Acid Ceramidase, Ceramide and Farber Disease

酸性神经酰胺酶、神经酰胺和法伯病

基本信息

批准号：
8224238
负责人：
EDWARD H. SCHUCHMAN
金额：
$ 37.97万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-01-15 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8224238
关键词：
16 year old 4-Hydroxy-Tamoxifen Adult Animals Apoptosis Assisted Reproductive Technology Birth Breeding Cattle Cells Ceramides Complementary DNA Data Development Discipline Disease Embryo Embryonic Development Enzymes Event Farber&apos s lipogranulomatosis Fertility Fertilization Fertilization in Vitro Funding GDF9 gene Genes Goals Human In Vitro Injection of therapeutic agent Knock-in Mouse Knock-out Knockout Mice Length Metabolism Modeling Morphology Mus Mutation Oocytes Organ Outcome Ovarian Tissue Pathogenesis Patients Phenotype Process Proteins Public Health Purkinje Cells Reagent Recombinants Regulation Request for Applications Research Research Personnel Residual state Role Signal Transduction Sphingolipids Staging Study models Superovulation Tamoxifen Transgenic Mice Work base cDNA Expression clinically relevant folliculogenesis galactosylgalactosylglucosylceramidase human disease in vivo inhibitor/antagonist insight macrophage mouse model new technology novel older patient oocyte maturation prevent promoter public health relevance pup recombinase research study tool trafficking translational medicine uptake zona pellucida glycoprotein

项目摘要

DESCRIPTION (provided by applicant): This application requests continued funding for our project to investigate the role of acid ceramidase (AC) in mammalian development and disease pathogenesis. During the previous funding period we have: a) shown that AC is essential for embryo survival beyond the 2-cell stage, b) demonstrated that AC is a critical component of oocyte development, and can be used to prevent oocyte and embryo apoptosis in vitro, c) revealed a novel, autocatalytic mechanism of AC processing and activation, and identified a new class of AC inhibitors, and d) constructed "floxed" AC conditional knockout mice carrying an inducible Cre recombinase (ACcKO/CreTM), and shown that viable ACKO-/- mice can be produced from these animals following tamoxifen injection. In the upcoming funding period we will extend these findings by pursuing the following two, interrelated aims: 1) Construct & characterize the first viable mouse models of AC deficiency. We will continue to use the ACcKO/CreTM mice to induce the ubiquitous knockout of AC activity at different stages of embryogenesis and after birth. We will also breed ACcKO mice to cell-specific (macrophage and Purkinje cell) Cre mice, and use these animals to evaluate the cell-specific functions of AC. The resulting AC deficient mice and embryos will be characterized pathologically, biochemically, and (for those that survive) clinically. In the event that the mice we obtain from these experiments do not have a phenotype that is clinically relevant to human AC deficiency (Farber disease), we will also create "hypomorph" mice using a mutation "knock-in" strategy. The goal of these studies is to gain further insights into the role of AC in mammalian development and disease pathogenesis, and to provide researchers with the first viable models of AC deficiency. 2) Further explore the in vivo role of AC in oocyte development. Oocytes are an excellent model for the study of mammalian apoptosis, and our work during the previous funding period has shown that AC is critical to this process. To pursue these findings further, three different Cre mice (GDF9-Cre, ZP3-Cre and Msx2-Cre) will be used to inactivate AC at different stages of folliculogenesis after breeding to the ACcKO mice produced in aim 1. Follicle morphology, as well as the number of oocytes retrieved following superovulation and their stage of development (GV, MI, MII), fertilization capacity, and apoptosis rates will be determined and compared. We will also use TM to inactivate AC in ovarian tissue, and study the uptake and trafficking of recombinant AC by mature oocytes. The goal of these studies is to gain a more complete understanding of AC's role in oocyte development, and to provide a deeper mechanistic appreciation of how AC influences in vitro maturation and fertilization. PUBLIC HEALTH RELEVANCE: AC is a key enzyme in the regulation of sphingolipid metabolism and signaling, and abnormal AC expression has been observed in many human diseases. Our previous work has provided many of the essential research tools needed to study this enzyme, and revealed its critical role in early development. The research we are proposing in the upcoming funding period will continue to provide important new information about this protein, and should impact a wide range of translational medicine disciplines (e.g., lysosomal disease research, fertility, sphingolipid signaling, etc).

描述（由申请人提供）：本申请要求继续资助我们的项目，以研究酸性神经酰胺酶（AC）在哺乳动物发育和疾病发病机制中的作用。在之前的资助期间，我们：a) 证明 AC 对于 2 细胞阶段之后的胚胎存活至关重要，b) 证明 AC 是卵母细胞发育的关键组成部分，可用于在体外预防卵母细胞和胚胎凋亡，c）揭示了一种新的AC加工和激活的自催化机制，并鉴定了一类新的AC抑制剂，d）构建了携带可诱导Cre重组酶（ACcKO/CreTM）的“floxed”AC条件敲除小鼠，并显示注射他莫昔芬后可以从这些动物中产生可存活的 ACKO-/- 小鼠。在即将到来的资助期内，我们将通过追求以下两个相互关联的目标来扩展这些发现：1）构建并表征第一个可行的 AC 缺乏小鼠模型。我们将继续使用 ACcKO/CreTM 小鼠在胚胎发生的不同阶段和出生后诱导普遍存在的 AC 活性敲除。我们还将把 ACcKO 小鼠培育成细胞特异性（巨噬细胞和浦肯野细胞）Cre 小鼠，并使用这些动物来评估 AC 的细胞特异性功能。由此产生的 AC 缺陷小鼠和胚胎将在病理学、生化和（对于存活的小鼠）临床上进行表征。如果我们从这些实验中获得的小鼠不具有与人类 AC 缺陷（法伯病）临床相关的表型，我们还将使用突变“敲入”策略创建“亚型”小鼠。这些研究的目的是进一步了解 AC 在哺乳动物发育和疾病发病机制中的作用，并为研究人员提供第一个可行的 AC 缺乏模型。 2）进一步探讨AC在卵母细胞发育中的体内作用。卵母细胞是研究哺乳动物细胞凋亡的优秀模型，我们在上一个资助期间的工作表明 AC 对此过程至关重要。为了进一步研究这些发现，三种不同的 Cre 小鼠（GDF9-Cre、ZP3-Cre 和 Msx2-Cre）将在与目标 1 中产生的 ACcKO 小鼠繁殖后，用于在卵泡发生的不同阶段灭活 AC。确定并比较超排后取出的卵母细胞数量及其发育阶段（GV、MI、MII）、受精能力和细胞凋亡率。我们还将利用TM灭活卵巢组织中的AC，并研究成熟卵母细胞对重组AC的摄取和运输。这些研究的目的是更全面地了解 AC 在卵母细胞发育中的作用，并更深入地了解 AC 如何影响体外成熟和受精。公共健康相关性：AC 是调节鞘脂代谢和信号转导的关键酶，在许多人类疾病中都观察到 AC 表达异常。我们之前的工作提供了研究这种酶所需的许多重要研究工具，并揭示了它在早期发育中的关键作用。我们在即将到来的资助期内提出的研究将继续提供有关这种蛋白质的重要新信息，并且应该会影响广泛的转化医学学科（例如，溶酶体疾病研究、生育能力、鞘脂信号传导等）。