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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.

Cellular prion protein (PrPC) protects neurons against oxidative stress damage. This role is lost upon its misfolding into insoluble prions in prion diseases, and correlated with cytoskeletal breakdown and neurophysiological deficits. Here we used mouse neuronal models to assess how PrPC protects the neuronal cytoskeleton, and its role in network communication, from oxidative stress damage. Oxidative stress was induced extrinsically by potassium superoxide (KO2) or intrinsically by Mito-Paraquat (MtPQ), targeting the mitochondria. In mouse neural lineage cells, KO2 was damaging to the cytoskeleton, with cells lacking PrPC (PrP-/-) damaged more than wild-type (WT) cells. In hippocampal slices, KO2 acutely inhibited neuronal communication in WT controls without damaging the cytoskeleton. This inhibition was not observed in PrP-/- slices. Neuronal communication and the cytoskeleton of PrP-/- slices became progressively disrupted and degenerated post-recovery, whereas the dysfunction in WT slices recovered in 5 days. This suggests that the acute inhibition of neuronal activity in WT slices in response to KO2 was a neuroprotective role of PrPC, which PrP-/- slices lacked. Heterozygous expression of PrPC was sufficient for this neuroprotection. Further, hippocampal slices from mice expressing PrPC without its GPI anchor (PrPGPI-/-) displayed acute inhibition of neuronal activity by KO2. However, they failed to restore normal activity and cytoskeletal formation post-recovery. This suggests that PrPC facilitates the depressive response to KO2 and its GPI anchoring is required to restore KO2-induced damages. Immuno spin-trapping showed increased radicals formed on the filamentous actin of PrP-/- and PrPGPI-/- slices, but not WT and PrP+/- slices, post-recovery suggesting ongoing dysregulation of redox balance in the slices lacking GPI-anchored PrPC. The MtPQ treatment of hippocampal slices temporarily inhibited neuronal communication independent of PrPC expression. Overall, GPI-anchored PrPC alters synapses and neurotransmission to protect and repair the neuronal cytoskeleton, and neuronal communication, from extrinsically induced oxidative stress damages.

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.

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.

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.

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.

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.