THE MOLECULAR BASIS OF PROGRESSIVE MYOCLONUS EPILEPSY: A MULTIPROTEIN COMPLEX WITH CYSTATIN B

Progressive myoclonous epilepsy (EPM1) is a degenerative disease of the central nervous system, caused by mutations of the cystatin B gene. In vitro, cystatin B interacts with and inhibits proteins of the cathepsin family of proteases. The study of the sub-cellular localization of cystatin B and cathepsin B by confocal microscopy, shows that, in vivo, the two proteins are located in different cellular compartments. Cystatin B, in proliferating cells, is mainly nuclear while in differentiated cells is found in the nucleus and in the cytoplasm. Cathepsin B is essentially cytoplasmic in either case. Cystatin B is ubiquitary but the damage caused by EPM1 is in the central nervous system. We have found interaction of cystatin B with at least two proteins specific of the central nervous system and shown the presence of a multiprotein complex which may be altered in the EPM1 disease. We are now proposing the following set of experiments to further characterize the molecular mechanisms underlying the cystatin B function and the EPM1 disease. 1. A detailed study of the nature of the cystatin B multiprotein complex to find out whether, in addition to cystatin B, the assembled proteins are involved with the disease. 2. The study, in vitro and in vivo, of the expression of the cystatin B natural mutants described in EPM1 patients, and of their interaction with the partner proteins. 3. The generation of a transgenic mouse expressing a cystatin B substitution mutant that will be crossed with homozygous cystb-/- mice, in order to generate animals over-expressing a cystatin B mutant on a homozygous cystb-/- background. Such mouse should represent a EPM1 animal model with characteristics closely resembling the genetic background of the human disease.