Cell expression of a four extra octarepeat mutated PrP^C modifies cell structure and cell cycle regulation

Abstract

RK13 cell lines generated to express bovine PrP C with a four extra octarepeat insertional mutation (Bo10ORPrP C ) show partially insoluble PrP C and lower rates of cell growth when compared to either the same cells expressing wild type C or the original RK13 cell line. The expression of C in cell cultures was also associated with changes in cell size and reorganization of the actin cytoskeleton. This last process was reversed by Clostridium difficile toxin-B, a specific inhibitor of small GTPase proteins. Further, in clones expressing Bo-10ORPrP C , increased proportions of cells at cell cycle stage G2/M were observed. Proteasome inhibitors caused a further expansion of G2/M-stage cells that was more marked in cell lines expressing Bo-10ORPrP C than those expressing Bo-6ORPrP C , while this effect was minimal or null in the original RK13 cell line. Hence, the presence of Bo10ORPrP C in RK13 cells promotes cell cycle arrest at G2/M, and the effect is amplified by proteasome inhibition. These findings suggest a role for PrP C in cell morphology and cell cycle regulation, and open new avenues for understanding the mechanisms underlying PrP mutation-associated diseases.

Protein Disulfide Isomerase Regulates Endoplasmic Reticulum Stress and the Apoptotic Process during Prion Infection and PrP Mutant-Induced Cytotoxicity

“…Previous studies have repeatedly confirmed that transient expressions of PrP-KEDL and PrPs with extra-octarepeat insertions cause cell apoptosis and pathological abnormality in animal models or cell lines [17], [18], [19]. Under these experimental conditions, expressions of PrP-KEDL and PrP-PG15 in 293-T cells significantly decreased cell viability (Fig.…”

Proteomic consequences of expression and pathological conversion of the prion protein in inducible neuroblastoma N2a cells

“…44 Modification of cytoskeleton upon PrP expression may also reveal a role of the protein in cell morphology. 45 As often in 2DE differential analysis, proteins involved in energy metabolism were detected. In our case, the most remarkable observation was a clear increase upon prion infection of several proteins such as: pyruvate kinase, alpha enolase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, ATP synthase or lactate dehydrogenase.…”

Effect of Scrapie on the Stability of Housekeeping Genes

“…29 Moreover, the interaction between PrP C and cytoskeletal components, such as b-actin and a-tubulin, has been reported, 30 and more recently, a role for PrP C in cell morphology was suggested. 31 Our analysis found ACTB to be the most stable HKG in the medulla oblongata of healthy sheep; however, it was the less adequate gene with regards to stability in the same region obtained from scrapie-infected sheep. The possible implication of b-actin in the neuropathology associated with prion disease could explain the loss of stability observed for this gene in the affected medulla oblongata.…”

“…Moreover, ACTB could be involved in the neuropathology of scrapie. 29,31 Therefore, our previously selected reference genes would not be the most appropriate Figure 3 Expression of BAX and BCL2 in the medulla oblongata of scrapie-infected (black bar) and healthy (grey bar) sheep using one housekeeping gene for normalization. Y axis shows the normalized expression values.…”