ABSTRACT
Parkinson's disease is one of several neurodegenerative diseases associated with a misfolded, aggregated and pathological protein. In Parkinson's disease this protein is alpha-synuclein and its neuronal deposits in the form of Lewy bodies are considered a hallmark of the disease. In this review we describe the clinical and experimental data that have led to think of alpha-synuclein as a prion-like protein and we summarize data from in vitro, cellular and animal models supporting this view.
Subject(s)
Parkinson Disease/metabolism , alpha-Synuclein/metabolism , Animals , Humans , Parkinson Disease/genetics , Prions/chemistry , Prions/genetics , Prions/metabolism , Protein Folding , alpha-Synuclein/chemistry , alpha-Synuclein/geneticsABSTRACT
Filaments made of α-synuclein form the characteristic Lewy pathology in Parkinson and other diseases. The formation of α-synuclein filaments can be reproduced in vitro by incubation of recombinant protein, but the filament growth is very slow and highly variable and so unsuitable for fast high throughput anti-aggregation drug screening. To overcome this obstacle we have investigated whether the protein misfolding cyclic amplification (PMCA) technique, used for fast amplification of prion protein aggregates, could be adapted for growing α-synuclein aggregates and thus suitable for screening of drugs to affect α-synuclein aggregation for the treatment of the yet incurable α-synucleinopathies. Circular dichroism, electron microscopy, and native and SDS-polyacrylamide gels were used to demonstrate α-synuclein aggregate formation by PMCA, and the strain imprint of the α-synuclein fibrils was studied by proteinase K digestion. We also demonstrated that α-synuclein fibrils are able to seed new α-synuclein PMCA reactions and to enter and aggregate in cells in culture. In particular, we have generated a line of "chronically infected" cells, which transmit α-synuclein aggregates even after multiple passages. To evaluate the sensitivity of the PMCA system as an α-synuclein anti-aggregating drug screening assay a panel of 10 drugs was tested. Anti-amyloid compounds proved efficient in inhibiting α-synuclein fibril formation induced by PMCA. Our results show that α-synuclein PMCA is a fast and reproducible system that could be used as a high throughput screening method for finding new α-synuclein anti-aggregating compounds.
Subject(s)
Amyloid/metabolism , Protein Folding , alpha-Synuclein/antagonists & inhibitors , Cell Line , Humans , Recombinant Proteins/metabolism , Reproducibility of Results , alpha-Synuclein/metabolismABSTRACT
The specific characteristics of Transmissible Spongiform Encephalopathy (TSE) strains may be altered during passage across a species barrier. In this study we investigated the biochemical and biological characteristics of Bovine Spongiform Encephalopathy (BSE) after transmission in both natural host species (cattle, sheep, pigs and mice) and in transgenic mice overexpressing the corresponding cellular prion protein (PrPC) in comparison with other non-BSE related prions from the same species. After these passages, most features of the BSE agent remained unchanged. BSE-derived agents only showed slight modifications in the biochemical properties of the accumulated PrPSc, which were demonstrated to be reversible upon re-inoculation into transgenic mice expressing bovine-PrPC. Transmission experiments in transgenic mice expressing bovine, porcine or human-PrP revealed that all BSE-derived agents were transmitted with no or a weak transmission barrier. In contrast, a high species barrier was observed for the non-BSE related prions that harboured an identical PrP amino acid sequence, supporting the theory that the prion transmission barrier is modulated by strain properties (presumably conformation-dependent) rather than by PrP amino acid sequence differences between host and donor. As identical results were observed with prions propagated either in natural hosts or in transgenic mouse models, we postulate that the species barrier and its passage consequences are uniquely governed by the host PrPC sequence and not influenced by other host genetic factors. The results presented herein reinforce the idea that the BSE agent is highly promiscuous, infecting other species, maintaining its properties in the new species, and even increasing its capabilities to jump to other species including humans. These data are essential for the development of an accurate risk assessment for BSE.
Subject(s)
Encephalopathy, Bovine Spongiform/metabolism , Prion Diseases/metabolism , Prions/metabolism , Animals , Cattle , Disease Models, Animal , Mice , Mice, Transgenic , Sheep , SwineABSTRACT
PrP(C), a host protein which in prion-infected animals is converted to PrP(Sc), is linked to the cell membrane by a GPI anchor. Mice expressing PrP(C) without GPI anchor (tgGPIâ» mice), are susceptible to prion infection but accumulate anchorless PrP(Sc) extra-, rather than intracellularly. We investigated whether tgGPIâ» mice could faithfully propagate prion strains despite the deviant structure and location of anchorless PrP(Sc). We found that RML and ME7, but not 22L prions propagated in tgGPIâ» brain developed novel cell tropisms, as determined by the Cell Panel Assay (CPA). Surprisingly, the levels of proteinase K-resistant PrP(Sc) (PrP(res)) in RML- or ME7-infected tgGPIâ» brain were 25-50 times higher than in wild-type brain. When returned to wild-type brain, ME7 prions recovered their original properties, however RML prions had given rise to a novel prion strain, designated SFL, which remained unchanged even after three passages in wild-type mice. Because both RML PrP(Sc) and SFL PrP(Sc) are stably propagated in wild-type mice we propose that the two conformations are separated by a high activation energy barrier which is abrogated in tgGPIâ» mice.
Subject(s)
PrPC Proteins/chemistry , PrPSc Proteins/chemistry , Scrapie/transmission , Animals , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Membrane Glycoproteins/deficiency , Mice , Mice, Inbred C57BL , Mice, Transgenic , PrPC Proteins/metabolism , PrPSc Proteins/metabolism , Protein Conformation , Scrapie/metabolismABSTRACT
Prion diseases, which are mostly represented in humans by Creutzfeldt-Jakob disease, are transmissible neurodegenerative disorders characterized by vacuolization and neuronal loss, as well as by the accumulation of an abnormal form of the prion protein. These disorders have yet no effective treatment, and drugs that block prion replication in vitro do not significantly slow down the progression of the disease when used in vivo at late stages. Cell therapy that has been already tested in other neurodegenerative disorders therefore represents an interesting alternative approach. In this study, we showed for the first time in prion diseases that intracerebral transplantation of fetal neural stem cells significantly extended both incubation and survival time. This result was dependant on the time window chosen for the engraftment and was obtained with both genetically modified and wild-type stem cells, therefore forging a path toward efficient stem cell therapy for human prion diseases.
Subject(s)
Brain Tissue Transplantation , Fetal Tissue Transplantation , Infectious Disease Incubation Period , Neural Stem Cells/transplantation , Prion Diseases/therapy , Animals , Brain/pathology , Female , Mice , Mice, Inbred BALB C , Models, Animal , Neural Stem Cells/metabolism , PrPC Proteins/metabolism , PrPSc Proteins/metabolism , Prion Diseases/metabolism , Prion Diseases/pathology , Time FactorsABSTRACT
Until now only a few cell lines have been proved able to propagate prions and only limited prion strains have been replicated in cell models. Neurosphere lines isolated from the brains of mice at embryonic day 14 grow as aggregates and contain CNS stem cells. Others authors have previously reported that cultured neurospheres expressing cellular prion protein (PrP(C)) can be infected with prions. As potential neural progenitors the neurosphere cultures are supposed to differentiate into neurons and astrocytes which represent the main cell types infected by prions in vivo. Here we study the ability of undifferentiated and differentiated neurospheres to replicate several prion strains. Neurosphere cultures were isolated from 129/ola, FVB, Prnp(0/0) and Tga20 mice, which over-express murine PrP. We were not able to detect PrP(res) accumulation in dividing neurosphere cultures after prion exposure to two different mouse adapted scrapie inocula (RML and 22L). In contrast, with differentiated neurosphere cultures expressing PrP(C) (129/ola, FVB and Tga20) a successful PrP(Res) amplification was observed in very short time experiments when infected with the same inocula, implying that cell differentiation improve prion replication in these cultured cells. The mouse BSE adapted inocula (301C) was not amplified in these neurosphere cultures neither before nor after differentiation, suggesting that these cell cultures showed a differential prion strain susceptibility. These results suggest that differentiated neurosphere cultures can complement prion bioassays in mouse models.
Subject(s)
Cell Differentiation/genetics , Neurons/metabolism , PrPC Proteins/genetics , PrPC Proteins/metabolism , Spheroids, Cellular/metabolism , Amino Acid Sequence/genetics , Animals , Cell Culture Techniques , Cells, Cultured , Infections/genetics , Infections/metabolism , Mice , Mice, Transgenic , Models, Biological , Molecular Biology , PrPSc Proteins/genetics , PrPSc Proteins/metabolism , Prion Diseases/genetics , Prion Diseases/metabolism , Prion Diseases/physiopathology , Species Specificity , Spheroids, Cellular/cytologyABSTRACT
How susceptible pigs are to infection with sheep prions is unknown. We show, through transmission experiments in transgenic mice expressing porcine prion protein (PrP), that the susceptibility of this mouse model to bovine spongiform encephalopathy (BSE) can be enhanced after its passage in ARQ sheep, indicating that the pathogenicity of the BSE agent is modified after passage in sheep. Transgenic mice expressing porcine PrP were, nevertheless, completely resistant to infection with a broad panel of classical scrapie isolates from different sheep PrP genotypes and with different biochemical characteristics. The atypical (Nor98 like) isolate (SC-PS152) was the only scrapie isolate capable of transmission in these mice, although with a marked transmission barrier. Unexpectedly, the atypical scrapie agent appeared to undergo a strain phenotype shift upon transmission to porcine-PrP transgenic mice and acquired new strain properties, suggesting that atypical scrapie agent may exhibit different phenotypes depending on the host cellular PrP or other genetic factors.
Subject(s)
Encephalopathy, Bovine Spongiform/etiology , Prions/genetics , Prions/pathogenicity , Scrapie/etiology , Animals , Brain/pathology , Brain Chemistry , Cattle , Communicable Diseases, Emerging/etiology , Communicable Diseases, Emerging/pathology , Communicable Diseases, Emerging/transmission , Communicable Diseases, Emerging/veterinary , Disease Models, Animal , Encephalopathy, Bovine Spongiform/pathology , Encephalopathy, Bovine Spongiform/transmission , Mice , Mice, Transgenic , Prions/chemistry , Prions/isolation & purification , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Scrapie/pathology , Scrapie/transmission , Sheep , Sheep Diseases/etiology , Sheep Diseases/pathology , Sheep Diseases/transmission , Species Specificity , SwineABSTRACT
Faeces from infected animals have been suggested as a potential source of contamination and transmission of prion diseases in the environment. This work describes the development of a procedure for the detection of PrP(res) in stools which is based on a detergent-based extraction and immunoprecipitation (IP). The procedure was evaluated by analyzing TSE-spiked sheep and mice faeces, and proved to be specific for PrP(res) with sensitivities of 5-10 microg of infected brain tissue. In order to analyze the shedding of prions, we studied stools from orally inoculated mice over 4-days post-inoculation and also stools from terminally sick scrapie-infected mice. PrP(res) was only detected in stools shortly after the oral ingestion of TSE agents. The procedure described could be a useful tool for studying the excretion of prions and for evaluating potential environmental contamination by prions.
Subject(s)
Encephalopathy, Bovine Spongiform/metabolism , Feces/chemistry , Prions/isolation & purification , Scrapie/metabolism , Animals , Blotting, Western , Cattle , Disease Models, Animal , Mice , Mice, Transgenic , PrPSc Proteins/isolation & purification , Species Specificity , Time FactorsABSTRACT
Sheep can be experimentally infected with bovine spongiform encephalopathy (BSE), and the ensuing disease is similar to scrapie in terms of pathogenesis and clinical signs. BSE infection in sheep is an animal and human health concern. In this study, the transmission in BoPrP-Tg110 mice of prions from BSE-infected sheep was examined and compared to the transmission of original cattle BSE in cattle and sheep scrapie prions. Our results indicate no transmission barrier for sheep BSE prions to infect BoPrP-Tg110 mice, but the course of the disease is accelerated compared to the effects of the original BSE isolate. The shortened incubation period of sheep BSE in the model was conserved in subsequent passage in BoPrP-Tg110 mice, indicating that it is not related to infectious titer differences. Biochemical signature, lesion profile, and PrP(Sc) deposition pattern of both cattle and sheep BSE were similar. In contrast, all three sheep scrapie isolates tested showed an evident transmission barrier and further adaptation in subsequent passage. Taken together, those data indicate that BSE agent can be altered by crossing a species barrier, raising concerns about the virulence of this new prion towards other species, including humans. The BoPrP-Tg110 mouse bioassay should be considered as a valuable tool for discriminating scrapie and BSE in sheep.