Infectious Prions Accumulate to High Levels in Non Proliferative C2C12 Myotubes

Abstract

Prion diseases are driven by the strain-specific, template-dependent transconformation of the normal cellular prion protein (PrPC) into a disease specific isoform PrPSc. Cell culture models of prion infection generally use replicating cells resulting in lower levels of prion accumulation compared to animals. Using non-replicating cells allows the accumulation of higher levels of PrPSc and, thus, greater amounts of infectivity. Here, we infect non-proliferating muscle fiber myotube cultures prepared from differentiated myoblasts. We demonstrate that prion-infected myotubes generate substantial amounts of PrPSc and that the level of infectivity produced in these post-mitotic cells, 105.5 L.D.50/mg of total protein, approaches that observed in vivo. Exposure of the myotubes to different mouse-adapted agents demonstrates strain-specific replication of infectious agents. Mouse-derived myotubes could not be infected with hamster prions suggesting that the species barrier effect is intact. We suggest that non-proliferating myotubes will be a valuable model system for generating infectious prions and for screening compounds for anti-prion activity.

The prion protein is embedded in a molecular environment that modulates transforming growth factor β and integrin signaling

“…1a ) 18 . The parental wild-type cell lines are familiar to the prion research community due to their distinct properties with regard to PrP: (1) NMuMG cells exhibit a more than five-fold increase in their PrP protein levels when EMT was induced by the addition of Tgfb1 (attempts to infect these cells with prions have been unsuccessful but were also not exhaustive) 19 ; (2) C2C12 cells are the only muscle cell model currently known to be susceptible to prion infection 20 , 21 ; (3) N2a neuroblastoma cells may be the most often used cell model in prion research and can readily be infected with mouse-adapted Rocky Mountain Laboratory (RML) prions; and (4) CAD5 catecholaminergic cells exhibit susceptibility to infection with several prion strains 22 . To stabilize existing protein-protein interactions, cells were subjected to mild formaldehyde crosslinking prior to cell lysis (Fig.…”

The Prion Protein Controls Polysialylation of Neural Cell Adhesion Molecule 1 during Cellular Morphogenesis

“…This experiment revealed no apparent differences in the average steady-state PSA-NCAM1 levels in PrP-deficient tissues ( panel a in S2 Fig ), arguing that the effect of PrP on NCAM1 polysialylation might be unique to NMuMG cells, or could be restricted to cells undergoing specific cellular morphogenesis programs. Thus, before moving to further mechanistic investigations, analyses were extended to C2C12 cells, a muscle cell morphogenesis model, known to express both PSA-NCAM1 and PrP during myotube formation [ 36 , 37 ]. In support of the notion that PrP regulates NCAM1 polysialylation more broadly during morphogenesis, yet contrasting observations made in NMuMG cells, CRISPR-Cas9-generated PrP ko C2C12 myocytes and myotubes exhibited a profound increase in PSA-NCAM1 levels relative to wt C2C12 cells ( Fig 5b ).…”

CRISPR-Cas9-Based Knockout of the Prion Protein and Its Effect on the Proteome

“…The most often used and arguably best understood cell model for studying the cellular biology of PrP is the mouse neuroblastoma cell line Neuro-2a (N2a) [7] , [8] . Recently, mouse C2C12 cells, a cell line of myoblasts origins, were reported to provide an attractive experimental paradigm for studying the cellular biology of PrP [9] . In light of previous reports that document a role for PrP in morphogenetic rearrangements underlying epithelial-to-mesenchymal transition (EMT) during zebrafish development [10] , [11] , it would further be of interest to explore the possible involvement of PrP in signaling pathways known to play a role in EMT in a mouse epithelial cell line.…”