Prion protein is ubiquitinated after developing protease resistance in the brains of scrapie-infected mice

Although the key event in the pathology of prion diseases is thought to be the conversion of cellular prion protein (PrP(C)) to the protease-resistant scrapie species termed PrP(Sc), the factors that contribute to neurodegeneration in scrapie-infected animals are poorly understood. One probable determinant could be when the accumulation of PrP(Sc) in infected brain overwhelms the ubiquitin-proteasome system and triggers the degenerative cascade. In the present study, it was found that in mouse brains infected with the ME7 scrapie strain, the level of ubiquitin protein conjugates increased significantly at approximately 144 days post-infection (pi) when clinical signs first become apparent. This elevation correlated with the detection of protease-resistant PrP(Sc) and a decline in two endopeptidase activities associated with proteasome function. However, ubiquitination of PrP was only detected at the terminal stage, 3 weeks after the development of clinical symptoms (approximately 165 days pi). These results suggest that ubiquitination of PrP is a late event phenomenon and this conjugation occurs after the formation of protease-resistant PrP(Sc). Whether this post-translational modification and the impairment of proteasome function are pivotal events in the pathogenesis of prion diseases remains to be determined.     

The scrapie prion protein is present in flotillin-1-positive vesicles in central- but not peripheral-derived neuronal cell lines

Abstract Transmissible prion diseases are fatal neurodegenerative diseases associated with the conversion of the normal host prion protein (PrP c) into an abnormal isoform (PrP Sc) that accumulates in brain. This pathology affects neurons of the central nervous system whereas no clear toxic effect has been reported for peripheral neurons. We examined the subcellular distribution of PrP c and PrP Sc in the scrapie-infected mouse neuronal cell lines GT1-7 and N2a, derived, respectively, from the central and peripheral nervous system. We observed that in both cell types, PrP c is present in the endocytic compartment, mainly in LAMP-1-positive late endosomes, but excluded from LYAAT-1-lysosomes. In contrast, PrP Sc was distributed differently in the two cell lines. In infected N2a, PrP Sc and PrP c had comparable distribution patterns. In infected GT1-7, PrP Sc is present in an additional vesicular compartment which is flotillin-1-positive. The level of expression of flotillin-1 is higher in GT1-7 than in N2a cells, but no difference is observed between infected and noninfected cells. In Alzheimer's disease patients, it has been reported that flotillin-1 is abundant in brain areas containing the beta-amyloid protein, which accumulates in endosomal vesicles in primary neurons. We propose that the flotillin compartment could store aggregated proteins and play a role in these neurodegenerative pathologies.     

Neuropil and neuronal changes in hippocampal NADPH-diaphorase histochemistry in the ME7 model of murine prion disease

Nitric oxide (NO) has been implicated in neurotoxicity and cerebral blood flow changes in chronic neurodegeneration, but its activity in the mammalian prion diseases has not been studied in detail. Nicotine adenine dinucleotide phosphate (NADPH)-diaphorase (NADPH-d) histochemistry is a simple and robust histochemical procedure that allows localization of the tissue distribution of NO synthases. The aim of the present study is to assess whether NADPH-d histochemical activity is altered in the hippocampus in the ME7 model of prion disease in C57BL/6J mice. At early and late stages after the initiation of the disease we assessed features of the NADPH-d positive cells and the neuropil histochemical activity in CA1 and dentate gyrus using densitometric analysis. In C57BL/6J mice 13 weeks postinjection of the prion agent ME7, when behavioural changes first become apparent, neuropil NADPH-d histochemical staining increases, whereas at late stages it decreases dramatically. Both type I and type II NADPH-d positive cells were found to survive throughout the hippocampal formation into the late stages of the disease, but diaphorase activity was reduced in dendritic branches and abnormal varicosities were present in both dendritic and axonal processes of NADPH-d positive type I cells. The pathophysiological implications of the results remain to be investigated but both blood flow alteration and NO neurotoxicity may be features of the disease.     

Complete Freund's adjuvant immunization prolongs survival in experimental prion disease in mice

We recently reported that immunization of mice with certain self-prion protein peptides induced specific T-cell and B-cell immune responses; importantly, this immunization was associated with a decrease in the number of protease-resistant PrP(Sc) particles recoverable in a transplanted, scrapie-infected syngeneic tumor. The present study was carried out to determine whether immunization with the immunogenic PrP peptides might influence the natural history of experimental scrapie in mice. We immunized C57BL/6 mice with self-prion peptides in complete Freund's adjuvant (CFA) or with CFA alone as a control and then infected the mice with mouse-adapted scrapie by injection either intraperitoneally or intracerebrally. We report here that immunization with CFA, irrespective of whether prion peptides were present in the inoculum, resulted in marked prolongation of survival of the mice, whether the challenge was intracerebral or intraperitoneal. Mice in the immunized and control groups that died contained equivalent amounts of PrP(Sc). Thus, CFA immunization has a therapeutic effect in experimental scrapie in mice, possibly by reducing the rate of PrP(Sc) accumulation in the brain.     

Assessment of the potential of plasma fractionation processes to remove causative agents of transmissible spongiform encephalopathy

Although there is no evidence that classical CJD (cCJD) can be transmitted by human blood or blood products in clinical practice, uncertainties surrounding new variant CJD (nvCJD) have led to the safety of plasma products derived from UK donors being questioned. To better define whether or not there is a risk of nvCJD being transmitted it is necessary to determine how the causative agent would partition across the separations processes used in the preparation of plasma products.
The abnormal prion protein which is associated with transmissible spongiform encephalopathies (TSEs), such as CJD, has a low solubility, a high tendency to form aggregates and adheres to surfaces readily. If the physicochemical properties of the agent of nvCJD are similar to those of abnormal prion protein then nvCJD may be removed by precipitation and adsorption technologies used in plasma fractionation.
Available data on the removal of TSE agents by such bioprocess technologies have been used to estimate the potential degree of reduction expected from each step in the plasma fractionation processes used by the SNBTS. The overall process reduction factors estimated are: 10¹³ (albumin), 10⁹ (immunoglobulins), 10⁷ (factor IX, thrombin), 10⁵ (fibrinogen), 10⁴ (factor VIII) and 10³ (factor II, IX and X); however, it will be necessary to establish the accuracy of these estimates by practical validation studies.     

Antigen retrieval using sodium hydroxide for prion immunohistochemistry in bovine spongiform encephalopathy and scrapie

Formalin-fixed and paraffin wax-embedded (FFPE) tissue sections are usually used for histopathological and immunohistochemical analyses in prion diseases in animals and man. However, formalin fixation cross-links proteins, reducing disease-associated prion protein (PrP^Sc) immunolabelling. To detect PrP^Sc in animals naturally affected with bovine spongiform encephalopathy (BSE) and scrapie, we applied minimal pretreatment with sodium hydroxide (NaOH). This simple pretreatment, combined with enzymatic digestion using proteinase K (PK), was equally effective in the detection of PrP^Sc in FFPE tissue, and superior in terms of speed, compared with the usual autoclaving method. The most effective results, without any section loss, were obtained with 10 μg/ml PK in phosphate buffered saline containing 0.1% Triton-X at room temperature for 10 min and 150 mM NaOH at 60°C for 10 min. By this simple procedure, PrP^Sc was visualized in the brain of animals with BSE and scrapie using a range of anti-PrP primary antibodies.     

Prion strain discrimination in cell culture: the cell panel assay

Prions are thought to consist mainly or entirely of misfolded PrP, a constitutively expressed host protein. Prions associated with the same PrP sequence may occur in the form of different strains; the strain phenotype is believed to be encoded by the conformation of the PrP. Some cell lines can be persistently infected by prions and, interestingly, show preference for certain strains. We report that a cloned murine neuroblastoma cell population, N2a-PK1, is highly heterogeneous in regard to its susceptibility to RML and 22L prions. Remarkably, sibling subclones may show very different relative susceptibilities to the two strains, indicating that the responses can vary independently. We have assembled four cell lines, N2a-PK1, N2a-R33, LD9 and CAD5, which show widely different responses to prion strains RML, 22L, 301C, and Me7, into a panel that allows their discrimination in vitro within 2 weeks, using the standard scrapie cell assay (SSCA).     

Spontaneous generation of mammalian prions

Prions are transmissible agents that cause lethal neurodegeneration in humans and other mammals. Prions bind avidly to metal surfaces such as steel wires and, when surface-bound, can initiate infection of brain or cultured cells with remarkable efficiency. While investigating the properties of metal-bound prions by using the scrapie cell assay to measure infectivity, we observed, at low frequency, positive assay results in control groups in which metal wires had been coated with uninfected mouse brain homogenate. This phenomenon proved to be reproducible in rigorous and exhaustive control experiments designed to exclude prion contamination. The infectivity generated in cell culture could be readily transferred to mice and had strain characteristics distinct from the mouse-adapted prion strains used in the laboratory. The apparent ”spontaneous generation” of prions from normal brain tissue could result if the metal surface, possibly with bound cofactors, catalyzed de novo formation of prions from normal cellular prion protein. Alternatively, if prions were naturally present in the brain at levels not detectable by conventional methods, metal surfaces might concentrate them to the extent that they become quantifiable by the scrapie cell assay.     

Classic scrapie in sheep with the ARR/ARR prion genotype in Germany and France

In the past, natural scrapie and bovine spongiform encephalopathy (BSE) infections have essentially not been diagnosed in sheep homozygous for the A136R154R171 haplotype of the prion protein. This genotype was therefore assumed to confer resistance to BSE and classic scrapie under natural exposure conditions. Hence, to exclude prions from the human food chain, massive breeding efforts have been undertaken in the European Union to amplify this gene. We report the identification of 2 natural scrapie cases in ARR/ARR sheep that have biochemical and transmission characteristics similar to cases of classic scrapie, although the abnormally folded prion protein (PrP(Sc)) was associated with a lower proteinase-K resistance. PrP(Sc) was clearly distinct from BSE prions passaged in sheep and from atypical scrapie prions. These findings strongly support the idea that scrapie prions are a mosaic of agents, which harbor different biologic properties, rather than a unique entity.