Acid Ceramidase, Ceramide and Farber Disease

酸性神经酰胺酶、神经酰胺和法伯病

• 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
• 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' 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

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。在上一个资助期内，我们有：a）表明AC对于超过2-细胞期的胚胎存活是必需的，B）证明AC是卵母细胞发育的关键组分，并且可以用于体外防止卵母细胞和胚胎凋亡，c）揭示了AC加工和活化的新的自催化机制，并且鉴定了一类新的AC抑制剂，和d）构建的携带诱导型Cre重组酶（ACcKO/CreTM）的“floxed”AC条件性敲除小鼠，并显示在他莫昔芬注射后可从这些动物产生存活的ACKO-/-小鼠。在即将到来的资助期间，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）构建和表征第一个AC缺乏症的可行小鼠模型。我们将继续使用ACcKO/CreTM小鼠在胚胎发育的不同阶段和出生后诱导AC活性的普遍敲除。我们还将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。将对所得AC缺陷小鼠和胚胎进行病理学、生物化学和（对于存活的）临床表征。如果我们从这些实验中获得的小鼠不具有与人类AC缺乏症（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一个很好的模型，我们在上一个资助期的工作表明，AC对这一过程至关重要。为了进一步研究这些发现，将使用三种不同的Cre小鼠（GDF 9-Cre、ZP 3-Cre和Msx 2-Cre）在与目的1中产生的ACcKO小鼠交配后的卵泡发生的不同阶段对AC进行传代。将测定并比较卵泡形态、超数排卵后获得的卵母细胞数量及其发育阶段（GV、MI、MII）、受精能力和凋亡率。我们还将使用TM在卵巢组织中重组AC，并研究成熟卵母细胞对重组AC的摄取和运输。这些研究的目的是获得一个更完整的了解AC在卵母细胞发育中的作用，并提供一个更深层次的机制如何AC影响体外成熟和受精的赞赏。