MYELINATION--ASSEMBLY OF LIPIDS AND PROTEINS

髓鞘形成——脂质和蛋白质的组装

• 批准号: 3395111
• 负责人: JOYCE A BENJAMINS
• 金额: $ 14.35万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-04-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3395111
• 关键词: Golgi apparatus; cell membrane; developmental neurobiology; galactolipids; histochemistry /cytochemistry; immunochemistry; immunologic techniques; laboratory mouse; laboratory rabbit; laboratory rat; lipid biosynthesis; lipogenesis inhibitor; membrane lipids; membrane proteins; monensin; myelin; myelin proteolipid; myelination; neuronal transport; oligodendroglia; paclitaxel; protein biosynthesis; protein transport; radiotracer; tissue /cell culture; western blottings

The objective of this proposal is to define the mechanisms regulating intracellular transport of lipids and proteins to myelin. One set of experiments characterizes transport of proteolipid protein (PLP) and sulfatide from their sites of synthesis to assembly into myelin or oligodendroglial (OL) plasma membrane. The hypothesis that PLP and sulfatide are co-transported will be investigated by comparing distribution and metabolism of PLP with sulfatide in intracellular pools. Sulfatide will be compared to PLP with regard to (1) sites of accumulation when transport is blocked by ionophores or agents which disrupt cytoskeleton: (2) orientation in intracellular vesicles and OL plasma membrane; and (3) long-term turnover in extra-myelin pools. These studies will utilize in vivo and in vitro labelling with (3H)leucine and (35S)sulfate, and subcellular fractionation of whole tissue and OL isolated from rat brainstem. Immunocytochemical staining of OL and their membrane sheets in culture will directly visualize these intracellular pools. The second set of experiments characterizes intracellular transport of galactocerebroside:Ab complexes in cultured OL and subsequent effects on OL membranes and metabolism. The effects of GalC Ab on OL will be compared to the effects of Ab to other glycolipids and the lectin peanut agglutinin. Ab to GalC in the presence of second Ab causes patching of GalC on OL membranes, and the GalC:Ab complexes are rapidly internalized. Sensitivity of these events to metabolic inhibitors, ionophores and agents which disrupt cytoskeleton will be examined. Metabolism of GalC will be measured by incorporation of (3H)galactose to determine if its synthesis is stimulated by internalization of GalC:Ab complexes. Exposure of OL to GalC Ab for 2-24 hours results in reversible condensation of membrane sheets; the role of cytoskeleton in this change will be investigated by immunocytochemical and biochemical methods. The hypothesis that continued exposure to GalC Ab alters proliferation and maturation of OL will be investigated by comparing incorporation of (3H)thymidine and time of appearance of GalC, 2'3'-CNP, basic protein and PLP in treated and control cultures. These studies are relevant to further understanding and eventual treatment of diseases involving myelin. Characterization of mechanisms controlling myelin assembly and OL function will provide significant information about regulation of myelination in normal development and remyelination following damage.

该提议的目的是定义机制 调节脂质和蛋白质的细胞内转运 髓线。 一组实验表征了运输的 蛋白脂蛋白蛋白（PLP）和硫酸硫化物的位置 合成组装到髓磷脂或寡头（OL）等离子体中 膜。 PLP和亚磺胺是共同传输的假设 将通过比较分布和代谢来研究 PLP在细胞内池中用亚硫酸盐。 亚硫酸盐将是 与（1）累积位点相比 运输被破坏的离子载体或试剂阻塞 细胞骨架：（2）细胞内囊泡和OL的方向 质膜； （3）在膜林池外的长期营业额。 这些研究将使用体内和体外标记 （3H）亮氨酸和（35s）硫酸盐，以及亚细胞分级 从大鼠脑干中分离出的整体组织和OL。 OL及其膜片的免疫细胞化学染色 文化将直接可视化这些细胞内池。 这 第二组实验表征了细胞内转运 半乳脑尿素：培养的OL和随后的AB复合物 对OL膜和代谢的影响。 galc ab对 将OL与AB对其他糖脂的影响进行比较 凝集素花生凝集素。 在第二次存在的情况下AB到Galc AB会导致galc在OL膜上的修补，而GALC：AB：AB 复合物是迅速内在化的。 这些事件的敏感性 代谢抑制剂，离子载体和破坏药物 将检查细胞骨架。 将测量GALC的代谢 通过掺入（3H）半乳糖来确定其合成是否为 通过galc的内在化刺激：AB复合物。 的接触 OL到Galc AB持续2-24小时导致可逆的凝结 膜片；细胞骨架在这一变化中的作用将是 通过免疫细胞化学和生化方法研究。 这 持续暴露于galc ab的假设改变了增殖 通过比较，将研究OL的成熟 （3H）胸苷掺入和GALC的外观时间， 2'3'-CNP，经过处理和控制培养物中的基本蛋白质和PLP。 这些研究与进一步的理解和最终有关 治疗涉及髓鞘的疾病。 表征 控制髓鞘组装和OL功能的机制将提供 有关正常髓鞘化调节的重要信息 损坏后的开发和再髓。