This proposal is part of a major initiative to establish a role for 14-3-3 in prion diseases; to develop isoform specific ELISA tests and to compare results with current diagnostic tools. The finding that 14-3-3 isoforms (spots 130 and/or 131) are a highly sensitive and specific surrogate marker in the two dimensional gel electrophoresis spinal fluid (CSF) test for the diagnosis in the clinical stages of Creutzfeldt-Jakob disease (CJD) does not reveal the biological mechanism of onset or transmissibility of the disease. Neither do we know precisely which of the seven distinct known mammalian gene products of 14-3-3 or their many alternative forms and post-translational modifications are involved nor why specific isoforms of 14-3-3 are present in spinal fluid. We wish to test changes in subcellular localisation and/or post-translational modification of 14-3-3 isoforms in neural tissue and the appearance in spinal fluid in the early stages of the disease before clinical symptoms are manifest. This will be addressed by determining appearance of 14-3-3 in spinal fluid above a measurable threshold using an established scrapie model in sheep and in mouse. We will also use the monoclonals and other antisera for analysing temporal changes in localisation of 14-3-3 isoforms by immuno-electron microscopy of 14-3-3. Developing an ELISA test would enable the widespread screening of patients suspected of having the disease. It will be invaluable for establishing the earliest diagnosis of prion disease, essential if the 14-3-3 spinal fluid assay is to be inked to therapy to arrest the progress of the disease while quality of life is still good.
Secondly, the ELISA test would also enable the widespread screening of cattle and sheep, particularly if a blood test could be developed. The earliest diagnosis will be of great value in agriculture, to alert the possibility of an outbreak of BSE or scrapie and to prevent contaminated animal products entering the food chain. Knockout mice deficient in specific isoforms of 14-3-3 in specific tissues will be produced. We will then infect the knockout mice with scrapie and test in an established model system whether susceptibility is altered. We will identify proteins that interact with 14-3-3 by transfection of 14-3-3 in neuroblastoma cell lines and look for these in CSF. 14-3-3 proteins mediate interactions between a wide variety of proteins. These interactions are regulated by phosphorylation of both 14-3-3 and its target proteins. We have shown that short phosphopeptides can block these interactions. In the long term there may be some application for drug development since it may be possible to design short peptide mimetics which will impede the formation of complexes specific for a particular 14-3-3 isoform(s) that could be involved in transport of prion proteins.
Funding SchemeCSC - Cost-sharing contracts