PrP Accumulation Research Articles

Scrapie-Specific Pathology of Sheep Lymphoid Tissues

Transmissible spongiform encephalopathies (TSEs) or prion diseases often result in accumulation of disease-associated PrP (PrPd) in the lymphoreticular system (LRS), specifically in association with follicular dendritic cells (FDCs) and tingible body macrophages (TBMs) of secondary follicles. We studied the effects of sheep scrapie on lymphoid tissue in tonsils and lymph nodes by light and electron microscopy. FDCs of sheep were grouped according to morphology as immature, mature or regressing. Scrapie was associated with FDC dendrite hypertrophy and electron dense deposit or vesicles. PrPd was located using immunogold labelling at the plasmalemma of FDC dendrites and, infrequently, mature B cells. Abnormal electron dense deposits surrounding FDC dendrites were identified as immunoglobulins suggesting that excess immune complexes are retained and are indicative of an FDC dysfunction. Within scrapie-affected lymph nodes, macrophages outside the follicle and a proportion of germinal centre TBMs accumulated PrPd within endosomes and lysosomes. In addition, TBMs showed PrPd in association with the cell membrane, non-coated pits and vesicles, and also with discrete, large and random endoplasmic reticulum networks, which co-localised with ubiquitin. These observations suggest that PrPd is internalised via the caveolin-mediated pathway, and causes an abnormal disease-related alteration in endoplasmic reticulum structure. In contrast to current dogma, this study shows that sheep scrapie is associated with cytopathology of germinal centres, which we attribute to abnormal antigen complex trapping by FDCs and abnormal endocytic events in TBMs. The nature of the sub-cellular changes in FDCs and TBMs differs from those of scrapie infected neurones and glial cells suggesting that different PrPd/cell membrane interactions occur in different cell types.

Retinal Cell Types are Differentially Affected in Sheep with Scrapie

Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases characterized microscopically by spongiform lesions (vacuolation) in the neuropil, neuronal loss, and gliosis. Accumulation of the abnormal form of the prion protein (PrP(Sc)) has been demonstrated in the retina of natural and non-natural TSE-affected hosts, with or without evidence of microscopically detectable retinal pathology. This study was conducted to investigate the effect of PrP(Sc) accumulation on retinal neurons in a natural host lacking overt microscopical evidence of retinal degeneration by comparing the distribution of retinal cell type-specific markers in control and scrapie-affected sheep. In retinas with PrP(Sc)-immunoreactivity, there was disruption of the normal immunoreactivity patterns of the alpha isoform of protein kinase C (PKCalpha) and vesicular glutamate transporter 1 (VGLUT1), markers of retinal bipolar cells. Altered immunoreactivity was also observed for microtubule-associated protein 2 (MAP2), a marker of a subset of retinal ganglion cells, and glutamine synthetase (GS), a marker of Müller glia. These results demonstrate alterations of immunoreactivity patterns for proteins associated with specific cell types in retinas with PrP(Sc) accumulation, despite an absence of microscopical evidence of retinal degeneration.

Gene expression profile of quinacrine‐cured prion‐infected mouse neuronal cells

Prion diseases are transmissible fatal neurodegenerative diseases of humans and animals, characterised by the presence of an abnormal isoform (scrapie prion protein; PrP(Sc)) of the endogenous cellular prion protein (PrP(C)). The pathological mechanisms at the basis of prion diseases remain elusive, although the accumulation of PrP(Sc) has been linked to neurodegeneration. Different genomic approaches have been applied to carry out large-scale expression analysis in prion-infected brains and cell lines, in order to define factors potentially involved in pathogenesis. However, the general lack of overlap between the genes found in these studies prompted us to carry an analysis of gene expression using an alternative approach. Specifically, in order to avoid the complexities of shifting gene expression in a heterogeneous cell population, we used a single clone of GT1 cells that was de novo infected with mouse prion-infected brain homogenate and then treated with quinacrine to clear PrP(Sc). By comparing the gene expression profiles of about 15 000 genes in quinacrine-cured and not cured prion-infected GT1 cells, we investigated the influence of the presence or the absence of PrP(Sc). By real-time PCR, we confirmed that the gene encoding for laminin was down-regulated as a consequence of the elimination of PrP(Sc) by the quinacrine treatment. Thus, we speculate that this protein could be a specific candidate for further analysis of its role in prion infection and pathogenesis.

Copaxone interferes with the PrPSc–GAG interaction

The hallmark of prion disease-induced neurodegeneration is the accumulation of PrP(Sc), a misfolded form of PrP(C). In addition, several lines of evidence indicate a role for the immune system and, in particular, inflammation in prion disease pathogenesis. In this work, we tested whether Copaxone, an immunomodulatory agent currently used for the treatment of multiple sclerosis, can affect prion disease manifestation in scrapie-infected hamsters. We show here that Copaxone exerted no effect on prion disease incubation time when treatment commenced 2 weeks after i.p. prion infection. However, when Copaxone was mixed with the initial prion inoculum or administered to hamsters weekly starting on the day of infection, prion disease incubation time was prolonged by 30 days. This suggests that Copaxone may affect the initial infection process. In vitro experiments indicate that Copaxone significantly reduced PrP(Sc) binding to both Chinese hamster ovary (CHO) cells and heparin beads and also binds to heparin by itself. Interestingly, Copaxone also abolished PrP(Sc) accumulation in scrapie-infected cells. We propose that Copaxone delays prion infection by competing with the PrP(Sc)-glycosaminoglycans interaction. Whether the immunomodulating activity of Copaxone is related to its heparin binding and anti-prion properties remains to be established.

Increased PrP mRNA expression in lymphoid follicles of the ileal Peyer's patch of sheep experimentally exposed to the scrapie agent

To understand the functional role of cellular prion protein (PrP(C)) in the initiation and maintenance of prion disease within the host, it is important to obtain a more detailed understanding of PrP(C) transcription in tissues during the development of disease. Using an experimental model with oral infection, we examined the effect of scrapie and the accumulation of the scrapie related form of the prion protein (PrP(Sc)) on the expression level of PrP mRNA in the ileal Peyer's patch of sheep. In the early phase of infection, prior to PrP(Sc) accumulation, no effect on the PrP expression was detected. However, it was found that lambs with PrP genotypes associated with high susceptibility for scrapie generally had higher PrP mRNA levels than lambs with less susceptible genotypes. Further, in highly susceptible VRQ/VRQ sheep at a stage of disease with high accumulation of PrP(Sc), real-time RT-PCR and microdissection were used to investigate levels of PrP mRNA in four different tissue compartments. An increased level of PrP mRNA was found in lymphoid follicles of infected sheep compared with controls, indicating upregulation of PrP expression in the follicles to compensate for the loss of PrP(C) converted to PrP(Sc), or that PrP(Sc) accumulation directly or indirectly influences the PrP expression. Still, the PrP expression level in the follicles was low compared with the other compartments investigated, suggesting that although increased PrP expression could contribute to PrP(Sc) accumulation, other factors are also important in the processes leading to accumulation of PrP(Sc) in the follicles.

Galectin-3 expression is correlated with abnormal prion protein accumulation in murine scrapie

To investigate the involvement of galectin-3 in the process of neurodegeneration in prion diseases, the expression and cellular localization of galectin-3 in the brain were studied in scrapie, a mouse model of prion disease. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses showed that the expression of galectin-3 protein and mRNA was induced in scrapie-affected brains, particularly at the time when the abnormal prion protein PrP(Sc) began to accumulate in the brains. Immunohistochemically, immunostaining for galectin-3 was found mainly in B4-isolectin-positive cells (presumably activated microglia/macrophages), but not in astrocytes. Galectin-3 immunoreactivity was localized mainly in areas of PrP(Sc) accumulation and neuronal death in scrapie-infected brains. These findings suggest that the expression of galectin-3 by activated microglia/macrophages in prion disease correlates with abnormal prion protein accumulation.

Selective re‐routing of prion protein to proteasomes and alteration of its vesicular secretion prevent PrPSc formation

Conversion of the cellular prion protein (PrP(C)) into the abnormal scrapie isoform (PrP(Sc)) is the hallmark of prion diseases, which are fatal and transmissible neurodegenerative disorders. ER-retained anti-prion recombinant single-chain Fv fragments have been proved to be an effective tool for inhibition of PrP(C) trafficking to the cell surface and antagonize PrP(Sc) formation and infectivity. In the present study, we have generated the secreted version of 8H4 intrabody (Sec-8H4) in order to compel PrP(C) outside the cells. The stable expression of the Sec-8H4 intrabodies induces proteasome degradation of endogenous prion protein but does not influence its glycosylation profile and maturation. Moreover, we found a dramatic diverting of PrP(C) traffic from its vesicular secretion and, most importantly, a total inhibition of PrP(Sc) accumulation in NGF-differentiated Sec-8H4 PC12 cells. These results confirm that perturbing the intracellular traffic of endogenous PrP(C) is an effective strategy to inhibit PrP(Sc) accumulation and provide convincing evidences for application of intracellular antibodies in prion diseases.

Molecular biology of prion protein and its first homologous protein

Conformational conversion of the normal cellular isoform of prion protein, PrP(C), a glycoprotein anchored to the cell membrane by a glycosylphosphatidylinositol moiety, into the abnormally folded, amyloidogenic prion protein, PrP(Sc), plays a pivotal role in the pathogenesis of prion diseases. It has been suggested that PrP(C) might be functionally disturbed by constitutive conversion to PrP(Sc) due to either the resulting depletion of PrP(C) or the dominant negative effects of PrP(Sc) on PrP(C) or both. Consistent with this, we and others showed that mice devoid of PrP(C) (PrP-/-) spontaneously developed abnormal phenotypes very similar to the neurological abnormalities of prion diseases, supporting the concept that functional loss of PrP(C) might at least be partly involved in the pathogenesis of the diseases. However, no neuronal cell death could be detected in PrP-/- mice, indicating that the functional loss of PrP(C) alone might not be enough to induce neuronal cell death, one of major pathological hallmarks of prion diseases. Interestingly, it was recently shown that the first identified PrP-like protein, termed PrPLP/Doppel (Dpl), is neurotoxic in the absence of PrP(C), causing Purkinje cell degeneration in the cerebellum of mice. Although it is not understood if PrP(Sc) could have a neurotoxic potential similar to PrPLP/Dpl, it is very interesting to speculate that accumulation of PrP(Sc) and the functional disturbance of PrP(C), both of which are caused by constitutive conversion, might be required for the neurodegeneration in prion diseases.

Prevention of Prion Propagation by Dehydrocholesterol Reductase Inhibitors in Cultured Cells and a Therapeutic Trial in Mice

In prion diseases, the normal cellular form of prion protein (PrP(C)) is converted into the disease-associated isoforms (PrP(Sc)) which accumulate in the infected tissues. Although the precise mechanism of this conversion remains unsolved, drugs of various categories have been reported to reduce the accumulation of PrP(Sc) in prion-infected cultured cells. We here show that AY-9944 (a 7-dehydrocholesterol reductase inhibitor) and U18666A (a 24-dehydrocholesterol reductase inhibitor) prevent PrP(Sc) from accumulating in prion-infected mouse neuroblastoma cells (ScN2a), with an ED50 of about 0.5 microM and 10 nM, respectively. In order to evaluate the efficacy of these two inhibitors in vivo, C57BL/6J mice inoculated with mouse-adapted scrapie-prion received repetitive intraperitoneal injections of U18666A (10 mg/kg) or a mixture of U18666A (10 mg/kg) and AY-9944 (12 mg/kg). By contrast to the potent anti-prion effects observed in ScN2a cells, the in vivo trial was abortive with neither drug halting the progression of the disease.

Activation of Prn‐p gene and stable transfection of Prn‐p cDNA in leukemia MEL and neuroblastoma N2a cells increased production of PrPC but not prevented DNA fragmentation initiated by serum deprivation

Prion protein (PrP(C)) via its isoform PrP(SC) is involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). We observed that murine erythroleukemia (MEL) cells arrested in phase G(1) undergo transcriptional activation of Prn-p gene. Here, we explored the potential role of activation of Prn-p gene and cytosolic accumulation of PrP(C) in growth arrest, differentiation, and apoptotic DNA fragmentation by stably transfecting MEL and N2a cells with Prn-p cDNA. Stably transfected MEL cells (clones # 6, 12, 20, 38, and 42) were assessed for growth and differentiation, while clones N2a13 and N2a8 of N2a cells for growth and apoptosis by flow cytometry using Annexin V and propidium iodide (PI). Our results indicate that (a) Induction of terminal differentiation of stably transfected MEL cells led to growth arrest, activation of Prn-p gene, concomitant expression of transfected Prn-p cDNA, suppression of bax gene, cytosolic accumulation of PrP(C), and DNA fragmentation. The latter was also induced in non-differentiated MEL cells growing under serum-free conditions; (b) similarly, serum deprivation promoted growth arrest, apoptosis/necrosis associated with DNA fragmentation in parental N2a and N2a13 cells that produced relative high level of PrP(C) and not PrP(SC). These data indicate that activation of Prn-p gene and expression of transfected Prn-p cDNA in cells of both hematopoietic and neuronal origin occurred concomitantly, and led to cytosolic accumulation of PrP(C) and DNA damage induced by serum deprivation. PrP(C) production failed to protect DNA fragmentation induced by serum deprivation. The question how does PrP(C) contribute to growth arrest and DNA fragmentation is discussed.

Efficient in vitro amplification of a mouse-adapted scrapie prion protein

Protein misfolding cyclic amplification (PMCA) is a highly sensitive technique used to detect minute amounts of scrapie prion protein (PrP Sc), a major protein component of the infectious agents associated with prion diseases. Although exponential in vitro amplification of hamster scrapie PrP Sc has been established, the PMCA used was unsuccessful in achieving good amplification of PrP Sc from other animals. Here, we have investigated the cause of the insufficient PrP Sc amplification in mice and have developed an improved method suitable for amplification of the PrP Sc of the mouse-adapted scrapie prion strain Chandler. Mouse PrP C, the cellular form of the prion protein, tends to become resistant to proteases during incubation independent of sonication. By adding digitonin to the reaction buffer as a lipid detergent, accumulation of the protease-resistant PrP C was inhibited; hence, mouse PrP Sc could be amplified to infinite levels. The present study is the first report describing effective amplification of PrP Sc of the mouse-adapted scrapie prion and this improved PMCA technique will contribute to prion research that uses mice as experimental animals.

Accelerated Accumulation of Misfolded Prion Protein and Spongiform Degeneration in aDrosophilaModel of Gerstmann–Sträussler–Scheinker Syndrome

Prion diseases are CNS disorders that can occur in sporadic, infectious, and inherited forms. Although all forms of prion disease are associated with the accumulation of pathogenic conformers of the prion protein, collectively termed PrP(Sc), the mechanisms by which PrP(Sc) molecules form and cause neuronal degeneration are unknown. Using the bipartite galactosidase-4-upstream activating sequence expression system, we generated transgenic Drosophila melanogaster heterologously expressing either wild-type (WT) or mutant, disease-associated (P101L) mouse PrP molecules in cholinergic neurons. Transgenic flies expressing neuronal P101L PrP molecules exhibited severe locomotor dysfunction and premature death as larvae and adults. These striking clinical abnormalities were accompanied by age-dependent accumulation of misfolded PrP molecules, intracellular PrP aggregates, and neuronal vacuoles. In contrast, transgenic flies expressing comparable levels of WT PrP displayed no clinical, pathological, or biochemical abnormalities. These results indicate that transgenic Drosophila expressing neuronal P101L PrP specifically exhibit several hallmark features of human Gerstmann-Sträussler-Scheinker (GSS) syndrome. Because the rates of abnormal PrP accumulation and clinical progression are highly accelerated in Drosophila compared with the rates of these processes in rodents or humans, the P101L mutant may be used for future genetic and pharmacologic studies as a novel invertebrate model of GSS.

Lymphoid follicles of the ileal Peyer's patch of lambs express low levels of PrP, as demonstrated by quantitative real-time RT-PCR on microdissected tissue compartments, in situ hybridization and immunohistochemistry

The expression level of normal cellular prion protein (PrP(C)) is thought to influence the transmission of transmissible spongiform encephalopathies (TSEs) from the peripheral entry site to the site of pathological changes in the central nervous system. In many TSEs, the clinical disease is preceded by a period in which the agent accumulates in lymphoid organs, particularly in association with follicular dendritic cells of lymphoid follicles. As the probable route of entry of the TSE agent is via the gut, the expression profile of PrP was examined in well-developed gut-associated lymphoid tissue of lambs, the ileal Peyer's patch, by laser microdissection and real-time RT-PCR. Lymphoid follicles were found to have very low levels of expression, whilst highest levels were detected in the outer submucosa and the muscular layer. These findings were supported by in situ hybridization and immunohistochemistry, which showed specific labelling in nerve cells in ganglia of the submucosal (Meissner's) and myenteric (Auerbach's) plexi of the enteric nervous system. Based on the assumption that potential sites for conversion to the scrapie-related prion protein (PrP(Sc)) should display high levels of expression of PrP(C), this study suggests that the accumulation of PrP(Sc) in the lymphoid follicles of the Peyer's patch is not preceded by PrP conversion in the same tissue compartment.

PrP(d) accumulation in organs of ARQ/ARQ sheep experimentally infected with BSE by peripheral routes.

To study the pathogenesis of bovine spongiform encephalopathy infection in small ruminants, two Lacaune sheep with the AA136RR154QQ171 and one with the AA136RR154RR171 genotype for the prion protein, were inoculated with a brain homogenate from a French cattle BSE case by peripheral routes. Sheep with the ARQ/ARQ genotype are considered as susceptible to prion diseases contrary to those with the ARR/ARR genotype. The accumulation of disease-associated prion protein (PrP(d)) was analysed by biochemical and immunohistochemical methods. No PrP(d) accumulation was detected in samples from the ARR/ARR sheep 2 years post inoculation. In the two ARQ/ARQ sheep that had scrapie-like clinical symptoms, PrP(d) was found in the central, sympathetic and enteric nervous systems and in lymphoid organs. Remarkably, PrP(d) was also detected in some muscle types as well as in all peripheral nerves that had not been reported previously thus revealing a widespread distribution of BSE-associated PrP(d) in sheep tissues.

Cerebrovascular P-glycoprotein expression is decreased in Creutzfeldt–Jakob disease

The abnormal conformation and assembly of proteins in the central nervous system is increasingly thought to be a critical pathogenic mechanism in neurodegenerative disorders such as Creutzfeldt-Jakob disease (CJD) and Alzheimer's disease (AD). CJD is marked primarily by the buildup of misfolded prion protein (PrP(Sc)) in brain, whereas the accrual of beta-amyloid protein (Abeta) and tau protein are characteristic for AD. Prior studies have shown that the ATP-binding cassette transporter P-glycoprotein (P-gp) is a cellular efflux pump for Abeta, and that age-associated deficits in P-gp may be involved in the pathogenesis of Alzheimer's disease. In the present study, we investigated the relationship between P-gp and idiopathic CJD, and found that CJD, like AD, is associated with a decrease in the expression of cerebrovascular P-gp. In some instances, Abeta and PrP deposits coexist in cases of CJD, suggesting the possibility of pathogenic interactions. Since there is, to date, no evidence that PrP itself is a substrate for P-gp, we hypothesize that the age-related deficits in P-gp could promote the accumulation of PrP(Sc) either by promoting the buildup of Abeta (which could act as a seed for the aggregation of PrP(Sc)), or by overloading the ubiquitin-proteasomal catabolic system, and thereby facilitating the accumulation of PrP. Alternatively, the loss of P-gp could be a non-specific response to neurodegenerative changes in the central nervous system. In either case, dysfunction of this critical toxin-elimination pathway in CJD and AD suggests that selectively increasing cerebrovascular P-gp function could open new therapeutic pathways for the prevention and/or treatment of a number of proteopathic disorders of the central nervous system.

Immunohistochemical Features of PrPd Accumulation in Natural and Experimental Goat Transmissible Spongiform Encephalopathies

Scrapie is a transmissible spongiform encephalopathy (TSE) or prion disease, which naturally affects sheep and goats. Immunohistochemical epitope mapping of abnormal PrP accumulations (PrP(d)) in brain can help in characterizing sheep TSE sources or strains and in identifying potential bovine spongiform encephalopathy (BSE) infections of sheep. Natural and experimental TSE infections of goats were examined to determine whether the epitope mapping approach could also be applied to aid recognition of BSE infection in goats. Goats experimentally infected with the SSBP/1 or CH1641 sheep scrapie strains or with cattle BSE, together with four field cases of natural TSE in goats, were examined immunohistochemically with six different antibodies. CH1641 and SSBP/1 infections in goats, as in sheep, showed PrP(d) accumulations which were mainly intracellular. Some differences in targeting, particularly of Purkinje cells, was evident in inter-species comparisons of CH1641 and SSBP/1. PrP(d) labelling of goat BSE experimental cases showed extensive intracellular and extracellular accumulations, also similar to those in sheep BSE. Intra-neuronal PrP(d) in both goat and sheep BSE was labelled only by antibodies recognizing epitopes located C-terminally of residue His99, whereas in natural sheep TSE sources, and in sheep and goat SSBP/1, PrP(d) was also detected by antibodies to epitopes located between residues Trp93 and His99. Testing of four natural goat TSE samples showed one case in which epitope mapping characteristics and the overall patterns of PrP(d) accumulation was identical with those of experimental goat BSE. The four natural goat scrapie cases examined showed some degree of immunohistochemical phenotype variability, suggesting that multiple strains exist within the relatively small UK goat population.

Inhibition of Protease-Resistant Prion Protein Formation in a Transformed Deer Cell Line Infected with Chronic Wasting Disease

Chronic wasting disease (CWD) is an emerging transmissible spongiform encephalopathy (prion disease) of North American cervids, i.e., mule deer, white-tailed deer, and elk (wapiti). To facilitate in vitro studies of CWD, we have developed a transformed deer cell line that is persistently infected with CWD. Primary cultures derived from uninfected mule deer brain tissue were transformed by transfection with a plasmid containing the simian virus 40 genome. A transformed cell line (MDB) was exposed to microsomes prepared from the brainstem of a CWD-affected mule deer. CWD-associated, protease-resistant prion protein (PrP(CWD)) was used as an indicator of CWD infection. Although no PrP(CWD) was detected in any of these cultures after two passes, dilution cloning of cells yielded one PrP(CWD)-positive clone out of 51. This clone, designated MDB(CWD), has maintained stable PrP(CWD) production through 32 serial passes thus far. A second round of dilution cloning yielded 20 PrP(CWD)-positive subclones out of 30, one of which was designated MDB(CWD2). The MDB(CWD2) cell line was positive for fibronectin and negative for microtubule-associated protein 2 (a neuronal marker) and glial fibrillary acidic protein (an activated astrocyte marker), consistent with derivation from brain fibroblasts (e.g., meningeal fibroblasts). Two inhibitors of rodent scrapie protease-resistant PrP accumulation, pentosan polysulfate and a porphyrin compound, indium (III) meso-tetra(4-sulfonatophenyl)porphine chloride, potently blocked PrP(CWD) accumulation in MDB(CWD) cells. This demonstrates the utility of these cells in a rapid in vitro screening assay for PrP(CWD) inhibitors and suggests that these compounds have potential to be active against CWD in vivo.

Pathogenesis and prevalence of variant Creutzfeldt–Jakob disease

In the late 1980s and early 1990s, there was widespread exposure of the UK population to bovine spongiform encephalopathy (BSE)-contaminated food products, which has led to over 150 deaths from variant Creutzfeldt-Jakob disease (vCJD). Although the pathogenesis in humans is not fully understood, data from animal models and, to a lesser extent, patients with vCJD suggest that oral exposure to BSE is rapidly followed by accumulation of PrP(res) in gut-associated lymphoid tissue, then, after haematogenous spread, throughout the lymphoreticular system. Spread to the central nervous system may not occur for several years, but blood from individuals in the pre-clinical phase appears to be able to transmit disease. The incidence of vCJD has remained low and is in decline, but it is known from iatrogenic CJD and kuru that human prion disease can have incubation periods of up to 40 years. Cases of vCJD are therefore likely to occur for many more years and alternative phenotypes may develop in individuals with different PRNP genotypes to those seen to date. Studies in transgenic mice have shown that sub-clinical infection is frequent following oral exposure to BSE and a study looking at the accumulation of PrP in anonymized human lymphoid tissue samples found positive cases. There are likely to be a number of asymptomatic 'carriers' of disease within the UK and although it is unclear whether these individuals will develop clinical disease, there is a potential for iatrogenic spread to others. These uncertainties highlight the importance of developing a reliable blood test for vCJD and the continued need for surveillance.

Ovine Infection with the Agents of Scrapie (CH1641 Isolate) and Bovine Spongiform Encephalopathy: Immunochemical Similarities can be Resolved by Immunohistochemistry

Immunochemical ("rapid") tests, which recognize a partly protease-resistant conformer of the prion protein (PrP(res)) are now widely used in Europe for the diagnosis of transmissible spongiform encephalopathies (TSEs). Some of these tests can be used to distinguish natural scrapie from experimental bovine spongiform encephalopathy (BSE) in sheep, on the basis of migration pattern differences of PrP(res) in Western immunoblots. However, PrP(res) from sheep inoculated with CH1641 scrapie gives an immunoblot profile similar to that of sheep inoculated with BSE. Therefore, field scrapie strains similar to CH1641 might be misclassified as ovine BSE in the rapid tests currently employed. This study confirmed that the Western blot similarities (size of the unglycosylated band and distinct reactivity with 6H4 and P4 antibodies) between CH1641 and BSE remained consistent regardless of the PrP genotype of the sheep, but the two infections resulted in accumulation of disease-associated PrP (PrP(d)) that could easily be distinguished by the immunohistochemical "peptide mapping" method. This method, which reveals conformational differences of PrP(d) by the use of a panel of antibodies, indicated that PrP(d) from the CH1641 isolate was truncated further upstream in the N terminus than was PrP(d) from other ovine TSEs, including experimental BSE. In addition, the immunohistochemical "PrP(d) profile method", which defines the phenotype of PrP(d) accumulation in the brain of affected sheep, showed that CH1641 infection leads to much more intra-neuronal and considerably less extracellular PrP(d) than does experimental BSE. The overall results demonstrate that a combined Western blotting and immunohistochemical approach is required to discriminate between different TSE strains in sheep.

The metabolism of glycosaminoglycans is impaired in prion diseases

It is well established that the conversion of PrP(C) to PrP(Sc) is the key event in prion disease biology. In addition, several lines of evidence suggest that glycosaminoglycans (GAGs) and in particular heparan sulfate (HS) may play a role in the PrP(C) to PrP(Sc) conversion process. It has been proposed that PrP(Sc) accumulation in prion diseases may induce aberrant activation of lysosomal activity, which has been shown to result in neurodegeneration in a number of diseases, especially lysosomal storage disorders. Among such diseases, only the ones resulting from defects in GAGs degradation are accompanied by secretion of large amounts of GAG metabolites in urine. In this work, we show that GAGs are secreted in the urine of prion-infected animals and humans, and surprisingly, also in the urine of mice ablated for the PrP gene. We hypothesize that both the presence of PrP(Sc) or the absence of PrP(C) may alter the metabolism of GAGs.