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

BACKGROUND INFORMATION

Cellular prion protein (PrP ) is infamous for its role in prion diseases. The physiological function of PrP remains enigmatic, but several studies point to its involvement in cell differentiation processes. To test this possibility, we monitored PrP changes during the differentiation of prion-susceptible CAD 5 cells, and then we analysed the effect of PrP 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 PrP expression and enhanced N-terminal β-cleavage of the protein, which suggests a role for the PrP in the differentiation process. Moreover, the majority of PrP in differentiated cells is inside the cell, and a large proportion of the protein does not associate with membrane lipid rafts. In contrast, PrP in proliferating cells is found mostly on the cytoplasmic membrane and is predominantly associated with lipid rafts. To determine the importance of PrP in cell differentiation, a CAD 5 PrP cell line with ablated PrP 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

PrP characteristics, such as cell localisation, level of expression and posttranslational modifications, change during CAD 5 cell differentiation, but PrP ablation does not change the course of the differentiation process.

SIGNIFICANCE

Ablation of PrP expression does not affect CAD 5 cell differentiation, although we observed many intriguing changes in PrP features during the process. Our study does not support the concept that PrP is important for neuronal cell differentiation, at least in simple in vitro conditions.