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Prion diseases are fatal infectious neurodegenerative disorders in human and animals caused by misfolding of the cellular prion protein (PrP C ) into the pathological isoform PrP Sc Elucidating the molecular and cellular mechanisms underlying prion propagation may help to develop disease interventions. Cell culture systems for prion propagation have greatly advanced molecular insights into prion biology, but translation of in vitro to in vivo findings is often disappointing. A wider range of cell culture systems might help overcome these shortcomings. Here, we describe an immortalized mouse neuronal astrocyte cell line (C8D1A) that can be infected with murine prions. Both PrP C protein and mRNA levels in astrocytes were comparable with those in neuronal and non-neuronal cell lines permitting persistent prion infection. We challenged astrocytes with three mouse-adapted prion strains (22L, RML, and ME7) and cultured them for six passages. Immunoblotting results revealed that the astrocytes propagated 22L prions well over all six passages, whereas ME7 prions did not replicate, and RML prions replicated only very weakly after five passages. Immunofluorescence analysis indicated similar results for PrP Sc Interestingly, when we used prion conversion activity as a readout in real-time quaking-induced conversion assays with RML-infected cell lysates, we observed a strong signal over all six passages, comparable with that for 22L-infected cells. These data indicate that the C8D1A cell line is permissive to prion infection. Moreover, the propagated prions differed in conversion and proteinase K-resistance levels in these astrocytes. We propose that the C8D1A cell line could be used to decipher prion strain biology., Competing Interests: Conflict of interest—Conflict of interest statement: The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Tahir et al.)

Prion diseases are fatal infectious neurodegenerative disorders in human and animals caused by misfolding of the cellular prion protein (PrP C ) into the infectious isoform PrP Sc . These diseases have the potential to transmit within or between species, and no cure is available to date. Targeting the unfolded protein response (UPR) as an anti-prion therapeutic approach has been widely reported for prion diseases. Here, we describe the anti-prion effect of the chemical compound Sephin1 which has been shown to protect in mouse models of protein misfolding diseases including amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) by selectively inhibiting the stress-induced regulatory subunit of protein phosphatase 1, thus prolonging eIF2α phosphorylation. We show here that Sephin1 dose and time dependently reduced PrP Sc in different neuronal cell lines which were persistently infected with various prion strains. In addition, prion seeding activity was reduced in Sephin1-treated cells. Importantly, we found that Sephin1 significantly overcame the endoplasmic reticulum (ER) stress induced in treated cells, as measured by lower expression of stress-induced aberrant prion protein. In a mouse model of prion infection, intraperitoneal treatment with Sephin1 significantly prolonged survival of prion-infected mice. When combining Sephin1 with the neuroprotective drug metformin, the survival of prion-infected mice was also prolonged. These results suggest that Sephin1 could be a potential anti-prion drug selectively targeting one component of the UPR pathway.

Prion diseases are fatal infectious neurodegenerative disorders in humans and other animals and are caused by misfolding of the cellular prion protein (PrP C ) into the pathological isoform PrP Sc These diseases have the potential to transmit within or between species, including zoonotic transmission to humans. Elucidating the molecular and cellular mechanisms underlying prion propagation and transmission is therefore critical for developing molecular strategies for disease intervention. We have shown previously that impaired quality control mechanisms directly influence prion propagation. In this study, we manipulated cellular quality control pathways in vitro by stably and transiently overexpressing selected quality control folding (ERp57) and cargo (VIP36) proteins and investigated the effects of this overexpression on prion propagation. We found that ERp57 or VIP36 overexpression in persistently prion-infected neuroblastoma cells significantly reduces the amount of PrP Sc in immunoblots and prion-seeding activity in the real-time quaking-induced conversion (RT-QuIC) assay. Using different cell lines infected with various prion strains confirmed that this effect is not cell type- or prion strain-specific. Moreover, de novo prion infection revealed that the overexpression significantly reduced newly formed PrP Sc in acutely infected cells. ERp57-overexpressing cells significantly overcame endoplasmic reticulum stress, as revealed by expression of lower levels of the stress markers BiP and CHOP, accompanied by a decrease in PrP aggregates. Furthermore, application of ERp57-expressing lentiviruses prolonged the survival of prion-infected mice. Taken together, improved cellular quality control via ERp57 or VIP36 overexpression impairs prion propagation and could be utilized as a potential therapeutic strategy., (© 2018 Thapa et al.)

Chronic wasting disease (CWD) is the most contagious prion disease. It is expanding rapidly in North America, was found recently in Europe, and the potential for transmission to humans cannot be excluded yet. We hypothesized that it is possible to prevent peripheral CWD infection and CWD prion shedding by inducing auto-antibodies against the cellular prion protein (PrP C ) by active vaccination. Our objective is to overcome self-tolerance against PrP by using a multimeric recombinant PrP (recPrP) as an immunogen. We expressed in E. coli, purified and refolded four immunogens: cervid and murine recPrP in monomeric and dimeric form. Testing immunogenicity in sera of the vaccinated transgenic mice expressing cervid PrP revealed that all four immunogens effectively overcame self-tolerance against the prion protein as shown by high antibody titers. Confocal microscopy analysis revealed effective binding of post-immune sera to surface-located PrP C in both murine and cervid PrP expressing cultured cells. Remarkably, the post-immune auto-antibodies effectively inhibited CWD-induced prion conversion in RT-QuIC assay when incubated with either PrP substrate or CWD seed. Furthermore, they mitigated prion propagation in CWD-infected cervid-PrP expressing RK13 cells. Together, multimeric recombinant cervid PrP effectively overcomes self-tolerance to PrP and induces auto-antibodies that interfere with CWD conversion in vitro.