Exploring the role of the prion protein in muscle paradigms and in primary cultured cerebellar granules cells

The elusive function of PrPC hampers the understanding of the molecular mechanism at the basis of prion diseases, and the development of suitable therapeutic protocols. A wealth of evidence has nonetheless suggested that the function of the protein, ranging from cell signalling to differentiation to protection against oxidative and apoptotic injuries, is beneficial to the cell, but the detailed comprehension of its physiology remains poor. We have undertaken the functional analysis of PrPC using experimental paradigms novel to the prion field, composed of ex vivo, in vivo and in vitro models derived from wild-type, PrP-knockout, and PrP-overexpressing (about 4-fold the physiologic levels) congenic mice. Because among extra-neural tissues, muscles have been correlated to PrPC patho-physiology, we have exploited an ex vivo muscle paradigm consisting of isolated hearts subjected to ischemia/reperfusion protocols that involve oxidative challenge, and an in vivo paradigm provided by the hind-limb muscle subjected to degeneration/regeneration protocol. By subjecting these muscles to acute stress protocols, and by analysing the response in relation to the different PrP genotypes, we have assessed the involvement of PrPC in the cell defence against oxidative injury and in adult tissue morphogenesis, respectively. Ca2+ is the most important second messenger of the cell, and governs several physiologic events, including cell differentiation, while the derangement of Ca2+ homeostasis is known to severely contribute to oxidative stress injuries. Several reports have indicated that PrPC is intimately related to the Ca2+ homeostasis, and on this basis we have monitored the local Ca2+ fluxes in cerebellar granule cells (CGC) - derived from the same murine lines previously described - using recombinant Ca2+-sensitive photo-proteins, aequorins, genetically targeted to different cell domains/organelles. Our results indicate that in the absence of PrPC modifications of Ca2+ movements occur in the cytosolic domains beneath the plasma membrane, in the lumen of the endoplasmic reticulum and in the mitochondrial matrix. All these results strongly indicate a close link between PrPC and local Ca2+ movements, thereby strengthening the hypothesis that Ca2+ might be the cellular effector of cell signalling pathways mediated by PrPC.