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Chronic wasting disease (CWD) is a cervid prion disease with unknown zoonotic potential that might pose a risk to humans who are exposed. To assess the potential of CWD to infect human neural tissue, we used human cerebral organoids with 2 different prion genotypes, 1 of which has previously been associated with susceptibility to zoonotic prion disease. We exposed organoids from both genotypes to high concentrations of CWD inocula from 3 different sources for 7 days, then screened for infection periodically for up to 180 days. No de novo CWD propagation or deposition of protease-resistant forms of human prions was evident in CWD-exposed organoids. Some persistence of the original inoculum was detected, which was equivalent in prion gene knockout organoids and thus not attributable to human prion propagation. Overall, the unsuccessful propagation of CWD in cerebral organoids supports a strong species barrier to transmission of CWD prions to humans.

Abstract Background Sporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease, is a fatal neurodegenerative disease with currently no treatment options. Stem cell therapy for neurodegenerative diseases is emerging as a possible treatment option. However, while there are a few clinical trials for other neurodegenerative disorders such as Parkinson’s disease, prion disease cell therapy research has so far been confined to animal models. Methods Here, we use a novel approach to study cell therapies in sCJD using a human cerebral organoid model. Cerebral organoids can be infected with sCJD prions allowing us to assess how neural precursor cell (NPC) therapy impacts the progression of sCJD. After 90 days of sCJD or mock infection, organoids were either seeded with NPCs or left unseeded and monitored for cellular composition changes, prion infection parameters and neuroelectrophysiological function at 180 days post-infection. Results Our results showed NPCs integrated into organoids leading to an increase in neuronal markers and changes in cell signaling irrespective of sCJD infection. Although a small, but significant, decrease in protease-resistant PrP deposition was observed in the CJD-infected organoids that received the NPCs, other disease-associated parameters showed minimal changes. However, the NPCs had a beneficial impact on organoid function following infection. sCJD infection caused reduction in neuronal spike rate and mean burst spike rate, indicative of reduced action potentials. NPC seeding restored these electrophysiological parameters to the uninfected control level. Conclusions Together with the previous animal studies, our results support that cell therapy may have some functional benefit for the treatment of human prion diseases.

Prions or prion-like aggregates such as those composed of PrP, α-synuclein, and tau are key features of proteinopathies such as prion, Parkinson's and Alzheimer's diseases, respectively. Their presence on solid surfaces may be biohazardous under some circumstances. PrP prions bound to solids are detectable by ultrasensitive real-time quaking-induced conversion (RT-QuIC) assays if the solids can be immersed in assay wells or the prions transferred to pads. Here we show that prion-like seeds can remain detectable on steel wires for at least a year, or even after enzymatic cleaning and sterilization. We also show that contamination of larger objects with pathological seeds of α-synuclein, tau, and PrP can be detected by simply assaying a sampling medium that has been transiently applied to the surface. Human α-synuclein seeds in dementia with Lewy bodies brain tissue were detected by α-synuclein RT-QuIC after drying of tissue dilutions with concentrations as low as 10-6 onto stainless steel. Tau RT-QuIC detected tau seeding activity on steel exposed to Alzheimer's disease brain tissue diluted as much as a billion fold. Prion RT-QuIC assays detected seeding activity on plates exposed to brain dilutions as extreme as 10-5-10-8 from prion-affected humans, sheep, cattle and cervids. Sampling medium collected from surgical instruments used in necropsies of sporadic Creutzfeldt-Jakob disease-infected transgenic mice was positive down to 10-6 dilution. Sensitivity for prion detection was not sacrificed by omitting the recombinant PrP substrate from the sampling medium during its application to a surface and subsequent storage as long as the substrate was added prior to performing the assay reaction. Our findings demonstrate practical prototypic surface RT-QuIC protocols for the highly sensitive detection of pathologic seeds of α-synuclein, tau, and PrP on solid objects.

Prion diseases are transmissible, fatal neurologic diseases of mammals caused by the accumulation of mis-folded, disease associated prion protein (PrPd). Creutzfeldt-Jakob Disease (CJD) is the most common human prion disease and can occur by sporadic onset (sCJD) (~85% of CJD cases), genetic mutations in the prion protein gene (10-15%) or iatrogenic transmission (rare). PrPd is difficult to inactivate and many methods to reduce prion infectivity are dangerous, caustic, expensive, or impractical. Identifying viable and safe methods for decontamination of CJD exposed materials is critically important for medical facilities and research institutions. Previous research has shown that concentrated sodium hypochlorite (bleach) was effective at inactivation of CJD prions derived from brains of mice or guinea pigs. Unfortunately, human prions adapted to rodents may mis-fold differently than in humans, and the rodent adapted prions may not have the same resistance or susceptibility to inactivation present in bona fide CJD prions. To confirm that bleach was efficacious against human sourced CJD prions, we exposed different subtypes of sCJD-infected human brain homogenates to different concentrations of bleach for increasing exposure times. Initial and residual prion seeding activity following inactivation were measured using Real-Time Quaking Induced Conversion. In addition, we tested how passage of human sCJD into either transgenic mice that expressed human prion protein, or transmission of CJD to human cerebral organoids (CO), two common laboratory practices, may affect CJD prions' susceptibility to bleach inactivation. Our results show that bleach is effective against human sourced sCJD prions, and both treatment time and concentration of bleach were important factors for CJD inactivation. CJD derived from human brains, transgenic mouse brains or CO were all susceptible to inactivation with as low as a 10 percent bleach solution with a 30-minute exposure time or a 50 percent bleach solution with as little as a 1-minute exposure time.

The PINK1/Parkin pathway of mitophagy has been implicated in the pathogenesis of Parkinson's disease. In prion diseases, a transmissible neurodegenerative disease caused by the misfolded and infectious prion protein (PrPSc), expression of both PINK1 and Parkin are elevated, suggesting that PINK1/Parkin mediated mitophagy may also play a role in prion pathogenesis. Using mice in which expression of either PINK1 (PINK1KO) or Parkin (ParkinKO) has been ablated, we analyzed the potential role of PINK1 and Parkin in prion pathogenesis. Prion infected PINK1KO and ParkinKO mice succumbed to disease more rapidly (153 and 150 days, respectively) than wild-type control C57Bl/6 mice (161 days). Faster incubation times in PINK1KO and ParkinKO mice did not correlate with altered prion pathology in the brain, altered expression of proteins associated with mitochondrial dynamics, or prion-related changes in mitochondrial respiration. However, the expression level of mitochondrial respiration Complex I, a major site for the formation of reactive oxygen species (ROS), was higher in prion infected PINK1KO and ParkinKO mice when compared to prion infected control mice. Our results demonstrate a protective role for PINK1/Parkin mitophagy during prion disease, likely by helping to minimize ROS formation via Complex I, leading to slower prion disease progression.

Abstract Human cerebral organoids (COs) are three-dimensional self-organizing cultures of cerebral brain tissue differentiated from induced pluripotent stem cells. We have recently shown that COs are susceptible to infection with different subtypes of Creutzfeldt–Jakob disease (CJD) prions, which in humans cause different manifestations of the disease. The ability to study live human brain tissue infected with different CJD subtypes opens a wide array of possibilities from differentiating mechanisms of cell death and identifying neuronal selective vulnerabilities to testing therapeutics. However, the question remained as to whether the prions generated in the CO model truly represent those in the infecting inoculum. Mouse models expressing human prion protein are commonly used to characterize human prion disease as they reproduce many of the molecular and clinical phenotypes associated with CJD subtypes. We therefore inoculated these mice with COs that had been infected with two CJD subtypes (MV1 and MV2) to see if the original subtype characteristics (referred to as strains once transmitted into a model organism) of the infecting prions were maintained in the COs when compared with the original human brain inocula. We found that disease characteristics caused by the molecular subtype of the disease associated prion protein were similar in mice inoculated with either CO derived material or human brain material, demonstrating that the disease associated prions generated in COs shared strain characteristics with those in humans. As the first and only in vitro model of human neurodegenerative disease that can faithfully reproduce different subtypes of prion disease, these findings support the use of the CO model for investigating human prion diseases and their subtypes.

Abstract Chronic wasting disease (CWD) is a prion disease of cervids including deer, elk, reindeer, and moose. Human consumption of cervids is common, therefore assessing the risk potential of CWD transmission to humans is critical. In a previous study, we tested CWD transmission via intracerebral inoculation into transgenic mice (tg66 and tgRM) that over-expressed human prion protein. Mice screened by traditional prion detection assays were negative. However, in a group of 88 mice screened by the ultrasensitive RT-QuIC assay, we identified 4 tg66 mice that produced inconsistent positive RT-QuIC reactions. These data could be false positive reactions, residual input inoculum or indicative of subclinical infections suggestive of cross species transmission of CWD to humans. Additional experiments were required to understand the nature of the prion seeding activity in this model. In this manuscript, second passage experiments using brains from mice with weak prion seeding activity showed they were not infectious to additional recipient tg66 mice. Clearance experiments showed that input CWD prion seeding activity was eliminated by 180 days in tg66 mice and PrPKO mice, which are unable to replicate prion protein, indicating that the weak positive levels of seeding activity detected at later time points was not likely residual inoculum. The failure of CWD prions to cause disease in tg66 after two sequential passages suggested that a strong species barrier prevented CWD infection of mice expressing human prion protein.

The authors report the near-atomic structure of the ex vivo aRML prion fibril. The comparison between this structure and the previously solved 263K prion fibril shows that both common and divergent features characterize these prion strains and their respective conformational replication templates.

Prion diseases are transmissible, fatal neurologic diseases that include Creutzfeldt-Jakob Disease (CJD) in humans, chronic wasting disease (CWD) in cervids, bovine spongiform encephalopathy (BSE) in cattle and scrapie in sheep. Prions are extremely difficult to inactivate and established methods to reduce prion infectivity are often dangerous, caustic, expensive, or impractical. Identifying viable and safe methods for treating prion contaminated materials is important for hospitals, research facilities, biologists, hunters, and meat-processors. For three decades, some prion researchers have used a phenolic product called Environ LpH (eLpH) to inactivate prions. ELpH has been discontinued, but a similar product, Wex-cide 128, containing the similar phenolic chemicals as eLpH is now available. In the current study, we directly compared the anti-prion efficacy of eLpH and Wex-cide 128 against prions from four different species (hamster 263K, cervid CWD, mouse 22L and human CJD). Decontamination was performed on either prion infected brain homogenates or prion contaminated steel wires and mouse bioassay was used to quantify the remaining prion infectivity. Our data show that both eLpH and Wex-cide 128 removed 4.0-5.5 logs of prion infectivity from 22L, CWD and 263K prion homogenates, but only about 1.25-1.50 logs of prion infectivity from human sporadic CJD. Wex-cide 128 is a viable substitute for inactivation of most prions from most species, but the resistance of CJD to phenolic inactivation is a concern and emphasizes the fact that inactivation methods should be confirmed for each target prion strain.

Abstract Background Past experiments studying innate immunity in the central nervous system (CNS) utilized microglia obtained from neonatal mouse brain, which differ developmentally from adult microglia. These differences might impact our current understanding of the role of microglia in CNS development, function, and disease. Methods Cytokine protein secretion was compared in ex vivo P3 and adult microglial cultures after exposure to agonists for three different toll-like receptors (TLR4, lipopolysaccharide [LPS]; TLR7, imiquimod [IMQ]; and TLR9, CpG Oligodeoxynucleotide [CpG-ODN] 1585). In addition, changes in inflammatory gene expression in ex vivo adult microglia in response to the TLR agonists was assessed. Furthermore, in vivo experiments evaluated changes in gene expression associated with inflammation and TLR signaling in brains of mice with or without treatment with PLX5622 to reduce microglia. Results Ex vivo adult and P3 microglia increased cytokine secretion when exposed to TLR4 agonist LPS and to TLR7 agonist IMQ. However, adult microglia decreased expression of numerous genes after exposure to TLR 9 agonist CpG-ODN 1585. In contrast, in vivo studies indicated a core group of inflammatory and TLR signaling genes increased when each of the TLR agonists was introduced into the CNS. Reducing microglia in the brain led to decreased expression of various inflammatory and TLR signaling genes. Mice with reduced microglia showed extreme impairment in upregulation of genes after exposure to TLR7 agonist IMQ. Conclusions Cultured adult microglia were more reactive than P3 microglia to LPS or IMQ exposure. In vivo results indicated microglial influences on neuroinflammation were agonist specific, with responses to TLR7 agonist IMQ more dysregulated in mice with reduced microglia. Thus, TLR7-mediated innate immune responses in the CNS appeared more dependent on the presence of microglia. Furthermore, partial responses to TLR4 and TLR9 agonists in mice with reduced microglia suggested other cell types in the CNS can compensate for their absence.

Prion strains in a given type of mammalian host are distinguished by differences in clinical presentation, neuropathological lesions, survival time, and characteristics of the infecting prion protein (PrP) assemblies. Near-atomic structures of prions from two host species with different PrP sequences have been determined but comparisons of distinct prion strains of the same amino acid sequence are needed to identify purely conformational determinants of prion strain characteristics. Here we report a 3.2 Å resolution cryogenic electron microscopy-based structure of the 22L prion strain purified from the brains of mice engineered to express only PrP lacking glycophosphatidylinositol anchors [anchorless (a) 22L]. Comparison of this near-atomic structure to our recently determined structure of the aRML strain propagated in the same inbred mouse reveals that these two mouse prion strains have distinct conformational templates for growth via incorporation of PrP molecules of the same sequence. Both a22L and aRML are assembled as stacks of PrP molecules forming parallel in-register intermolecular β-sheets and intervening loops, with single monomers spanning the ordered fibril core. Each monomer shares an N-terminal steric zipper, three major arches, and an overall V-shape, but the details of these and other conformational features differ markedly. Thus, variations in shared conformational motifs within a parallel in-register β-stack fibril architecture provide a structural basis for prion strain differentiation within a single host genotype.

Abstract Efforts to contain the spread of chronic wasting disease (CWD), a fatal, contagious prion disease of cervids, would be aided by the availability of additional diagnostic tools. RT-QuIC assays allow ultrasensitive detection of prion seeds in a wide variety of cervid tissues, fluids and excreta. The best documented antemortem diagnostic test involving RT-QuIC analysis targets lymphoid tissue in rectal biopsies. Here we have tested a more easily accessed specimen, ear pinna punches, using an improved RT-QuIC assay involving iron oxide magnetic extraction to detect CWD infections in asymptomatic mule and white-tailed deer. Comparison of multiple parts of the ear pinna indicated that a central punch spanning the auricular nerve provided the most consistent detection of CWD infection. When compared to results obtained from gold-standard retropharyngeal lymph node specimens, our RT-QuIC analyses of ear samples provided apparent diagnostic sensitivity (81%) and specificity (91%) that rivaled, or improved upon, those observed in previous analyses of rectal biopsies using RT-QuIC. These results provide evidence that RT-QuIC analysis of ear pinna punches may be a useful approach to detecting CWD infections in cervids.

Abstract Creutzfeldt–Jakob Disease (CJD) is a fatal, currently incurable, neurodegenerative disease. The search for candidate treatments would be greatly facilitated by the availability of human cell-based models of prion disease. Recently, an induced pluripotent stem cell derived human cerebral organoid model was shown to take up and propagate human CJD prions. This model offers new opportunities to screen drug candidates for the treatment of human prion diseases in an entirely human genetic background. Here we provide the first evidence that human cerebral organoids can be a viable model for CJD drug screening by using an established anti-prion compound, pentosan polysulfate (PPS). PPS delayed prion propagation in a prophylactic-like treatment paradigm and also alleviated propagation when applied following establishment of infection in a therapeutic-like treatment paradigm. This study demonstrates the utility of cerebral organoids as the first human 3D cell culture system for screening therapeutic drug candidates for human prion diseases.

Abstract Accumulation of misfolded host proteins is central to neuropathogenesis of numerous human brain diseases including prion and prion-like diseases. Neurons of retina are also affected by these diseases. Previously, our group and others found that prion-induced retinal damage to photoreceptor cells in mice and humans resembled pathology of human retinitis pigmentosa caused by mutations in retinal proteins. Here, using confocal, epifluorescent and electron microscopy we followed deposition of disease-associated prion protein (PrPSc) and its association with damage to critical retinal structures following intracerebral prion inoculation. The earliest time and place of retinal PrPSc deposition was 67 days post-inoculation (dpi) on the inner segment (IS) of cone photoreceptors. At 104 and 118 dpi, PrPSc was associated with the base of cilia and swollen cone inner segments, suggesting ciliopathy as a pathogenic mechanism. By 118 dpi, PrPSc was deposited in both rods and cones which showed rootlet damage in the IS, and photoreceptor cell death was indicated by thinning of the outer nuclear layer. In the outer plexiform layer (OPL) in uninfected mice, normal host PrP (PrPC) was mainly associated with cone bipolar cell processes, but in infected mice, at 118 dpi, PrPSc was detected on cone and rod bipolar cell dendrites extending into ribbon synapses. Loss of ribbon synapses in cone pedicles and rod spherules in the OPL was observed to precede destruction of most rods and cones over the next 2–3 weeks. However, bipolar cells and horizontal cells were less damaged, indicating high selectivity among neurons for injury by prions. PrPSc deposition in cone and rod inner segments and on the bipolar cell processes participating in ribbon synapses appear to be critical early events leading to damage and death of photoreceptors after prion infection. These mechanisms may also occur in human retinitis pigmentosa and prion-like diseases, such as AD.

Abstract Objective The detection of prion seeding activity in CSF and olfactory mucosal brushings using real‐time quaking‐induced conversion assays allows highly accurate clinical diagnosis of sporadic Creutzfeldt–Jakob disease. To gauge transmission risks associated with these biospecimens and their testing, we have bioassayed prion infectivity levels in patients’ brain tissue, nasal brushings, and CSF, and assessed the pathogenicity of amplified products of real‐time quaking‐induced conversion assays seeded with Creutzfeldt–Jakob disease prions. Methods We obtained olfactory mucosal brushings and CSF from patients with a final diagnosis of sporadic Creutzfeldt–Jakob disease subtype MM1 (n = 3). Samples were inoculated intracerebrally into Tg66 transgenic mice that overexpress the homologous human 129M prion protein. The mice were evaluated for clinical, neuropathological, and biochemical evidence of prion infection. Results Patients’ brain tissue at 102 to 105 fold dilutions affected 47/48 Tg66 mice. In contrast, maximum acutely tolerable doses of insoluble pellets from their olfactory mucosa brushings caused evidence of prion disease in only 4/28 inoculated mice, and no effects were seen with 10‐fold dilutions. No clinical prion disease was observed in mice inoculated with antemortem CSF samples or prion‐seeded real‐time quaking‐induced conversion assay products. Interpretation Pellets from patients’ olfactory mucosa brushings had ≥10,000‐fold lower infectivity per unit volume than brain tissue, while CSF lacked detectable infectivity. Nonetheless, the results suggest that appropriate precautions may be warranted in surgical interventions involving the olfactory areas. The lack of pathogenic infectivity in the real‐time quaking‐induced conversion assay products provides evidence that the assay does not replicate biohazardous prions in vitro.

Abstract Background Prion diseases and prion-like disorders, including Alzheimer’s disease and Parkinson’s disease, are characterized by gliosis and accumulation of misfolded aggregated host proteins. Ablating microglia in prion-infected brain by treatment with the colony-stimulating factor-1 receptor (CSF-1R) inhibitor, PLX5622, increased accumulation of misfolded prion protein and decreased survival time. Methods To better understand the role of glia during neurodegeneration, we used RNA-seq technology, network analysis, and hierarchical cluster analysis to compare gene expression in brains of prion-infected versus mock-inoculated mice. Comparisons were also made between PLX5622-treated prion-infected mice and untreated prion-infected mice to assess mechanisms involved in disease acceleration in the absence of microglia. Results RNA-seq and network analysis suggested that microglia responded to prion infection through activation of integrin CD11c/18 and did not adopt the expression signature associated with other neurodegenerative disease models. Instead, microglia acquired an alternative molecular signature late in the disease process. Furthermore, astrocytes expressed a signature pattern of genes which appeared to be specific for prion diseases. Comparisons were also made with prion-infected mice treated with PLX5622 to assess the impact of microglia ablation on astrocyte gene expression during prion infection. In the presence of microglia, a unique mix of transcripts associated with A1- and A2-reactive astrocytes was increased in brains of prion-infected mice. After ablation of microglia, this reactive astrocyte expression pattern was enhanced. Thus, after prion infection, microglia appeared to decrease the overall A1/A2-astrocyte responses which might contribute to increased survival after infection. Conclusions RNA-seq analysis indicated dysregulation of over 300 biological processes within the CNS during prion disease. Distinctive microglia- and astrocyte-associated expression signatures were identified during prion infection. Furthermore, astrogliosis and the unique astrocyte-associated expression signature were independent of microglial influences. Astrogliosis and the unique astrocyte-associated gene expression pattern were increased when microglia were ablated. Our findings emphasize the potential existence of alternative pathways for activating the A1/A2 paradigm in astrocytes during neurodegenerative disease.

Microglia (MG) are critical to host defense during prion infection, but the mechanism(s) of this neuroprotection are poorly understood. To better examine the influence of MG during prion infection, we reduced MG in the brains of C57BL/10 mice using PLX5622 and assessed prion clearance and replication using multiple approaches that included bioassay, immunohistochemistry, and Real-Time Quaking Inducted Conversion (RT-QuIC). We also utilized a strategy of intermittent PLX5622 treatments to reduce MG and allow MG repopulation to test whether new MG could alter prion disease progress. Lastly, we investigated the influence of MG using tga20 mice, a rapid prion model that accumulates fewer pathological features and less PrPres in the infected brain. In C57BL/10 mice we found that MG were excluded from the inoculation site early after infection, but Iba1 positive infiltrating monocytes/macrophage were present. Reducing MG in the brain prior to prion inoculation did not increase susceptibility to prion infection. Short intermittent treatments with PLX5622 in prion infected C57BL/10 mice after 80 dpi were unsuccessful at altering the MG population, gliosis, or survival. Additionally, MG depletion using PLX5622 in tga20 mice had only a minor impact on prion pathogenesis, indicating that the presence of MG might be less important in this fast model with less prion accumulation. In contrast to the benefits of MG against prion disease in late stages of disease, our current experiments suggest MG do not play a role in early prion pathogenesis, clearance, or replication.

ABSTRACT Chronic wasting disease (CWD) is a transmissible prion disease first observed in the 1960s in North America. This invariably fatal disease affects multiple cervid species in the wild and in captivity. In addition to the several known transmission pathways involving cervid host species, prions have been detected in the feces of crows and coyotes after consumption of experimentally spiked tissues. This raises questions about the role of cervid consumers in the perpetuation of CWD. Mountain lions have been shown to preferentially select CWD-infected prey and are also apparently resistant to infection. In this study, two captive mountain lions were fed ground mule deer muscle tissue spiked with brain-derived CWD prions, and lion feces were collected for 1 week afterward. The input brain and resulting fecal materials were analyzed using the highly sensitive real-time quaking-induced conversion (RT-QuIC) assay to quantify prion seeding activity. We recovered only 2.8 to 3.9% of input CWD prions after passage through the mountain lions’ gastrointestinal tracts. Interestingly, CWD prions were shed only in the first defecation following consumption. Our data support the possibility that mountain lions feeding upon infected carcasses could excrete CWD prions in their feces over a short period of time but also suggest that most of the ingested prions are eliminated or sequestered by this large predator. IMPORTANCE CWD prions appear to spread naturally among susceptible cervid species in captivity and in the wild. A better understanding of all the ways these prions move, persist, and subsequently infect target species through the environment is critical to developing comprehensive disease control strategies. In our study, we show limited, transient pass-through of CWD prions in an apex predator, the mountain lion, using the highly sensitive RT-QuIC assay on feces collected after lions were fed prion-spiked muscle tissue. Prions were detected in feces only in the first defecation after exposure. Moreover, the amount of CWD prions recovered in feces was reduced by >96% after passing through the lion digestive system. This indicates that mountain lions may have some potential to distribute CWD prions within their home ranges but that they also effectively eliminate most of the CWD prions they consume.

Abstract For the transmissible, neurogenerative family of prion diseases, few human models of infection exist and none represent structured neuronal tissue. Human cerebral organoids are self-organizing, three-dimensional brain tissues that can be grown from induced pluripotent stem cells. Organoids can model aspects of neurodegeneration in Alzheimer’s Disease and Down’s Syndrome, reproducing tau hyperphosphorylation and amyloid plaque pathology. To determine whether organoids could be used to reproduce human prion infection and pathogenesis, we inoculated organoids with two sporadic Creutzfeldt-Jakob Disease prion subtypes. Organoids showed uptake, followed by clearance, of the infectious inoculum. Subsequent re-emergence of prion self-seeding activity indicated de novo propagation. Organoid health assays, prion titer, prion protein electrophoretic mobility and immunohistochemistry demonstrated inoculum-specific differences. Our study shows, for the first time, that cerebral organoids can model aspects of human prion disease and thus offer a powerful system for investigating different human prion subtype pathologies and testing putative therapeutics.

Abstract Degeneration of photoreceptors in the retina is a major cause of blindness in humans. Often retinal degeneration is due to inheritance of mutations in genes important in photoreceptor (PR) function, but can also be induced by other events including retinal trauma, microvascular disease, virus infection or prion infection. The onset of apoptosis and degeneration of PR neurons correlates with invasion of the PR cellular areas by microglia or monocytes, suggesting a causal role for these cells in pathogenesis of PR degenerative disease. To study the role of microglia in prion-induced retinal disease, we fed prion-infected mice a CSF-1 receptor blocking drug, PLX5622, to eliminate microglia in vivo, and the effects on retinal degeneration were analyzed over time. In mice not receiving drug, the main inflammatory cells invading the degenerating PR areas were microglia, not monocytes. Administration of PLX5622 was highly effective at ablating microglia in retina. However, lack of microglia during prion infection did not prevent degeneration of PR cells. Therefore, microglia were not required for the PR damage process during prion infection. Indeed, mice lacking microglia had slightly faster onset of PR damage. Similar results were seen in C57BL/10 mice and transgenic mice expressing GFP or RFP on microglia and monocytes, respectively. These results were supported by experiments using prion-infected Cx3cr1 knockout mice without PLX5622 treatment, where microglial expansion in retina was delayed, but PR degeneration was not. Contrary to predictions, microglia were not a causative factor in retinal damage by prion infection. Instead, newly generated PrPSc accumulated around the inner segment region of the PR cells and appeared to correlate with initiation of the pathogenic process in the absence of microglia.

There are currently no validated methods for the diagnosis of prion disease at the preclinical stage. Here the authors show that serial protein misfolding cyclic amplification and real-time quaking-induced conversion can be used to detect prions in the skin of prion-inoculated hamsters and humanized transgenic mice at early preclinical stages.

Abstract Chronic wasting disease (CWD) is a fatal prion disease which infects deer, elk and moose. CWD was first described as a wasting syndrome in captive deer in Colorado and Wyoming wildlife facilities from 1967 to 1979. Currently, CWD has been reported in 26 states of the USA, three Canadian provinces, South Korea, Norway and Finland. Since human consumption of cervids is common, it is critical to determine if CWD can infect humans. Published research, including epidemiologic studies and transmission studies using animal models, including transgenic mice that express human prion protein, have suggested existence of a strong species barrier between cervid CWD and humans. In the current study, we tested CWD transmission into two additional strains of transgenic mice (tg66 and tgRM). These mice over-express human prion protein at high levels and are highly sensitive to infection by human-tropic prions. One hundred and eight mice were inoculated intracerebrally with three different sources of CWD. After long periods of observation, brain tissues from CWD-inoculated mice were screened for evidence of prion infection by RT-QuIC, immunohistochemistry (IHC) and immunoblot. No IHC or immunoblot evidence was found to suggest transmission had occurred, and most mice were negative by RT-QuIC assay. However, four mice with inconsistent positive RT-QuIC reactions were detected. The seeding activity detected in these mice may represent a low level of CWD agent, suggesting a possible transfer of CWD infection. Alternatively, these results might be due to false positive reactions or residual CWD inoculum.

Prion diseases are progressive, neurodegenerative diseases affecting humans and animals. Also known as the transmissible spongiform encephalopathies, for the hallmark spongiform change seen in the brain, these diseases manifest increased oxidative damage early in disease and changes in antioxidant enzymes in terminal brain tissue. Superoxide dismutase 2 (SOD2) is an antioxidant enzyme that is critical for life. SOD2 knock-out mice can only be kept alive for several weeks post-birth and only with antioxidant therapy. However, this results in the development of a spongiform encephalopathy. Consequently, we hypothesized that reduced levels of SOD2 may accelerate prion disease progression and play a critical role in the formation of spongiform change. Using SOD2 heterozygous knock-out and litter mate wild-type controls, we examined neuronal long-term potentiation, disease duration, pathology, and degree of spongiform change in mice infected with three strains of mouse adapted scrapie. No influence of the reduced SOD2 expression was observed in any parameter measured for any strain. We conclude that changes relating to SOD2 during prion disease are most likely secondary to the disease processes causing toxicity and do not influence the development of spongiform pathology.

Tau aggregates consisting of hyperphosphorylated tau fibrils are associated with many neurodegenerative diseases, including Alzheimer's disease, Pick's disease, frontotemporal dementia, and progressive supranuclear palsy. Tau may contribute to the pathogenesis of these diseases, collectively referred to as tauopathies. In human genetic prion diseases, tau aggregates are detected in association with amyloid plaques consisting of prion protein (PrP). However, the role of abnormal tau aggregates in PrP amyloid disease remains unclear. Previously we inoculated scrapie prions into transgenic mice expressing human tau, mouse tau, glycophosphatidylinositol (GPI) anchored PrP, and anchorless PrP. These mice developed both spongiform vacuolar pathology and PrP amyloid pathology, and human tau was detected near PrP amyloid plaques. However, the presence of human tau did not alter the disease tempo or prion-induced neuropathology. In the present study, we tested mice which more closely modeled familial human prion disease. These mice expressed human tau but lacked both mouse tau and GPI-anchored PrP. However, they did produce anchorless PrP, resulting in perivascular PrP amyloid plaques, i.e. cerebral amyloid angiopathy (CAA), without spongiform degeneration. Typical of PrP amyloid disease, the clinical course was very slow in this model. Nevertheless, the accumulation of aggregated, phosphorylated human tau and its association with PrP amyloid plaques failed to alter the timing or course of the clinical disease observed. These data suggest that human tau does not contribute to the pathogenesis of mouse PrP amyloid brain disease and raise the possibility that tau may also not be pathogenic in human PrP amyloid disease.

Abstract Human familial prion diseases are associated with mutations at 34 different prion protein (PrP) amino acid residues. However, it is unclear whether infectious prions are found in all cases. Mutant PrP itself may be neurotoxic, or alternatively, PrP mutation might predispose to spontaneous formation of infectious PrP isoforms. Previous reports demonstrated transmission to animal models by human brain tissue expressing 7 different PrP mutations, but 3 other mutations were not transmissible. In the present work, we tested transmission using brain homogenates from patients expressing 3 untested PrP mutants: G131V, Y226X, and Q227X. Human brain homogenates were injected intracerebrally into tg66 transgenic mice overexpressing human PrP. Mice were followed for nearly 800 days. From 593 to 762 dpi, 4 of 8 mice injected with Y226X brain had PrPSc detectable in brain by immunostaining, immunoblot, and PrP amyloid seeding activity assayed by RT-QuIC. From 531 to 784 dpi, 11 of 11 G131V-injected mice had PrPSc deposition in brain, but none were positive by immunoblot or RT-QuIC assay. In contrast, from 529 to 798 dpi, no tg66 mice injected with Q227X brain had PrPSc or PrP amyloid seeding activity detectable by these methods. Y226X is the only one of 4 known PrP truncations associated with familial disease which has been shown to be transmissible. This transmission of prion infectivity from a patient expressing truncated human PrP may have implications for the spread and possible transmission of other aggregated truncated proteins in prion-like diseases such as Alzheimer’s disease, Parkinson’s disease and tauopathies.

In prion diseases, the spread of infectious prions (PrPSc) is thought to occur within nerves and across synapses of the central nervous system (CNS). However, the mechanisms by which PrPSc moves within axons and across nerve synapses remain undetermined. Molecular motors, including kinesins and dyneins, transport many types of intracellular cargo. Kinesin-1C (KIF5C) has been shown to transport vesicles carrying the normal prion protein (PrPC) within axons, but whether KIF5C is involved in PrPSc axonal transport is unknown. The current study tested whether stereotactic inoculation in the striatum of KIF5C knock-out mice (Kif5c−/−) with 0.5 µL volumes of mouse-adapted scrapie strains 22 L or ME7 would result in an altered rate of prion spreading and/or disease timing. Groups of mice injected with each strain were euthanized at either pre-clinical time points or following the development of prion disease. Immunohistochemistry for PrP was performed on brain sections and PrPSc distribution and tempo of spread were compared between mouse strains. In these experiments, no differences in PrPSc spread, distribution or survival times were observed between C57BL/6 and Kif5c−/− mice.

Prion protein (PrPC) is a protease-sensitive and soluble cell surface glycoprotein expressed in almost all mammalian cell types. PrPSc, a protease-resistant and insoluble form of PrPC, is the causative agent of prion diseases, fatal and transmissible neurogenerative diseases of mammals. Prion infection is initiated via either ingestion or inoculation of PrPSc or when host PrPC stochastically refolds into PrPSc. In either instance, the early events that occur during prion infection remain poorly understood. We have used transgenic mice expressing mouse PrPC tagged with a unique antibody epitope to monitor the response of host PrPC to prion inoculation. Following intracranial inoculation of either prion-infected or uninfected brain homogenate, we show that host PrPC can accumulate both intra-axonally and within the myelin membrane of axons suggesting that it may play a role in axonal loss following brain injury. Moreover, in response to the inoculation host PrPC exhibits an increased insolubility and protease resistance similar to that of PrPSc, even in the absence of infectious prions. Thus, our results raise the possibility that damage to the brain may be one trigger by which PrPC stochastically refolds into pathogenic PrPSc leading to productive prion infection.

Chronic wasting disease (CWD) is a fatal prion disease that can infect deer, elk and moose. CWD has now been detected in 26 states of the USA, 3 Canadian provinces, South Korea, Norway, Sweden and Finland. CWD continues to spread from endemic areas, and new foci of infections are frequently detected. As increasing numbers of cervids become infected, the likelihood for human exposure increases. To date, no cases of CWD infection in humans have been confirmed, but experience with the BSE zoonosis in the United Kingdom suggests exposure to CWD should be minimized. Specifically, hunters, meat processors and others in contact with tissues from potentially CWD-infected cervids need a practical method to decontaminate knives, saws and other equipment. Prions are notoriously difficult to inactivate, and most effective methods require chemicals or sterilization processes that are either dangerous, caustic, expensive or not readily available. Although corrosive, sodium hypochlorite (bleach) is widely available and affordable and has been shown to inactivate prion agents including those that cause scrapie, bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. In the current study, we confirm that bleach is an effective disinfectant for CWD prions and establish minimum times and bleach concentrations to eliminate prion seeding activity from stainless steel and infected brain homogenate solutions. We found that a five-minute treatment with a 40% dilution of household bleach was effective at inactivating CWD seeding activity from stainless-steel wires and CWD-infected brain homogenates. However, bleach was not able to inactivate CWD seeding activity from solid tissues in our studies.

Neuroinflammation and neurodegeneration are common during prion infection, but the mechanisms that underlie these pathological features are not well understood. Several components of innate immunity, such as Toll-like receptor (TLR) 4 and Complement C1q, have been shown to influence prion disease. To identify additional components of innate immunity that might impact prion disease within the central nervous system (CNS), we screened RNA from brains of pre-clinical and clinical 22L-infected mice for alterations in genes associated with innate immunity. Transcription of several genes encoding damage-associated molecular pattern (DAMP) proteins and receptors were increased in the brains of prion-infected mice. To investigate the role of some of these proteins in prion disease of the CNS, we infected mice deficient in DAMP receptor genes Tlr2, C3ar1, and C5ar1 with 22L scrapie. Elimination of TLR2 accelerated disease by a median of 10 days, while lack of C3aR1 or C5aR1 had no effect on disease tempo. Histopathologically, all knockout mouse strains tested were similar to infected control mice in gliosis, vacuolation, and PrPSc deposition. Analysis of proinflammatory markers in the brains of infected knockout mice indicated only a few alterations in gene expression suggesting that C5aR1 and TLR2 signaling did not act synergistically in the brains of prion-infected mice. These results indicate that signaling through TLR2 confers partial neuroprotection during prion infection.

Mammalian prion structures and replication mechanisms are poorly understood. Most synthetic recombinant prion protein (rPrP) amyloids prepared without cofactors are non-infectious or much less infectious than bona fide tissue-derived PrPSc. This effect has been associated with differences in folding of the aggregates, manifested in part by reduced solvent exclusion and protease-resistance in rPrP amyloids, especially within residues ~90-160. Substitution of 4 lysines within residues 101-110 of rPrP (central lysine cluster) with alanines (K4A) or asparagines (K4N) allows formation of aggregates with extended proteinase K (PK) resistant cores reminiscent of PrPSc, particularly when seeded with PrPSc. Here we have compared the infectivity of rPrP aggregates made with K4N, K4A or wild-type (WT) rPrP, after seeding with scrapie brain homogenate (ScBH) or normal brain homogenate (NBH). None of these preparations caused clinical disease on first passage into rodents. However, the ScBH-seeded fibrils (only) led to a subclinical pathogenesis as indicated by increases in prion seeding activity, neuropathology, and abnormal PrP in the brain. Seeding activities usually accumulated to much higher levels in animals inoculated with ScBH-seeded fibrils made with the K4N, rather than WT, rPrP molecules. Brain homogenates from subclinical animals induced clinical disease on second passage into "hamsterized" Tg7 mice, with shorter incubation times in animals inoculated with ScBH-seeded K4N rPrP fibrils. On second passage from animals inoculated with ScBH-seeded WT fibrils, we detected an additional PK resistant PrP fragment that was similar to that of bona fide PrPSc. Together these data indicate that both the central lysine cluster and scrapie seeding of rPrP aggregates influence the induction of PrP misfolding, neuropathology and clinical manifestations upon passage in vivo. We confirm that some rPrP aggregates can initiate further aggregation without typical pathogenesis in vivo. We also provide evidence that there is little, if any, biohazard associated with routine RT-QuIC assays.

Chronic wasting disease is a prion disease of cervids. Assessment of its zoonotic potential is critical. To evaluate primate susceptibility, we tested monkeys from 2 genera. We found that 100% of intracerebrally inoculated and 92% of orally inoculated squirrel monkeys were susceptible, but cynomolgus macaques were not, suggesting possible low risk for humans.

The original version of this Article contained errors in the author affiliations. Affiliation 2 incorrectly read ‘Department of Neurology, The First Hospital of Jilin University, Changchun 130021 Jilin Province, China.’Affiliation 5 incorrectly read ‘Department of Otolaryngology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061 Shanxi Province, China’Affiliation 9 incorrectly read ‘State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.’This has now been corrected in both the PDF and HTML versions of the Article.

Hypochlorous acid (HOCl) is produced naturally by neutrophils and other cells to kill conventional microbes in vivo. Synthetic preparations containing HOCl can also be effective as microbial disinfectants. Here we have tested whether HOCl can also inactivate prions and other self-propagating protein amyloid seeds. Prions are deadly pathogens that are notoriously difficult to inactivate, and standard microbial disinfection protocols are often inadequate. Recommended treatments for prion decontamination include strongly basic (pH ≥~12) sodium hypochlorite bleach, ≥1 N sodium hydroxide, and/or prolonged autoclaving. These treatments are damaging and/or unsuitable for many clinical, agricultural and environmental applications. We have tested the anti-prion activity of a weakly acidic aqueous formulation of HOCl (BrioHOCl) that poses no apparent hazard to either users or many surfaces. For example, BrioHOCl can be applied directly to skin and mucous membranes and has been aerosolized to treat entire rooms without apparent deleterious effects. Here, we demonstrate that immersion in BrioHOCl can inactivate not only a range of target microbes, including spores of Bacillus subtilis, but also prions in tissue suspensions and on stainless steel. Real-time quaking-induced conversion (RT-QuIC) assays showed that BrioHOCl treatments eliminated all detectable prion seeding activity of human Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, cervine chronic wasting disease, sheep scrapie and hamster scrapie; these findings indicated reductions of ≥103- to 106-fold. Transgenic mouse bioassays showed that all detectable hamster-adapted scrapie infectivity in brain homogenates or on steel wires was eliminated, representing reductions of ≥~105.75-fold and >104-fold, respectively. Inactivation of RT-QuIC seeding activity correlated with free chlorine concentration and higher order aggregation or destruction of proteins generally, including prion protein. BrioHOCl treatments had similar effects on amyloids composed of human α-synuclein and a fragment of human tau. These results indicate that HOCl can block the self-propagating activity of prions and other amyloids.

Misfolding and aggregation of host proteins are important features of the pathogenesis of neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, frontotemporal dementia and prion diseases. In all these diseases, the misfolded protein increases in amount by a mechanism involving seeded polymerization. In prion diseases, host prion protein is misfolded to form a pathogenic protease-resistant form, PrPSc, which accumulates in neurons, astroglia and microglia in the CNS. Here using dual-staining immunohistochemistry, we compared the cell specificity of PrPSc accumulation at early preclinical times post-infection using three mouse scrapie strains that differ in brain regional pathology. PrPSc from each strain had a different pattern of cell specificity. Strain 22L was mainly associated with astroglia, whereas strain ME7 was mainly associated with neurons and neuropil. In thalamus and cortex, strain RML was similar to 22L, but in substantia nigra, RML was similar to ME7. Expression of 90 genes involved in neuroinflammation was studied quantitatively using mRNA from thalamus at preclinical times. Surprisingly, despite the cellular differences in PrPSc accumulation, the pattern of upregulated genes was similar for all three strains, and the small differences observed correlated with variations in the early disease tempo. Gene upregulation correlated with activation of both astroglia and microglia detected in early disease prior to vacuolar pathology or clinical signs. Interestingly, the profile of upregulated genes in scrapie differed markedly from that seen in two acute viral CNS diseases (LaCrosse virus and BE polytropic Friend retrovirus) that had reactive gliosis at levels similar to our prion-infected mice.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy, or prion disease, that affects deer, elk, and moose. Human susceptibility to CWD remains unproven despite likely exposure to CWD-infected cervids. We used 2 nonhuman primate species, cynomolgus macaques and squirrel monkeys, as human models for CWD susceptibility. CWD was inoculated into these 2 species by intracerebral and oral routes. After intracerebral inoculation of squirrel monkeys, 7 of 8 CWD isolates induced a clinical wasting syndrome within 33–53 months. The monkeys’ brains showed spongiform encephalopathy and protease-resistant prion protein (PrPres) diagnostic of prion disease. After oral exposure, 2 squirrel monkeys had PrPres in brain, spleen, and lymph nodes at 69 months postinfection. In contrast, cynomolgus macaques have not shown evidence of clinical disease as of 70 months postinfection. Thus, these 2 species differed in susceptibility to CWD. Because humans are evolutionarily closer to macaques than to squirrel monkeys, they may also be resistant to CWD.

Transmissible spongiform encephalopathy (TSE) diseases are known to cross species barriers, but the pathologic and biochemical changes that occur during transmission are not well understood. To better understand these changes, we infected 6 hamster species with 263K hamster scrapie strain and, after each of 3 successive passages in the new species, analyzed abnormal proteinase K (PK)–resistant prion protein (PrPres) glycoform ratios, PrPres PK sensitivity, incubation periods, and lesion profiles. Unique 263K molecular and biochemical profiles evolved in each of the infected hamster species. Characteristics of 263K in the new hamster species seemed to correlate best with host factors rather than agent strain. Furthermore, 2 polymorphic regions of the prion protein amino acid sequence correlated with profile differences in these TSE-infected hamster species.

ABSTRACT Aggregation of misfolded host proteins in the central nervous system is believed to be important in the pathogenic process in several neurodegenerative diseases of humans, including prion diseases, Alzheimer's disease, and Parkinson's disease. In these diseases, protein misfolding and aggregation appear to expand through a process of seeded polymerization. Prion diseases occur in both humans and animals and are experimentally transmissible orally or by injection, thus providing a controllable model of other neurodegenerative protein misfolding diseases. In rodents and ruminants, prion disease has a slow course, lasting months to years. Although prion infectivity has been detected in brain tissue at 3 to 4 weeks postinfection (p.i.), the details of early prion replication in the brain are not well understood. Here we studied the localization and quantitation of PrPSc generation in vivo starting at 30 min postmicroinjection of scrapie into the brain. In C57BL mice at 3 days p.i., generation of new PrPSc was detected by immunohistochemistry and immunoblot assays, and at 7 days p.i., new generation was confirmed by real-time quaking-induced conversion assay. The main site of new PrPSc generation was near the outer basement membrane of small and medium blood vessels. The finding and localization of replication at this site so early after injection have not been reported previously. This predominantly perivascular location suggested that structural components of the blood vessel basement membrane or perivascular astrocytes might act as cofactors in the initial generation of PrPSc. The location of PrPSc replication at the basement membrane also implies a role for the brain interstitial fluid drainage in the early infection process. IMPORTANCE Neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and prion diseases, of humans are characterized by misfolding and aggregation of certain proteins, resulting in the destruction of brain tissue. In these diseases, the damage process spreads progressively within the central nervous system, but only prion diseases are known to be transmissible between individuals. Here we used microinjection of infectious prion protein (PrPSc) into the mouse brain to model early events of iatrogenic prion transmission via surgical instruments or tissue grafts. At 3 and 7 days postinjection, we detected the generation of new PrPSc, mostly on the outer walls of blood vessels near the injection site. This location and very early replication were surprising and unique. Perivascular prion replication suggested the transport of injected PrPSc via brain interstitial fluid to the basement membranes of blood vessels, where interactions with possible cofactors made by astrocytes or endothelia might facilitate the earliest cycles of prion infection.

Different transmissible spongiform encephalopathy (TSE)-associated forms of prion protein (e.g. PrP(Sc)) can vary markedly in ultrastructure and biochemical characteristics, but each is propagated in the host. PrP(Sc) propagation involves conversion from its normal isoform, PrP(C), by a seeded or templated polymerization mechanism. Such a mechanism is also the basis of the RT-QuIC and eQuIC prion assays which use recombinant PrP (rPrP(Sen)) as a substrate. These ultrasensitive detection assays have been developed for TSE prions of several host species and sample tissues, but not for murine models which are central to TSE pathogenesis research. Here we have adapted RT-QuIC and eQuIC to various murine prions and evaluated how seeding activity depends on glycophosphatidylinositol (GPI) anchoring and the abundance of amyloid plaques and protease-resistant PrP(Sc) (PrP(Res)). Scrapie brain dilutions up to 10(-8) and 10(-13) were detected by RT-QuIC and eQuIC, respectively. Comparisons of scrapie-affected wild-type mice and transgenic mice expressing GPI anchorless PrP showed that, although similar concentrations of seeding activity accumulated in brain, the heavily amyloid-laden anchorless mouse tissue seeded more rapid reactions. Next we compared seeding activities in the brains of mice with similar infectivity titers, but widely divergent PrP(Res) levels. For this purpose we compared the 263K and 139A scrapie strains in transgenic mice expressing P101L PrP(C). Although the brains of 263K-affected mice had little immunoblot-detectable PrP(Res), RT-QuIC indicated that seeding activity was comparable to that associated with a high-PrP(Res) strain, 139A. Thus, in this comparison, RT-QuIC seeding activity correlated more closely with infectivity than with PrP(Res) levels. We also found that eQuIC, which incorporates a PrP(Sc) immunoprecipitation step, detected seeding activity in plasma from wild-type and anchorless PrP transgenic mice inoculated with 22L, 79A and/or RML scrapie strains. Overall, we conclude that these new mouse-adapted prion seeding assays detect diverse types of PrP(Sc).

Transmissible spongiform encephalopathies (TSE) or prion diseases are neurodegenerative disorders associated with conversion of normal host prion protein (PrP) to a misfolded, protease-resistant form (PrPres). Genetic variations of prion protein in humans and animals can alter susceptibility to both familial and infectious prion diseases. The N171S PrP polymorphism is found mainly in humans of African descent, but its low incidence has precluded study of its possible influence on prion disease. Similar to previous experiments of others, for laboratory studies we created a transgenic model expressing the mouse PrP homolog, PrP-170S, of human PrP-171S. Since PrP polymorphisms can vary in their effects on different TSE diseases, we tested these mice with four different strains of mouse-adapted scrapie. Whereas 22L and ME7 scrapie strains induced typical clinical disease, neuropathology and accumulation of PrPres in all transgenic mice at 99-128 average days post-inoculation, strains RML and 79A produced clinical disease and PrPres formation in only a small subset of mice at very late times. When mice expressing both PrP-170S and PrP-170N were inoculated with RML scrapie, dominant-negative inhibition of disease did not occur, possibly because interaction of strain RML with PrP-170S was minimal. Surprisingly, in vitro PrP conversion using protein misfolding cyclic amplification (PMCA), did not reproduce the in vivo findings, suggesting that the resistance noted in live mice might be due to factors or conditions not present in vitro. These findings suggest that in vivo conversion of PrP-170S by RML and 79A scrapie strains was slow and inefficient. PrP-170S mice may be an example of the conformational selection model where the structure of some prion strains does not favor interactions with PrP molecules expressing certain polymorphisms.

A major problem for the effective diagnosis and management of prion diseases is the lack of rapid high-throughput assays to measure low levels of prions. Such measurements have typically required prolonged bioassays in animals. Highly sensitive, but generally non-quantitative, prion detection methods have been developed based on prions' ability to seed the conversion of normally soluble protease-sensitive forms of prion protein to protease-resistant and/or amyloid fibrillar forms. Here we describe an approach for estimating the relative amount of prions using a new prion seeding assay called real-time quaking induced conversion assay (RT-QuIC). The underlying reaction blends aspects of the previously described quaking-induced conversion (QuIC) and amyloid seeding assay (ASA) methods and involves prion-seeded conversion of the alpha helix-rich form of bacterially expressed recombinant PrP(C) to a beta sheet-rich amyloid fibrillar form. The RT-QuIC is as sensitive as the animal bioassay, but can be accomplished in 2 days or less. Analogous to end-point dilution animal bioassays, this approach involves testing of serial dilutions of samples and statistically estimating the seeding dose (SD) giving positive responses in 50% of replicate reactions (SD(50)). Brain tissue from 263K scrapie-affected hamsters gave SD(50) values of 10(11)-10(12)/g, making the RT-QuIC similar in sensitivity to end-point dilution bioassays. Analysis of bioassay-positive nasal lavages from hamsters affected with transmissible mink encephalopathy gave SD(50) values of 10(3.5)-10(5.7)/ml, showing that nasal cavities release substantial prion infectivity that can be rapidly detected. Cerebral spinal fluid from 263K scrapie-affected hamsters contained prion SD(50) values of 10(2.0)-10(2.9)/ml. RT-QuIC assay also discriminated deer chronic wasting disease and sheep scrapie brain samples from normal control samples. In principle, end-point dilution quantitation can be applied to many types of prion and amyloid seeding assays. End point dilution RT-QuIC provides a sensitive, rapid, quantitative, and high throughput assay of prion seeding activity.

Prion diseases are fatal neurodegenerative diseases of humans and animals characterized by gray matter spongiosis and accumulation of aggregated, misfolded, protease-resistant prion protein (PrPres). PrPres can be deposited in brain in an amyloid-form and/or non-amyloid form, and is derived from host-encoded protease-sensitive PrP (PrPsen), a protein normally anchored to the plasma membrane by glycosylphosphatidylinositol (GPI). Previously, using heterozygous transgenic mice expressing only anchorless PrP, we found that PrP anchoring to the cell membrane was required for typical clinical scrapie. However, in the present experiments, using homozygous transgenic mice expressing two-fold more anchorless PrP, scrapie infection induced a new fatal disease with unique clinical signs and altered neuropathology, compared to non-transgenic mice expressing only anchored PrP. Brain tissue of transgenic mice had high amounts of infectivity, and histopathology showed dense amyloid PrPres plaque deposits without gray matter spongiosis. In contrast, infected non-transgenic mice had diffuse non-amyloid PrPres deposits with significant gray matter spongiosis. Brain graft studies suggested that anchored PrPsen expression was required for gray matter spongiosis during prion infection. Furthermore, electron and light microscopic studies in infected transgenic mice demonstrated several pathogenic processes not seen in typical prion disease, including cerebral amyloid angiopathy and ultrastructural alterations in perivascular neuropil. These findings were similar to certain human familial prion diseases as well as to non-prion human neurodegenerative diseases, such as Alzheimer's disease.

Distribution of prion infectivity in organs and tissues is important in understanding prion disease pathogenesis and designing strategies to prevent prion infection in animals and humans. Transmission of prion disease from cattle to humans resulted in banning human consumption of ruminant nervous system and certain other tissues. In the present study, we surveyed tissue distribution of prion infectivity in mice with prion disease. We show for the first time detection of infectivity in white and brown fat. Since high amounts of ruminant fat are consumed by humans and also incorporated into animal feed, fat-containing tissues may pose a previously unappreciated hazard for spread of prion infection.