Genetic Mapping of Activity Determinants within Cellular Prion Proteins

The PrP-like Doppel (Dpl) protein causes apoptotic death of cerebellar neurons in transgenic mice, a process prevented by expression of the wild type (wt) cellular prion protein, PrPC. Internally deleted forms of PrPC resembling Dpl such as PrPΔ32–121 produce a similar PrPC-sensitive pro-apoptotic phenotype in transgenic mice. Here we demonstrate that these phenotypic attributes of wt Dpl, wt PrPC, and PrPΔ132–121 can be accurately recapitulated by transfected mouse cerebellar granule cell cultures. This system was then explored by mutagenesis of the co-expressed prion proteins to reveal functional determinants. By this means, neuroprotective activity of wt PrPC was shown to be nullified by a deletion of the N-terminal charged region implicated in endocytosis and retrograde axonal transport (PrPΔ23–28), by deletion of all five octarepeats (PrPΔ51–90), or by glycine replacement of four octarepeat histidine residues required for selective binding of copper ions (Prnp“H/G”). In the case of Dpl, overlapping deletions defined a requirement for the gene interval encoding helices B and B′ (DplΔ101–125). These data suggest contributions of copper binding and neuronal trafficking to wt PrPC function in vivo and place constraints upon current hypotheses to explain Dpl/PrPC antagonism by competitive ligand binding. Further implementation of this assay should provide a fuller understanding of the attributes and subcellular localizations required for activity of these enigmatic proteins.