The long-term goal of this application is to identify the function of prion protein (PrP) in the central nervous system. Four octapeptide repeats in the N-terminus of PrP are homologous to the BH2 domain of Bcl2 family of proteins. Based on preliminary observations that PrP interacts with pro-apoptotic Bax proteins and protects yeast and primary human neurons against Bax-mediated cell death, the investigator proposes the hypothesis that interaction between PrP and Bax mediates neuronal survival, and that this mechanism may be implicated in prion disease associated neuronal loss. This hypothesis will be tested in the following Specific Aims.
Aim 1 will determine if PrP interaction with Bax is necessary for the neuroprotective function of PrP. PrP mutants will be generated that prevent PrP-Bax interaction and determining if co-expression of these mutants abolishes PrP's ability to inhibit Bax-mediated cell death. The function of these mutants will be tested in yeast by using galactose inducible constructs and micro-injection of eukaryotic expression constructs in human neurons.
Aim 2 will determine if human PrP mutations associated with prion diseases dysregulate PrP-Bax interaction and PrP neuroprotective function. Mutations of PrP that are known to cause disease will be tested for their ability to interact with Bax and protect yeast or neurons against Bax-mediated cell death.
Aim 3 will determine the location of neuroprotective PrP. The functional location of PrP and Bax will be determined by using mutant PrP and protein trafficking blocks. Primary cultures of human neurons will be used to examine protein localization by subcellular fractionation, beta-galactosidase complementation, pulse-chase experiments, immunofluorescence and confocal microscopy, and immuno-EM.
Aim 4 will determine the molecular mechanism of neuronal protection by PrP.
Specific Aim 5 will determine if PrP interacts with other pro-apoptotic proteins.
In Aim 6, the neuroprotective role of PrP and the possible elimination of this function in PrP mutations associated with familial prion diseases will be assessed in mice models of familial prion diseases. The result of these experiments will clearly define the neuroprotective role of PrP against Bax mediated cell death.
