Abstract
The study of prions and the discovery of candidate therapeutics for prion disease have been facilitated by the ability of prions to replicate in cultured cells. Paradigms in which prion proteins from different species are expressed in cells with low or no expression of endogenous prion protein (PrP) have expanded the range of prion strains that can be propagated. In these systems, cells stably expressing a PrP of interest are typically generated via coexpression of a selectable marker and treatment with an antibiotic. Here, we report the unexpected discovery that the aminoglycoside G418 (Geneticin) interferes with the ability of stably transfected cultured cells to become infected with prions. In G418-resistant lines of N2a or CAD5 cells, the presence of G418 reduced levels of protease-resistant PrP following challenge with the RML or 22L strains of mouse prions. G418 also interfered with the infection of cells expressing hamster PrP with the 263K strain of hamster prions. Interestingly, G418 had minimal to no effect on protease-resistant PrP levels in cells with established prion infection, arguing that G418 selectively interferes with de novo prion infection. As G418 treatment had no discernible effect on cellular PrP levels or its localization, this suggests that G418 may specifically target prion assemblies or processes involved in the earliest stages of prion infection.
Highlights
Myelin maintenance in the peripheral nervous system and regulation of neural cell adhesion molecule (NCAM) polysialylation [7, 8]
For both lines of CAD5-prion protein (PrP)−/−(MoPrP) cells, maintenance of the cells in higher concentrations of G418 resulted in higher levels of PrPC expression suggesting that, in the absence of selective agent, the composition of these polyclonal pools of cells drifts toward cells with lower levels of PrPC expression (Fig. 1A)
When 1.0 mg/ml G418 was used in an attempt to increase PrPC expression levels and maximize proteinase K (PK)-resistant PrP (PrPres) production [43], the efficiency of RML prion infection in CAD5-PrP−/− (MoPrP) cells was substantially reduced (Fig. 1B)
Summary
Myelin maintenance in the peripheral nervous system and regulation of neural cell adhesion molecule (NCAM) polysialylation [7, 8]. When CAD5-PrP−/−(MoPrP) cells were challenged with RML prions in the presence of 0.2 mg/ml G418, successful prion infection was observed, as indicated by the presence of PrPres in cellular lysates (Fig. 1B).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
