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Combined pharmacological, mutational and cell biology approaches indicate that p53‐dependent caspase 3 activation triggered by cellular prion is dependent on its endocytosis

We have previously established that cellular prion PrPc elicited p53‐dependent caspase 3 activation in various transfected cells and primary cultured neurons. Although we showed that PrPc modulates p53 expression at both transcriptional and post‐transcriptional levels, it remained unclear as to whether cellular prion signals at the membrane to trigger intracellular messages or if prion proapoptotic activity necessitated its translocation into the cytoplasm. Here, we compare the processing and cell death‐related functions of PrPc with those of a mutated PrPc protein (N‐3F4 MoPrPc) in which three basic N‐terminal residues responsible for PrPc internalization had been mutated. As expected, N‐3F4 MoPrPc remains exclusively located at the membrane, whereas PrPc partitions between membrane‐associated and intracellular compartments, but both, proteins undergo constitutive and protein kinase C‐regulated disintegrin‐mediated proteolysis, leading to N1 fragment production. Unlike PrPc, N‐3F4 MoPrPc expression does not induce caspase 3 activation after stimulation by staurosporine and was inert on p53 expression and promoter transactivation in both human cells and TSM1 mouse neurons. Interestingly, PrPc‐induced caspase 3 activation was closely linked to its endocytosis. This phenotype was enhanced by proteasomal inhibition and prevented by sucrose treatment. Accordingly, immunohistochemical analysis showed that protection towards degradation increased intracellular PrPc‐like immunoreactivity, while sucrose treatments fully abolished PrPc intracellular expression and co‐localization with transferrin. Altogether, we, establish here, using combined biochemical, mutational and cell biology approaches, that the caspase 3 activation associated with cellular prion is closely related to its ability to undergo endocytosis. This is, to our knowledge, the first direct description of an endocytosis‐dependent PrPc‐associated function.