Wiebke M. Wemheuer

Similarities between Forms of Sheep Scrapie and Creutzfeldt-Jakob Disease Are Encoded by Distinct Prion Types

Transmissible spongiform encephalopathies such as scrapie in sheep, Creutzfeldt-Jakob disease (CJD) in humans, and bovine sporadic encephalopathy in cattle are characterized by the accumulation of a misfolded protein: the pathological prion protein. Ever since bovine sporadic encephalopathy was discovered as the likely cause of the new variant of CJD in humans, parallels between human and animal transmissible spongiform encephalopathies must be viewed under the aspect of a disease risk for humans. In our study we have compared prion characteristics of different forms of sheep scrapie with those of different phenotypes of sporadic CJD. The disease characteristics of sporadic CJD depend considerably on the prion type 1 or 2. Our results show that there are obvious parallels between sporadic CJD type 1 and the so-called atypical/Nor98 scrapie. These parelleles apply to the deposition form of pathological prion protein in the brain, detected by the paraffin-embedded-tissue blot and the prion aggregate stability with regard to denaturation by the chaotropic salt guanidine hydrochloride. The same applies to sporadic CJD type 2 and classical scrapie. The observed parallels between types of sporadic CJD and types of sheep scrapie demonstrate that distinct groups of prion disease exist in different species. This should be taken into consideration when discussing interspecies transmission.

Presence and Seeding Activity of Pathological Prion Protein (PrPTSE) in Skeletal Muscles of White-Tailed Deer Infected with Chronic Wasting Disease

Chronic wasting disease (CWD) is a contagious, rapidly spreading transmissible spongiform encephalopathy (TSE), or prion disease, occurring in cervids such as white tailed-deer (WTD), mule deer or elk in North America. Despite efficient horizontal transmission of CWD among cervids natural transmission of the disease to other species has not yet been observed. Here, we report for the first time a direct biochemical demonstration of pathological prion protein PrPTSE and of PrPTSE-associated seeding activity, the static and dynamic biochemical markers for biological prion infectivity, respectively, in skeletal muscles of CWD-infected cervids, i. e. WTD for which no clinical signs of CWD had been recognized. The presence of PrPTSE was detected by Western- and postfixed frozen tissue blotting, while the seeding activity of PrPTSE was revealed by protein misfolding cyclic amplification (PMCA). Semi-quantitative Western blotting indicated that the concentration of PrPTSE in skeletal muscles of CWD-infected WTD was approximately 2000-10000 -fold lower than in brain tissue. Tissue-blot-analyses revealed that PrPTSE was located in muscle-associated nerve fascicles but not, in detectable amounts, in myocytes. The presence and seeding activity of PrPTSE in skeletal muscle from CWD-infected cervids suggests prevention of such tissue in the human diet as a precautionary measure for food safety, pending on further clarification of whether CWD may be transmissible to humans.

Sporadic Creutzfeldt-Jakob disease subtype-specific alterations of the brain proteome: impact on Rab3a recycling.

Sporadic Creutzfeldt–Jakob disease (sCJD) is characterized by wide clinical and pathological variability, which is mainly influenced by the conformation of the misfolded prion protein, and by the methionine and valine polymorphism at codon 129 of the prion protein gene. This heterogeneity likely implies differences in the molecular cascade that leads to the development of certain disease phenotypes. In this study, we investigated the proteome of the frontal cortex of patients with the two most common sCJD subtypes (MM1 and VV2) using 2D‐DIGE and MS. Analysis of 2D maps revealed that 46 proteins are differentially expressed in the sCJD. Common differential expression was detected for seven proteins, four showed opposite direction of differential expression, and the remaining ones displayed subtype‐specific alteration. The highest number of differentially expressed proteins was associated with signal transduction and neuronal activity. Moreover, functional groups of proteins involved in cell cycle and death, as well as in structure and motility included subtype‐specific expressed proteins exclusively. The expression of Rab GDP dissociation inhibitor alpha, which regulates Rab3a‐mediated neurotransmitter release, was affected in both sCJD subtypes that were analyzed. Therefore, we also investigated as to whether Rab3a recycling is altered. Indeed, we found an accumulation of the membrane‐associated form, thus the active one, which suggests that dysfunction of the Rab3a‐mediated exocytosis might be implicated in sCJD pathology.

Detection of classical and atypical/Nor98 scrapie by the paraffin-embedded tissue blot method

The paraffin-embedded tissue (PET) blot method was used to investigate sections of the central nervous system and lymphatic tissues from 24 cases of classical scrapie and 25 cases of atypical/Nor98 scrapie in sheep and four healthy control sheep. The PET blot detected deposits of PrPSc in the brain tissue of all 49 sheep with scrapie but no PrPSc labelling could be detected in the control sheep. By contrast, not all the atypical/Nor98 scrapie cases were detectable by immunohistochemistry. The high sensitivity of the PET blot method made it possible to observe that in some atypical/Nor98 cases, deposits of PrPSc may be restricted to supratentorial brain structures and that the diagnosis may be missed when only testing the obex area, where deposits are common in classical scrapie, and the cerebellar structures, where deposits are considered to be common in atypical/Nor98 cases.

Detergents modify proteinase K resistance of PrPSc in different transmissible spongiform encephalopathies (TSEs)

Prion diseases are diagnosed by the detection of their proteinase K-resistant prion protein fragment (PrP(Sc)). Various biochemical protocols use different detergents for the tissue preparation. We found that the resistance of PrP(Sc) against proteinase K may vary strongly with the detergent used. In our study, we investigated the influence of the most commonly used detergents on eight different TSE agents derived from different species and distinct prion disease forms. For a high throughput we used a membrane adsorption assay to detect small amounts of prion aggregates, as well as Western blotting. Tissue lysates were prepared using DOC, SLS, SDS or Triton X-100 in different concentrations and these were digested with various amounts of proteinase K. Detergents are able to enhance or diminish the detectability of PrP(Sc) after proteinase K digestion. Depending on the kind of detergent, its concentration - but also on the host species that developed the TSE and the disease form or prion type - the detectability of PrP(Sc) can be very different. The results obtained here may be helpful during the development or improvement of a PrP(Sc) detection method and they point towards a detergent effect that can be additionally used for decontamination purposes. A plausible explanation for the detergent effects described in this article could be an interaction with the lipids associated with PrP(Sc) that may stabilize the aggregates.

PrPSc spreading patterns in the brain of sheep linked to different prion types.

Scrapie in sheep and goats has been known for more than 250 years and belongs nowadays to the so-called prion diseases that also include e.g. bovine spongiform encephalopathy in cattle (BSE) and Creutzfeldt-Jakob disease in humans. According to the prion hypothesis, the pathological isoform (PrPSc) of the cellular prion protein (PrPc) comprises the essential, if not exclusive, component of the transmissible agent. Currently, two types of scrapie disease are known - classical and atypical/Nor98 scrapie. In the present study we examine 24 cases of classical and 25 cases of atypical/Nor98 scrapie with the sensitive PET blot method and validate the results with conventional immunohistochemistry. The sequential detection of PrPSc aggregates in the CNS of classical scrapie sheep implies that after neuroinvasion a spread from spinal cord and obex to the cerebellum, diencephalon and frontal cortex via the rostral brainstem takes place. We categorize the spread of PrPSc into four stages: the CNS entry stage, the brainstem stage, the cruciate sulcus stage and finally the basal ganglia stage. Such a sequential development of PrPSc was not detectable upon analysis of the present atypical/Nor98 scrapie cases. PrPSc distribution in one case of atypical/Nor98 scrapie in a presumably early disease phase suggests that the spread of PrPSc aggregates starts in the di- or telencephalon. In addition to the spontaneous generation of PrPSc, an uptake of the infectious agent into the brain, that bypasses the brainstem and starts its accumulation in the thalamus, needs to be taken into consideration for atypical/Nor98 scrapie.

Detection of Mycobacterium avium ssp. paratuberculosis in ileocaecal lymph nodes collected from elderly slaughter cows using a semi-nested IS900 polymerase chain reaction

The aim of this study was to investigate the occurrence of subclinical Mycobacterium avium spp. paratuberculosis (MAP) infections at slaughter by testing ileocaecal lymph nodes with a semi-nested IS900 PCR. Tissue samples were available within the framework of a parallel study investigating BSE-susceptibility factors in members of BSE-cohorts in the German Federal State of Lower Saxony. Ileocaecal lymph nodes were collected over a 2-year sampling period from 99 slaughter cattle of a mean age of 6.5 years (5.5-7.5 years). A recently developed IS900 semi-nested polymerase chain reaction (snPCR) assay offering a sensitivity of 1 genome equivalent was used for the detection of MAP-DNA. Based on this snPCR, 17 out of the 99 samples gave positive results, indicating a MAP occurrence of 17.17% in the random sample. All PCR products were sequenced for screening of polymorphisms. Nucleotide homologies of 98.5-100% were found with respect to the MAP K10 reference sequence IS900 (GenBank: AE16958). PCR analysis of ileocaecal lymph nodes collected from slaughter cattle proved to be a suitable technique to determine MAP occurrence in the local cattle population.

Filtration of Protein Aggregates Increases the Accuracy for Diagnosing Prion Diseases in Brain Biopsies

In brain biopsies taken from patients with rapidly progressive dementia, the first differential diagnoses to be ruled out are prion diseases. For safe diagnostic processing of tissue and instruments, a rapid, highly sensitive, and specific analysis for prion aggregates is necessary. Here, we examined 16 brain biopsies and brain samples (frontal cortex and cerebellum) from 65 autopsies by Western blot, paraffin-embedded tissue (PET) blot, immunohistochemistry, and the recently described membrane adsorption assay (MAA) for their suitability to detect pathologic prion protein. In our hands, the PET blot method provided the highest sensitivity in prion detection (biopsies, 100%; all autopsy sections, 96.3%), closely followed by the MAA (biopsies, 100%; all autopsy samples, 96%) and Western blot analysis (biopsies, 100%; all autopsy samples, 92%). Conventional immunohistochemistry is the least sensitive method (biopsies, 50%; all autopsy sections, 80%) and also gave 1 false-positive biopsy result. Consequently, our standard diagnostic protocol is to use the MAA as a first step for detecting or excluding a prion disease, followed by the PET blot for the prion deposition pattern, Western blotting for prion typing, and immunohistochemistry for differential diagnoses. With this standard and the availability of unfixed tissue, a diagnosis was possible in all 16 biopsies examined.

Subtype-Specific Synaptic Proteome Alterations in Sporadic Creutzfeldt-Jakob Disease

Sporadic Creutzfeldt-Jakob disease (sCJD) is characterized by wide clinical and pathological variability, which is mainly influenced by the conformation of the misfolded prion protein (PrPSc) and by methionine and valine polymorphism at codon 129 of the gene encoding PrP. This heterogeneity likely implies differences in the molecular cascades that lead to the development of certain disease phenotypes. Here, we investigated synaptic proteome patterns in two most common sCJD subtypes (MM1 and VV2) using 2D DIGE and mass spectrometry. We found that 23 distinct proteins were differentially expressed in at least one sCJD subtype when compared to age-matched controls. The majority of these proteins displayed significant subtype-specific alterations, with only up-regulated glial fibrillary acidic protein and down-regulated spectrin alpha chain in both sCJD subtypes. Differentially expressed proteins found in this study are mainly involved in synaptic structure and activity, mitochondrial function, or calcium metabolism. Moreover, several of them have been already linked to the pathophysiological processes occurring in Alzheimers disease.

Morgagnian cataract resulting from a naturally occurring nonsense mutation elucidates a role of CPAMD8 in mammalian lens development

To investigate the genetic basis of hereditary lens opacities we analyzed 31 cases of bilateral congenital cataract in Red Holstein Friesian cattle. A genome-wide association study revealed a significant association on bovine chromosome 7 at positions 6,166,179 and 12,429,691. Whole genome re-sequencing of one case and four relatives showed a nonsense mutation (g.5995966C>T) in the PZP-like, alpha-2-macroglobulin domain containing 8 (CPAMD8) gene leading to a premature stop codon (CPAMD8 p.Gln74*) associated with cataract development in cattle. With immunohistochemistry we confirmed a physiological expression of CPAMD8 in the ciliary body epithelium of the eye in unaffected cattle, while the protein was not detectable in the ciliary body of cattle with cataracts. RNA expression of CPAMD8 was detected in healthy adult, fetal and cataractous lenses.