Amplified immunohistochemical detection of PrPsc in animal transmissible spongiform encephalopathies using streptomycin.

Due to its sensitivity, immunohistochemistry (IHC) of abnormal prion protein (PrPsc) is used to study experimental and natural cases of transmissible spongiform encephalopathies (TSEs) such as Creutzfeldt-Jakob disease in humans or scrapie and bovine spongiform encephalopathy (BSE) in animals. The limits of detection are particularly critical when PrPsc IHC is used for diagnostic purposes. In this article, we describe for the first time the use of streptomycin sulfate in IHC, providing a novel original and easy way to amplify specifically PrPsc immunohistochemical detection in natural cases of BSE and scrapie, as well as in experimental TSEs in mice models using two different PrP antibodies.     

An alternative pretreatment procedure in animal transmissible spongiform encephalopathies diagnosis using PrPsc immunohistochemistry.

Because of its sensitivity, immunohistochemistry (IHC) of abnormal prion protein (PrPsc) is used more often in the diagnosis of transmissible spongiform encephalopathies (TSEs), such as scrapie and bovine spongiform encephalopathy (BSE). PrPsc IHC requires a combination of pretreatments (chemical, heating, and enzymatic). The method of application may depend on the anti-prion antibody considered. If these pretreatments are efficient for diagnostic purpose, it may, however, be interesting to use an alternative method to efficiently detect PrPsc IHC immunohistochemically using chemical pretreatments solely. Here we describe such pretreatments reporting the difficulty (section adhesion) but also the potential advantages of such methods (easy, quick, inexpensive, and amplifying effect).     

The cellular prion protein: biochemistry,topology, and physiologic functions

Studies on the transmission from man to animals of Creutzfeld-Jacob disease (CJD) led Prusiner to identify a proteinaceous infectious particle lacking nucleic acid, which was called prion. The identification of the infectious prion (PrPsc) then led to the discovery of the normal cellular counterpart (PrPc). One of the still enigmatic aspects regarding prion diseases is actually how, where, and when the transformation PrPc/PrPsc is occurring, this being due to the result of a large extent to the fact that so far most studies have been dedicated to the formation and transmission of PrPsc, whereas the understanding of physiologic roles of PrPc are in their infancy. In this review, we hope to identify the most reliable hypotheses for future experiments on PrPc. This is relevant not only for the understanding of PrPc functions but also to unravel the enigmatic nature of PrPc/PrPsc conversion.     

Inoculation of Scrapie with the Self-Assembling RADA-Peptide Disrupts Prion Accumulation and Extends Hamster Survival

Intracerebral inoculation of 263K Scrapie brain homogenate (PrPsc) with a self-assembling RADA-peptide (RADA) significantly delayed disease onset and increased hamster survival. Time of survival was dependent on the dose of RADA and pre-incubation with PrPsc prior to inoculation. RADA treatment resulted in the absence of detectable PrPsc at 40 d followed by an increased rate of PrPsc accumulation at 75 d up to sacrifice. In all PrPsc inoculated animals, clinical symptoms were observed ∼10 d prior to sacrifice and brains showed spongiform degeneration with Congo red positive plaques. A time-dependent increase in reactive gliosis was observed in both groups with more GFAP detected in RADA treated animals at all time points. The PrP protein showed dose-dependent binding to RADA and this binding was competitively inhibited by Congo Red. We conclude that RADA disrupts the efficacy of prion transmission by altering the rate of PrPsc accumulation. This is the first demonstration that a self-assembling biomolecular peptide can interact with PrPsc, disrupt the course of Scrapie disease process, and extend survival.     

Immunohistochemistry of PrPsc within bovine spongiform encephalopathy brain samples with graded autolysis.

Bovine spongiform encephalopathy (BSE) is a transmissible neurodegenerative disease of cattle. Clinical diagnosis can be confirmed by investigation of both spongiform changes and abnormal prion protein (PrPsc), a marker considered specific for the disease. Tissue autolysis, often unavoidable in routine field cases, is not compatible with histological examination of the brain even though PrPsc is still detectable by immunoblotting. To determine how autolysis might affect accurate diagnosis using PrPsc immunohistochemistry, we studied 50 field samples of BSE brainstem (obex) with various degrees of autolysis. We demonstrated that the antigen-unmasking pretreatments necessary for PrPsc immunohistochemistry were compatible with the preservation of autolyzed brain sections and that PrPsc detection was unaffected by autolysis, even though anatomic markers were sometimes lost. In tissue samples in which anatomic sites were still recognizable, PrPsc accumulation was detected in specific gray matter nuclei. In samples with advanced autolysis, PrPsc deposits were still observed, at least at the cellular level, as an intraneuronal pattern. We found that the sensitivity of PrPsc immunohistochemistry as a diagnostic method for BSE was undiminished even by severe tissue autolysis.     

Size distribution dependence of prion aggregates infectivity

We consider a model for the polymerization (fragmentation) process involved in infectious prion self-replication and study both its dynamics and non-zero steady state. We address several issues. Firstly, we extend a previous study of the nucleated polymerization model [M.L. Greer, L. Pujo-Menjouet, G.F. Webb, A mathematical analysis of the dynamics of prion proliferation, J. Theoret. Biol. 242 (2006) 598; H. Engler, J. Pruss, G.F. Webb, Analysis of a model for the dynamics of prions II, J. Math. Anal. Appl. 324 (2006) 98] to take into account size dependent replicative properties of prion aggregates. This is achieved by a choice of coefficients in the model that are not constant. Secondly, we show stability results for this steady state for general coefficients where reduction to a system of differential equations is not possible. We use a duality method based on recent ideas developed for population models. These results confirm the potential influence of the amyloid precursor production rate in promoting amyloidogenic diseases. Finally, we investigate how the converting factor may depend upon the aggregate size. Besides the confirmation that size-independent parameters are unlikely to occur, the present study suggests that the PrPsc aggregate size repartition is amongst the most relevant experimental data in order to investigate this dependence. In terms of prion strain, our results indicate that the PrPsc aggregate repartition could be a constraint during the adaptation mechanism of the species barrier overcoming, that opens experimental perspectives for prion amyloid polymerization and prion strain investigation.     

Efficient transmission of two different sheep scrapie isolates in transgenic mice expressing the ovine PrP gene

We produced transgenic mice expressing the sheep prion protein to obtain a sensitive model for sheep spongiform encephalopathies (scrapie). The complete open reading frame, with alanine, arginine, and glutamine at susceptibility codons 136, 154, and 171, respectively, was inserted downstream from the neuron-specific enolase promoter. A mouse line, Tg(OvPrP4), devoid of the murine PrP gene, was obtained by crossing with PrP knockout mice. Tg(OvPrP4) mice were shown to selectively express sheep PrP in their brains, as demonstrated in mRNA and protein analysis. We showed that these mice were susceptible to infection by sheep scrapie following intracerebral inoculation with two natural sheep scrapie isolates, as demonstrated not only by the occurrence of neurological signs but also by the presence of the spongiform changes and abnormal prion protein accumulation in their brains. Mean times to death of 238 and 290 days were observed with these isolates, but the clinical course of the disease was strikingly different in the two cases. One isolate led to a very early onset of neurological signs which could last for prolonged periods before death. Independently of the incubation periods, some of the mice inoculated with this isolate showed low or undetectable levels of PrPsc, as detected by both Western blotting and immunohistochemistry. The development of experimental scrapie in these mice following inoculation of the scrapie infectious agent further confirms that neuronal expression of the PrP open reading frame alone is sufficient to mediate susceptibility to spongiform encephalopathies. More importantly, these mice provide a new and promising tool for studying the infectious agents in sheep spongiform encephalopathies.     

Detection of PrPsc on lymphoid tissues from naturally affected scrapie animals: comparison of three visualization systems.

We assessed three different visualization systems used routinely in research and diagnosis of transmissable spongiform encephalopathies (TSEs) to demonstrate whether the methodology applied to immunohistochemical (IHC) examination may alter the results concerning detection of prion protein (PrPsc) in the lymphoreticular system (LRS): avidin-biotin-peroxidase (Vectastain ABC kit; Vector), Envision (DAKO), and catalyzed signal amplification (CSA; DAKO). The study aimed to determine which of these showed the highest sensitivity, with the hope of providing an accurate tool for pathogenesis and preclinical diagnosis research in TSEs. Histological sections from palatine tonsils, spleen, GALT (ileum and ileocecal valve), and lymph nodes from sheep belonging to a Spanish scrapie-positive flock were processed by IHC using L42 as primary antibody. As substrate chromogen, diaminobenzidine was used, and all slides were subjectively assessed by light microscopy. A further study using an image analyzer software system was carried out to confirm that the conclusion provided by microscopic examination was objective. The CSA system showed the highest sensitivity in all cases, increasing both variables assessed: the number of follicles that were PrPsc-positive was detected as well as the intensity of immunostaining in each of them.     

Resistance of neonatal mice to scrapie is associated with inefficient infection of the immature spleen

Previous studies demonstrated that neonatal mice up to about a week old are less susceptible than adult mice to infection by intraperitoneal inoculation with mouse-passaged scrapie. In peripherally inoculated adult mice, scrapie replicates in lymphoid tissues such as the spleen before invading the central nervous system. Here, we investigated scrapie susceptibility in neonatal mice in more detail, concentrating on spleen involvement. First, we demonstrated that neonatal mice are about 10 times less susceptible than adults to intraperitoneal scrapie inoculation. Then we injected mice intraperitoneally with a scrapie dose that produced disease in all mice inoculated at 10 days or older but in only about a third of neonatally inoculated mice. In this experiment, spleens collected 70 days after scrapie injection of mice 10 days old or older almost all contained pathological prion protein, PrPSc, and those that were bioassayed all contained high infectivity levels. In contrast, at this early stage, only two of six spleens from neonatally inoculated mice had detectable, low infectivity levels; no PrPSc was detected, even in the two spleens. Therefore, neonatal mice have an impaired ability to replicate scrapie in their spleens, suggesting that replication sites are absent or sparse at birth but mature within 10 days. The increase in susceptibility with age correlated with the first immunocytochemical detection of the normal cellular form of prion protein, PrPc, on maturing follicular dendritic cell networks. As lymphoid tissues are more mature at birth in sheep, cattle, and humans than in mice, our results suggest that in utero infection with scrapie-like agents is theoretically possible in these species.     

Pre-symptomatic detection of prions by cyclic amplification of protein misfolding

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders affecting humans and animals. At present, it is not possible to recognize individuals incubating the disease before the clinical symptoms appear. We investigated the effectiveness of the "Protein Misfolding Cyclic Amplification" (PMCA) technology to detect the protease-resistance disease-associated prion protein (PrP(res)) in pre-symptomatic stages. PMCA allowed detection of PrP(res) in the brain of pre-symptomatic hamsters, enabling a clear identification of infected animals as early as two weeks after inoculation. Furthermore, PMCA was able to amplify minute quantities of PrP(res) from a variety of experimental and natural TSEs. Finally, PMCA allowed the demonstration of PrP(res) in an experimentally infected cow 32 month post-inoculation, that did not show clinical signs and was negative by standard Western blot analysis. Our findings indicate that PMCA may be useful for the development of an ultra-sensitive diagnostic test to minimize the risk of further propagation of TSEs.     

Prion proteins and ECTO-NOX proteins exhibit similar oscillating redox activities

Both recombinant full-length mouse prion protein expressed in Escherichia coli and native prion protein (PrPsc) from mouse brain exhibited NADH oxidase and protein disulfide-thiol interchange activities similar to those formerly thought to be properties exclusive to the growth-related, cell surface ECTO-NOX proteins. The two activities exhibited the complex 2+3 pattern of oscillations characteristic of ECTO-NOX proteins where the two activities alternate to generate a period length of 24 min. The oscillations were augmented by copper and diminished by addition of the copper chelator bathocuproene. That the activity might be attributable to a contaminating protein was ruled out by experiments where the purified recombinant prion-containing extracts were resolved by SDS-PAGE and the activity was restricted to a single band corresponding to the predicted Mr of the recombinant prion as verified by Western blot analyses.     

Prion ‘virus’ particles caused by lysosomal storage disease?

Virus-like particles are commonly found in highly infectious scrapie brain fractions and cell lines, suggesting a viral source of prion diseases. However, new evidence indicates that these particles are likely the result of mucopolysaccharidosis; a lysosomal storage disease where similar particles are observed, along with neuronal degeneration. Heparan sulfate proteoglycans (HSPGs) have long been implicated in prion diseases including demonstrated impairment of glycosaminoglycan metabolism. In this work, unusual levels of glypican-1 and heparan sulfate in cell cultures treated with prion protein antibodies are disclosed, further evidence for a lysosomal storage disorder and suggestive of a HSPG origin for prion diseases.     

Phenotyping of protein-prion (PrPsc)-accumulating cells in lymphoid and neural tissues of naturally scrapie-affected sheep by double-labeling immunohistochemistry.

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases characterized by amyloid deposition of protein-prion (PrPsc), the pathogenic isoform of the host cellular protein PrPc, in the immune and central nervous systems. In the absence of definitive data on the nature of the infectious agent, PrPsc immunohistochemistry (IHC) constitutes one of the main methodologies for pathogenesis studies of these diseases. In situ PrPsc immunolabeling requires formalin fixation and paraffin embedding of tissues, followed by post-embedding antigen retrieval steps such as formic acid and hydrated autoclaving treatments. These procedures result in poor cellular antigen preservation, precluding the phenotyping of cells involved in scrapie pathogenesis. Until now, PrPsc-positive cell phenotyping relied mainly on morphological criteria. To identify these cells under the PrPsc IHC conditions, a new, rapid, and highly sensitive PrPsc double-labeling technique was developed, using a panel of screened antibodies that allow specific labeling of most of the cell subsets and structures using paraffin-embedded lymphoid and neural tissues from sheep, leading to an accurate identification of ovine PrPsc-accumulating cells. This technique constitutes a useful tool for IHC investigation of scrapie pathogenesis and may be applicable to the study of other ovine infectious diseases.     

Protein misfolding cyclic amplification as a rapid test for assessment of prion inactivation

Abnormal isoform of prion proteins (PrP(Sc)), which are infectious agents associated with prion diseases, retain infectivity after undergoing routine sterilization processes. A sensitive method to detect the infectivity is a bioassay, and it has been used for assessing prion inactivation. However, the result is obtained after several hundred days. Here, protein misfolding cyclic amplification (PMCA) in which PrP(Sc) can be amplified in vitro was applied for assessing prion inactivation by dry heating and autoclaving. Scrapie-infected hamster brains were inactivated under various conditions, and residual infectivity and PrP(Sc) were detected by the bioassay and PMCA, respectively. The PMCA results were in good agreement with those of the bioassay. In samples autoclaved at temperatures below 150 degrees C, while infected mice died in the bioassay, protease-resistant PrP (PrP(res)) signals were detected in the second or third round of PMCA. Three rounds of PMCA require only 6 days, which means that the PMCA method is much faster than the bioassay.     

Transmissible spongiform encephalopathy diagnosis using PrPsc immunohistochemistry on fixed but previously frozen brain samples.

The histological diagnosis of transmissible spongiform encephalopathies (TSEs), such as scrapie and bovine spongiform encephalopathy (BSE), relies on identification in the brain of spongiosis, gliosis, and neuron loss without inflammatory lesions. Because of its sensitivity, immunohistochemistry of abnormal prion protein (PrPsc) is of great help in this diagnosis and can be used on its own or complementary to the biochemical detection of PrPsc. However, in some cases no formalin-fixed material is available, rendering its use as a complementary method impossible. For that purpose, we studied the possibility of detecting PrPsc immunohistochemically in fixed brain samples that had been previously frozen and used for Western blotting analysis. We compared freshly and fixed-frozen brain samples originating from the same sheep, either affected or unaffected with scrapie. We also studied fixed-frozen brain samples from scrapie-affected goats and from cows showing BSE. We showed that in all the species tested, despite damage to the histological structures, PrPsc was still detectable in the fixed-frozen brain sections without unspecific background staining. Notwithstanding the limited number of cases thus far analyzed, we have already demonstrated the possibility of using PrPsc immunohistochemistry on fixed-frozen brain samples with very good efficacy, thus rendering possible its use for diagnostic purposes in TSEs.     

Detection of PrPCWD in feces from naturally exposed Rocky Mountain elk (Cervus elaphus nelsoni) using protein misfolding cyclic amplification

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy affecting captive and free-ranging cervids. Currently, tests for CWD in live animals involve relatively invasive procedures to collect lymphoid tissue biopsies and examine them for CWD-associated, protease-resistant cervid prion protein (PrP(CWD)) detected by immunohistochemistry (IHC). We adapted an ultrasensitive prion detection system, protein misfolding cyclic amplification (PMCA), to detect PrP(CWD) in Rocky Mountain elk (Cervus elaphus nelsoni) feces. Our PMCA reproducibly detected a 1.2 × 10(7) dilution of PrP(CWD) (a 10% infected brain homogenate diluted 1.2 × 10(6)-fold into 10% fecal homogenates), equivalent to approximately 100 pg of PrP(CWD)/g of feces. We developed a semiquantitative scoring system based on the first PMCA round at which PrP(CWD) was detected and fit a nonlinear regression curve to our serial dilutions to correlate PMCA scores with known PrP(CWD) concentrations. We used this PMCA scoring system to detect PrP(CWD) and estimate its concentration in feces from free-ranging elk from Rocky Mountain National Park, Colorado. We compared our results to PrP(CWD) IHC of rectoanal mucosa-associated lymphoid tissue and obex from the same animals. The PMCA successfully detected PrP(CWD) in feces from elk that were positive by IHC, with estimated prion loads from 100 to 5,000 pg PrP(CWD)/g of feces. These data show for the first time PrP(CWD) in feces from naturally exposed free-ranging elk and demonstrate the potential of PMCA as a new, noninvasive CWD diagnostic tool to complement IHC.     

High frequency occurrence of 1-OPRD variant of PRNP gene in gastric cancer cell lines and Chinese population with gastric cancer

The prion protein gene PRNP encodes PrPc and PrPsc, causing a number of neurological disorders. Approximately 10-15% of human prion disease is inherited and more than 20 pathogenic mutations have been found. Most of the genetic alterations are point mutations, with the exception of genetic insertions of one to nine extra octapeptide repeats occurring in the important octapeptide-coding region. Our previous work showed that PrPc was overexpressed in gastric cancer. We wondered whether mutations of PrPc existed in human gastric cancer. DNA sequencing and gel electrophoresis were used to determine the possible mutation of PrPc in patients and cell lines of gastric cancer. We found that 1-OPRD (one octapeptide-repeat deletion) homozygosity or heterozygosity exists in several gastric cancer cell lines, e.g. MKN28 and KatoIII are homozygous for 1-OPRD, and SGC7901 and BGC-823 are heterozygous for 1-OPRD. The mutation frequency in tissues of gastric cancer cases is significantly higher than that in the common population (p<0.05). All positive cases in gastric cancer were found to be heterozygous for 1-OPRD. Further study of the variant may be helpful in understanding the mechanisms of occurrence and development of clinical gastric carcinoma as well as the biology of the mysterious gene PRNP.     

Prion-Seeding Activity in Cerebrospinal Fluid of Deer with Chronic Wasting Disease

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are a uniformly fatal family of neurodegenerative diseases in mammals that includes chronic wasting disease (CWD) of cervids. The early and ante-mortem identification of TSE-infected individuals using conventional western blotting or immunohistochemistry (IHC) has proven difficult, as the levels of infectious prions in readily obtainable samples, including blood and bodily fluids, are typically beyond the limits of detection. The development of amplification-based seeding assays has been instrumental in the detection of low levels of infectious prions in clinical samples. In the present study, we evaluated the cerebrospinal fluid (CSF) of CWD-exposed (n=44) and naïve (n=4) deer (n=48 total) for CWD prions (PrP^d) using two amplification assays: serial protein misfolding cyclic amplification with polytetrafluoroethylene beads (sPMCAb) and real-time quaking induced conversion (RT-QuIC) employing a truncated Syrian hamster recombinant protein substrate. Samples were evaluated blindly in parallel with appropriate positive and negative controls. Results from amplification assays were compared to one another and to obex immunohistochemistry, and were correlated to available clinical histories including CWD inoculum source (e.g. saliva, blood), genotype, survival period, and duration of clinical signs. We found that both sPMCAb and RT-QuIC were capable of amplifying CWD prions from cervid CSF, and results correlated well with one another. Prion seeding activity in either assay was observed in approximately 50% of deer with PrP^d detected by IHC in the obex region of the brain. Important predictors of amplification included duration of clinical signs and time of first tonsil biopsy positive results, and ultimately the levels of PrP^d identified in the obex by IHC. Based on our findings, we expect that both sPMCAb and RT-QuIC may prove to be useful detection assays for the detection of prions in CSF.     

Involvement of astrocytes in transmissible spongiform encephalopathies: a confocal microscopy study

Astroglial proliferation associated with pathological prion protein (PrPsc) deposition is widely described in Transmissible Spongiform Encephalopathies (TSEs). However, little is known of the actual role played by glia in their pathogenesis. The aim of the study has been to determine whether PrPsc is located exclusively in neurons or in both neurons and glial cells present in the central nervous system in a natural Scrapie model. Samples of cerebellum from 25 Scrapie sheep from various flocks were sectioned. Following epitope retrieval with formic acid, proteinase K and heat treatment, primary antibody L42 and primary antibodies against glial fibrillary acidic protein were applied as prion- and astrocytic-specific markers, respectively. For visualization, a suitable mixture of fluorochrome-conjugated secondary antibodies was used. Relevant controls were processed in the same manner. As determined by confocal microscopy, PrPsc deposits co-localized with glial cells in all samples. Our results suggest that these cells can sustain active prion propagation, in agreement with similar findings from other studies of primary cell cultures and inoculated mice. Furthermore, despite ongoing debate regarding whether varied TSE sources show differences in their tropism for different cell lineages in the brains of affected animals, no differences in co-localization results were seen.     

Selective and efficient immunoprecipitation of the disease-associated form of the prion protein can be mediated by nonspecific interactions between monoclonal antibodies and scrapie-associated fibrils

Transmissible spongiform encephalopathies are characterized by the accumulation in brain tissues of an abnormal isoform of the prion protein named PrPsc, which is the only direct marker known for transmissible spongiform encephalopathies. Here we show that PrPsc can be specifically immunoprecipitated by using several monoclonal antibodies (mAbs) of various specificities independently of the properties of their binding site (paratope). These results strongly suggest that a significant proportion of mAbs can interact with PrPsc aggregates through nonspecific paratope-independent interactions allowing selective immunoprecipitation of PrPsc when these mAbs are immobilized on a polydisperse solid phase like microbeads.