FLUORESCENCE-BASED STUDIES OF SPHINGOMYELIN AND CERAMIDE

该提案旨在扩大爱德华·H·卡耐基博士的实验室之间的研究合作。纽约西奈山医学院人类遗传学系的Schuchman博士和以色列希伯来大学哈达萨医学院生物化学系的Shimon Gatt博士。主要目的是开发新的方法来研究两种鞘脂，鞘磷脂和神经酰胺，在正常细胞和两种遗传性疾病，尼曼-皮克病（由于酸性鞘磷脂酶[ASM]缺乏症）和法伯病（由于酸性神经酰胺酶[AC]缺乏症）患者的细胞中的代谢，以及开发治疗这些疾病的新方法。将开发基于受精的方法来确定植入前阶段的胚胎是否受到这些疾病的影响，和/或从携带者个体中选择正常的精子或卵子进行随后的体外受精和预防受影响儿童的出生。本研究涉及授予Schuchman博士的两项NIH赠款，R 01 HD 28607，题为“酸性鞘磷脂酶和尼曼-皮克病”，R 01 DK 54830，题为“神经酰胺酶、神经酰胺和法伯病”（估计开始日期12/1/99）。R 01 HD 28607将作为本研究的母基金。将开发几种治疗尼曼-匹克病（NPD）的方法。针对NPD的酶替代疗法，ASM将用荧光探针标记，并且运输（例如，摄取、组织分布等）将在细胞和动物中进行监测。荧光ASM已经为这些研究做好了准备，并使用体外酶测定系统显示保留了其催化活性。还将研究几种治疗NPD的新方法：1）鞘磷脂的抑制剂; 2）ASM的竞争性，旨在保护错误折叠的突变ASM免于蛋白水解;和3）为了改善NPD的酶替代疗法，ASM将以两种方式修饰：a）甘露糖将与ASM连接，旨在增加其被微囊和枯否细胞上的甘露糖受体的摄取;和B）带正电荷的（阳离子）基团将与ASM连接，目的是改善其在溶酶体内的位置。与我们开发NPD治疗方法的努力一致，还将进行研究以开发基于荧光的方法，以识别和选择用于体外受精的正常精子或卵子，预防或显着减少这种障碍个体生育受影响孩子的可能性。对于AC，将继续开发可靠的基于荧光的测定法，用于体外（即，使用纯酶或细胞和组织的提取物），和原位（即，在完整细胞中）分析。还将使用荧光程序来选择过表达AC的基因修饰的细胞，目的在于其纯化，并且将合成AC和神经酰胺合成酶的抑制剂以评估增加或相反地降低细胞神经酰胺水平对鞘磷脂和糖脂代谢以及对细胞活力的影响。

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.