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：
8661160
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/8661160
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 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 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.

描述（由申请人提供）：本申请要求为我们的项目继续资助，以研究酸性神经酰胺酶（AC）在哺乳动物发育和疾病发病机理中的作用。在上一个资金期间，我们有：a）表明AC对于超出2细胞阶段的胚胎生存至关重要，b）证明AC是卵母细胞发育的关键组成部分，可用于预防卵母细胞和胚胎细胞凋亡，c）揭示了一种新型的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 Recombinase（ACCKO/CRETM）的“ Floxed” AC条件敲除小鼠，并显示出可行的他莫昔芬注射后可以由这些动物产生Acko - / - 小鼠。在即将到来的资金期间，我们将通过追求以下两个相互关联的目的来扩展这些发现：1）构造和表征第一个可行的AC缺陷鼠标模型。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AC活性的普遍敲除。我们还将将ACCKO小鼠繁殖为细胞特异性（巨噬细胞和浦肯野细胞）Cre小鼠，并使用这些动物评估AC的细胞特异性功能。所得的AC缺乏的小鼠和胚胎将在病理，生化和（对于临床生存的患者）上进行表征。如果我们从这些实验中获得的小鼠没有与人类AC缺乏症相关的表型（Farber疾病），我们还将使用突变“敲入”策略创建“ hypomorph”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理中的作用，并为研究人员提供最初可行的AC缺乏模型。 2）进一步探索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绝佳模型，并且我们在上一个资金期间的工作表明，AC对这一过程至关重要。为了进一步追求这些发现，将三种不同的Cre小鼠（GDF9-CRE，ZP3-CRE和MSX2-CRE）用于在AIM 1的ACCKO小鼠繁殖后，在不同阶段的卵泡发生阶段灭活AC。随着超排卵及其发育阶段（GV，MI，MII）的卵母细胞数量，将确定和比较。我们还将使用TM在卵巢组织中灭活AC，并研究成熟卵母细胞对重组AC的摄取和运输。这些研究的目的是对AC在卵母细胞发育中的作用有了更完整的了解，并对AC在体外成熟和受精中的影响更深入地欣赏。