FLUORESCENCE-BASED STUDIES OF SPHINGOMYELIN AND CERAMIDE

这项提议旨在扩大纽约西奈山医学院人类遗传学系的爱德华·H·舒赫曼博士和以色列希伯来大学哈大沙医学院生物化学系的西蒙·加特博士之间的研究合作。主要目的是开发新的方法来研究两种神经鞘磷脂在正常细胞和两种遗传病患者的细胞中的代谢，尼曼-皮克病(由于酸性鞘磷脂酶[ASM]缺乏)和Farber病(由于酸性神经酰胺酶[AC]缺乏)，以及开发治疗这些疾病的新方法。将开发基于荧光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/或从携带者个人中选择正常的精子或卵子，用于随后的体外受精和预防受影响儿童的出生。这项研究涉及授予舒赫曼博士的两项基金资助，R01HD 28607，题为“酸性鞘磷脂酶和尼曼-皮克病”，R01 DK 54830，题为“神经酰胺酶，神经酰胺和法伯病”(预计开始日期为1999年12月1日)。R01 HD 28607将作为这项研究的父母补助金。将开发几种治疗尼曼-皮克病(NPD)的方法。针对NPD的酶替代疗法，将用荧光探针标记ASM，并将监测荧光酶在细胞和动物中的转运(例如，摄取、组织分布等)。已经为这些研究准备了荧光ASM，并在体外酶分析系统中显示出保持其催化活性。还将研究治疗NPD的几种新方法：1)鞘磷脂抑制剂；2)竞争ASM Will，旨在保护错误折叠的、突变的ASM免受蛋白质降解；以及3)为了改进NPD的酶替代疗法，ASM将通过两种方式进行修饰：a)将甘露糖连接到ASM，旨在增加其在巨噬细胞和库普弗细胞上的甘露糖受体的摄取；b)将正电荷基团连接到ASM，旨在改善其在溶酶体中的位置。与我们开发NPD治疗方法的努力相一致，我们还将开展研究，开发基于荧光的方法来识别和选择用于体外受精的正常精子或卵子，以防止或显著降低此类障碍个体感染儿童的可能性。对于AC，将继续开发可靠的、基于荧光的分析方法，无论是体外分析(即使用纯酶或细胞和组织的提取物)还是原位分析(即在完整的细胞中)。为了纯化AC，还将利用荧光程序选择过表达AC的基因修饰细胞，并将合成AC和神经酰胺合成酶的抑制剂，以评估增加或相反降低细胞神经酰胺水平对神经鞘蛋白和糖脂代谢以及细胞活力的影响。

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.