FLUORESCENCE-BASED STUDIES OF SPHINGOMYELIN AND CERAMIDE

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This proposal seeks to extend research collaborations between the laboratories of Dr. Edward H. Schuchman in the Department of Human Genetics, Mount Sinai School of Medicine in NY, and Dr. Shimon Gatt, in the Department of Biochemistry, Hebrew University-Hadassah School of Medicine in Israel. The main aim is the development of novel approaches for studying the metabolism of two sphingolipids, sphingomyelin and ceramide, in normal cells and cells from patients with two genetic diseases, Niemann-Pick disease (due to acid sphingomyelinase [ASM] deficiency) and Farber disease (due to acid ceramidase [AC] deficiency), as well as the development of novel approaches for the treatment of these disorders. Fluorescence-based methods will be developed to determine whether pre-implantation stage embryos are affected with these diseases and/or to select normal sperm or eggs from carrier individuals for subsequent in vitro fertilization and prevention of the birth of affected children. This research relates to two funded NIH grants awarded to Dr. Schuchman, R01HD 28607, entitled "Acid Sphingomyelinase and Niemann-Pick Disease," and R01 DK 54830, entitled "Ceramidases, Ceramide, and Farber Disease" (estimated start date 12/1/99). R01 HD 28607 will serve as the parent grant for this research. Several approaches for the treatment of Niemann-Pick disease (NPD) will be developed. Aiming towards enzyme replacement therapy for NPD, ASM will be labeled with fluorescent probes and the trafficking (e.g., uptake, tissue distribution, etc) of the fluorescent enzyme will be monitored in cells and animals. Fluorescent ASM has already been prepared for these studies and shown to retain its catalytic activity using in vitro enzyme assay systems. Several novel approaches for treatment of NPD also will be studied: 1) inhibitors of sphingomyelin; 2) competitive of ASM will , aiming at protecting misfolded, mutant ASM from proteolysis; and 3) for improving enzyme replacement therapy for NPD, ASM will be modified in two ways: a) mannose will be linked to ASM, aiming at its increased uptake by the mannose receptor on microphages and Kupffer cells; and b) positively-charged (cationic) groups will be linked to ASM, aiming at its improved location within the lysosomes. Concordant with our efforts to develop therapy for NPD, studies will also be carried out to develop fluorescence-based methods to identify and select normal sperm or eggs from used for in vitro fertilization, preventing or remarkably diminishing the possibility of such barrier individuals having an affected child. For AC, reliable, fluorescence-based assays will continue to be developed, both for in vitro (i.e., using pure enzyme or extracts of cells and tissues), and in situ (i.e., in intact cells) analysis. Fluorescent procedures also will be used for selecting gene- modified cells which over-express AC, aiming at its purification, and inhibitors of AC and ceramide synthetase will be synthesized for evaluating the effects of increasing, or conversely, decreasing levels of cellular ceramide on the metabolism of sphingomyelin and glycolipids, as well as on cell viability.