SC COBRE: REGULATION OF MAMMALIAN FATTY ACID ALPHA-HYDROXYLASE GENE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。在高等脊椎动物中，髓鞘极大地促进了神经传导，髓鞘是一种包裹在轴突周围的富含脂质的膜。已知许多破坏性神经退行性疾病会导致病理性脱髓鞘。髓鞘高度富含两种含半乳糖的神经鞘糖脂，即半乳糖神经酰胺(GalCer)和硫脂。这些髓鞘糖脂的一个独特特征是，GalCer和硫脂中大约一半的脂肪酸是2-羟基脂肪酸。没有其他哺乳动物组织含有如此高浓度的2-羟基脂肪酸，这表明2-羟基在髓鞘中具有独特的作用。虽然GalCer和硫脂在髓鞘形成中的作用已知很多，但这些糖鞘糖脂中2-羟基的具体功能尚不清楚。我们推测髓鞘糖脂的2-羟基化是髓鞘功能所必需的。为了验证我们的假设，我们克隆了人类脂肪酸2-羟基酶基因FA2H，并建立了一个基于GC/MS的高灵敏检测系统。这些工具将被用于实现以下特定目标：1)证明人类FA2H基因产物是一种脂肪酸2-羟基酶；2)证明FA2H在脑中高表达；3)构建靶向载体以产生FA2H基因敲除小鼠。我们已经证明了过量表达人FA2H的组织培养细胞中2-羟基脂肪酸和脂肪2-羟基酶活性增加。Northern印迹分析表明，FA2H在脑内高度表达。小鼠脑内2-羟基脂肪酸和脂肪酸2-羟基羧酸酶活性在活跃的髓鞘形成过程中急剧增加，并随着动物年龄的增长而下降。这些结果提供了FA2H依赖的脂肪酸2-羟基化是髓鞘中2-羟基鞘磷脂生物合成所必需的证据。目前正在构建产生FA2H基因敲除小鼠的靶向载体。利用FA2H基因敲除小鼠，我们将确定脂肪酸2-羟基酶在体内髓鞘形成中的作用

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In higher vertebrates, nerve conduction is greatly facilitated by myelin, a lipid-rich membrane that wraps around the axons. A number of devastating neurodegenerative diseases are known to cause pathological demyelination. Myelin is highly enriched with two galactose-containing sphingolipids, galactosylceramide (GalCer) and sulfatide. A unique feature of these myelin glycosphingolipids is that approximately one half of the fatty acids in GalCer and sulfatide are 2-hydroxy fatty acids. There are no other mammalian tissues that contain such high concentrations of 2-hydroxy fatty acids, suggesting that the 2-hydroxyl group has a unique role in myelin. Although much is known about the role of GalCer and sulfatide in myelination, specific functions of the 2-hydroxyl group in these glycosphingolipids are not known. We hypothesize that 2-hydroxylation of myelin glycosphingolipids is necessary for myelin function. To test our hypothesis, the human fatty acid 2-hydroxylase gene, FA2H, has been cloned, and a highly sensitive GC/MS-based assay system has been developed. These tools will be applied to pursue the following specific aims: 1) to prove that the human FA2H gene product is a fatty acid 2-hydroxylase; 2) to demonstrate that FA2H is highly expressed in brain; 3) to construct a targeting vector to generate FA2H-knockout mice. We have demonstrated increased 2-hydroxy fatty acids and fatty 2-hydroxylase activities in tissue culture cells overexpressing human FA2H. Northern blot analysis showed that FA2H is highly expressed in brain. Mouse brain 2-hydroxy fatty acids and fatty acid 2-hdyroyxlase activities sharply increased during active myelination and decreased as animals aged. These results provide evidence that FA2H-dependent fatty acid 2-hydroxylation is required for the biosynthesis of 2-hydroxy sphingolipids in myelin. Targeting vectors to generate FA2H-knockout mice are currently under construction. With FA2H-knockout mice, we will determine the role of fatty acid 2-hydroxylase in myelination in vivo