Conversion of PrPC into an alternatively folded isoform, PrPSc, is a fundamental event in transmissible spongiform encephalopathies or prion diseases (Prusiner 1998). PrPC is a ubiquitous cell-surface membrane protein, which is anchored to the membrane by a C-terminal glycosyl phosphatidyl inositol moiety, but can also acquire a transmembrane topology or be secreted (Hegde et al. 1998; Ermonval et al. 2003; for review). The chromosomal PrP gene, Prnp, is a member of the Prn gene family that also includes the gene Prnd encoding Dpl. Dpl shares ∼25% sequence similarity with PrP but lacks the N-terminally located octameric repeats and hydrophobic regions present in PrP (Moore et al. 1999). In contrast to PrP, Dpl has a very low expression in the adult brain, is expressed primarily in testis, and less so in other peripheral tissues (Li et al. 2000). The functions of PrP and Dpl have remained elusive. Mice with an ablated PrP gene (Prnp0/0) are resistant to infection with prions (Büeler et al. 1993; Prusiner et al. 1993), while showing normal development and behavior (Büeler et al. 1992). However, recent studies have revealed deficits in hippocampal-dependent spatial learning and hippocampal synaptic plasticity in Prnp0/0 mice (Criado et al. 2005). Deletion of the Prnd gene in mice results in male sterility but otherwise normal behavior (Behrens et al. 2002) and development of prion disease appears unabated (Behrens et al. 2001). Over-expression of Dpl in the brain of Prnp0/0 mice results in ataxia and cerebellar degeneration (Moore et al. 1999), a phenotype that is not observed in mice concomitantly expressing PrP (Nishida et al. 1999). Thus, the knock-out and transgenic mouse models have suggested intricate and perhaps antagonistic functional networks involving PrPC and Dpl.
One approach to elucidating the function of PrPC has been to identify interacting molecules. PrPC binds copper (Brown et al. 1997) and five octapeptide repeats are responsible for the coordination of bivalent copper ions (Burns et al. 2003). PrPC interacts or associates with a number of proteins including bacterial HSP-60 (Edenhofer et al. 1996), the 37-kDa/67-kDa laminin receptor (Rieger et al. 1997), laminin (Graner et al. 2000), the Grb2 protein, which is central in many signal transduction pathways (Spielhaupter and Schaltz 2001), the lipid raft protein caveolin 1 (Mouillet-Richard et al. 2000), and the neuronal cell adhesion molecule NCAM (Schmitt-Ulms et al. 2001). However, while these studies identified molecules interacting with PrPC, they did not clarify its function (Aguzzi and Hardt 2003; for review). Antibody-mediated cross-linking of PrP on the surface of differentiated 1C11 neuronal cells, mimicking interaction with a ligand, results in activation of p59Fyn kinase (Mouillet-Richard et al. 2000), production of NADPH oxidase-dependent reactive oxygen species and the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), suggesting a coupling of membrane-anchored PrPC to a signal transduction pathway (Schneider et al. 2003). Recently, cis- and trans-association of PrP with NCAM on the surface of mouse neurons was shown to result in recruitment of NCAM to lipid rafts and activation of p59Fyn (Santuccione et al. 2005). A role of PrPC in axon growth was proposed by Sales et al. (2002), who detected increased expression of the protein on the surface of elongating retinal axons in hamsters. A PrP-Fc fusion protein was shown to bind strongly to the granule cell layer of mouse cerebellum, suggesting the presence of a PrPC-interacting ligand (or ligands) in this region (Legname et al. 2002). In studies that did not distinguish axons and dendrites, it was shown that exposure of neonatal mouse cerebellar or hippocampal neurons to a PrP-Fc fusion protein enhanced the total length of neurites by a factor of ≤ 2 (Chen et al. 2003; Santuccione et al. 2005). Recent evidence suggests that the effect of PrP-Fc on neurite outgrowth in cultures of mouse neurons is mediated through its binding to NCAM (Santuccione et al. 2005).
In this study, we have developed an assay to study the effect of recPrP on fetal rat hippocampal neurons in culture. We report that in vitro, purified full-length recPrP with a conformation similar to native PrPC (Pan et al. 1993), dramatically enhances the development of neuronal polarity, including neurite definition and growth, axonal length, formation of neuronal networks and development of synaptic-like contacts. In the same assay, purified N-terminal or C-terminal domains of PrP fail to enhance polarization. Furthermore, purified recombinant Dpl (recDpl) does not enhance polarization and instead appears to be toxic to neurons.