The objectives of my proposed investigations are to define the mechanism by which prions are generated de-novo in the inherited prion diseases and to develop as a physician-scientist. I plan to study the molecular genetics of prion disease using several approaches. Denaturing gradient gel electrophoresis (DGGE) will be used to survey patients for mutations of the PrP gene that produce inherited neurological diseases, and for polymorphisms which may alter the phenotypic expression of CNS degeneration. All genetic alterations detected by DGGE will be assessed by DNA sequencing. I plan to determine if humans carrying a mutation of the PrP gene produce infectious prions that are detectable in their blood, cerebrospinal fluid or other body fluids prior to the onset of neurologic dysfunction. Transgenic (Tg) mice permissive for transmission of human (Hu) prions will be used in my studies. A comparison of the clinical and neuropathologic features of the neurological disease that occurs in mice harboring the mouse (Mo) PrP transgene which carries the P102L mutation is planned. I shall determine if these animals produce infectious prions that are detectable in the blood, cerebrospinal fluid or other body fluids prior to the onset of neurologic dysfunction. To investigate a possible therapeutic approach, I shall attempt to prevent transmission of human CJD prions to Tg mice by co-expressing HuPrPc and a C-terminal fragment of MoPrPpc. The C-terminus of PrPc is thought to bind to protein X, a putative molecular chaperone involved in the conversion of PrPc into PrPSc. Competitive inhibition of HuPrPpc binding to protein X by over expression of a MoPrPc C-terminal fragment, may prevent disease. In addition, propose to model sporadic and inherited prion disease in cultured human neuronal cells by infecting some with human prions and transfecting others with the HuPrP gene containing either the P102L or E200K mutation. If HuPrPSc is formed and Hu prion infectivity is produced in these cells, I shall attempt to reverse the infection by exogenous peptide administration. My studies will examine the phenotypic and genotypic diversity of prion disease, and investigate the biogenesis of prions in humans, transgenic animals, and post mitotic human neurons which carry a mutation of the PrP gene. Targeting protein X for gene therapy in prion diseases may offer a unique approach to the treatment of neurodegenerative disorders.
