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 PrP^TSE and of PrP^TSE-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 PrP^TSE was detected by Western- and postfixed frozen tissue blotting, while the seeding activity of PrP^TSE was revealed by protein misfolding cyclic amplification (PMCA). Semi-quantitative Western blotting indicated that the concentration of PrP^TSE in skeletal muscles of CWD-infected WTD was approximately 2000-10000 -fold lower than in brain tissue. Tissue-blot-analyses revealed that PrP^TSE was located in muscle-associated nerve fascicles but not, in detectable amounts, in myocytes. The presence and seeding activity of PrP^TSE 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.

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.

Fatal familial insomnia (FFI) belongs to the genetic human transmissible spongiform encephalopathies (TSE), such as genetic Creutzfeldt-Jakob disease (CJD) or Gerstmann-Straeussler-Scheinker syndrome (GSS). Here, we analyzed the properties of the pathological prion protein in six FFI cases by Western blot analysis, a protein aggregate stability assay, and aggregate deposition characteristics visualized with the paraffin-embedded tissue blot. While in all cases the unglycosylated fragment in Western blot analysis shared the same size with sporadic CJD prion type 2, the reticular/synaptic deposition pattern of the prion aggregates resembled the ones found in sporadic CJD type 1 (CJD types according to the Parchi classification from 1999). Regarding the conformational stability against denaturation with GdnHCl, FFI prion aggregates resembled CJD type 1 more than type 2. Our results suggest that the size of the proteinase-K-resistant fragments is not a valid criterion on its own. Additional criteria supplying information about conformational differences or similarities need to be taken into account. FFI may resemble a prion type with its own conformation sharing properties partly with type 1 and type 2 prions.

ARID1A is a subunit of the mammalian SWI/SNF complex, which is thought to regulate gene expression through restructuring chromatin structures. Its gene ARID1A is frequently mutated and ARID1A levels are lowered in several human cancers, especially gynecologic ones. A functional ARID1A loss may have prognostic or predictive value in terms of therapeutic strategies but has not been proposed based on a quantitative method. Hardly any literature is available on ARID1A levels in tumor samples. We developed an indirect enzyme-linked immunosorbent assay (ELISA) for ARID1A based on the current EMA and FDA criteria. We demonstrated that our ELISA provides the objective, accurate, and precise quantification of ARID1A concentrations in recombinant protein solutions, cell culture standards, and tissue lysates of tumors. A standard curve analysis yielded a ‘goodness of fit’ of R² = 0.99. Standards measured on several plates and days achieved an inter-assay accuracy of 90.26% and an inter-assay precision with a coefficient of variation of 4.53%. When tumor lysates were prepared and measured multiple times, our method had an inter-assay precision with a coefficient of variation of 11.78%. We believe that our suggested method ensures a high reproducibility and can be used for a high sample throughput to determine the ARID1A concentration in different tumor entities. The application of our ELISA on various tumor and control tissues will allow us to explore whether quantitative ARID1A measurements in tumor samples are of predictive value.

Chronic wasting disease (CWD) is an expanding prion disease of cervids. CWD prions persist in the environment, are shed in excreta, and accumulate in tissues of infected cervids, raising concerns about its zoonotic potential. Using cynomolgus macaques, we explored the zoonotic potential of CWD. Although most inoculated macaques remained asymptomatic, sensitive in vitro prion amplification assays revealed low levels of prions in macaque tissues. Inoculation of transgenic mice and bank voles with macaque tissues induced prion disease, achieving 100% transmission rates upon serial passage. One interpretation of these findings is that CWD prions retain infectivity across species and that primate infection may manifest atypically while still enabling transmission. Our results challenge earlier conclusions that minimize the zoonotic risk of CWD and underscore the need for continued surveillance.

A key process of neurodegeneration in Parkinson’s disease (PD) is the transneuronal spreading of α-synuclein. Alpha-synuclein is a presynaptic protein that is implicated in the pathogenesis of PD and other synucleinopathies, where it forms, upon intracellular aggregation, pathological inclusions. Other hallmarks of PD include neurodegeneration and microgliosis in susceptible brain regions. Whether it is primarily transneuronal spreading of α-synuclein particles, inclusion formation, or other mechanisms, such as inflammation, that cause neurodegeneration in PD is unclear. We used spreading/aggregation of α-synuclein induced by intracerebral injection of α-synuclein preformed fibrils into the mouse brain to address this question. We performed quantitative histological analysis for α-synuclein inclusions, neurodegeneration, and microgliosis in different brain regions, and a gene expression profiling of the ventral midbrain, at two different timepoints after disease induction. We observed significant neurodegeneration and microgliosis in brain regions not only with, but also without α-synuclein inclusions. We also observed prominent microgliosis in injured brain regions that did not correlate with neurodegeneration nor with inclusion load. In longitudinal gene expression profiling experiments, we observed early and unique alterations linked to microglial mediated inflammation that preceded neurodegeneration, indicating an active role of microglia in inducing neurodegeneration. Our observations indicate that α-synuclein inclusion formation is not the major driver in the early phases of PD-like neurodegeneration, but that diffusible, oligomeric α-synuclein species, which induce unusual microglial reactivity, play a key role in this process. Our findings uncover new features of α-synuclein induced pathologies, in particular microgliosis, and point to the necessity of a broader view of the process of “prion-like spreading” of that protein.