Changes in cellular prion protein expression, processing and localisation during differentiation of the neuronal cell line CAD 5

Background information Cellular prion protein (PrPC) is infamous for its role in prion diseases. The physiological function of PrPC remains enigmatic, but several studies point to its involvement in cell differentiation processes. To test this possibility, we monitored PrPC changes during the differentiation of prion‐susceptible CAD 5 cells, and then we analysed the effect of PrPC ablation on the differentiation process. Results Neuronal CAD 5 cells differentiate within 5 days of serum withdrawal, with the majority of the cells developing long neurites. This process is accompanied by an up to sixfold increase in PrPC expression and enhanced N‐terminal β‐cleavage of the protein, which suggests a role for the PrPC in the differentiation process. Moreover, the majority of PrPC in differentiated cells is inside the cell, and a large proportion of the protein does not associate with membrane lipid rafts. In contrast, PrPC in proliferating cells is found mostly on the cytoplasmic membrane and is predominantly associated with lipid rafts. To determine the importance of PrPC in cell differentiation, a CAD 5 PrP−/− cell line with ablated PrPC expression was created using the CRISPR/Cas9 system. We observed no considerable difference in morphology, proliferation rate or expression of molecular markers between CAD 5 and CAD 5 PrP−/− cells during the differentiation initiated by serum withdrawal. Conclusions PrPC characteristics, such as cell localisation, level of expression and posttranslational modifications, change during CAD 5 cell differentiation, but PrPC ablation does not change the course of the differentiation process. Significance Ablation of PrPC expression does not affect CAD 5 cell differentiation, although we observed many intriguing changes in PrPC features during the process. Our study does not support the concept that PrPC is important for neuronal cell differentiation, at least in simple in vitro conditions. Research article: Differentiation of neuronal CAD 5 cells is accompanied by significant increase in cellular prion protein (PrPC) expression and enhanced cleavage of its N‐terminus. In contrast to proliferating cells large amount of the protein has intracellular localization and does not associate with membrane lipid rafts. Despite these remarkable changes ablation of PrPC expression does not affect differentiation of CAD 5 cells demonstrating PrPC unimportance in the process.