present the track reconstruction in the pixel tracker and the track matching with the moving emulsion detector. The pixel detector performed as expected and it is shown that, after proper alignment, a vertex matching rate of 87% is achieved