MYELINATION--ASSEMBLY OF LIPIDS AND PROTEINS

髓鞘形成——脂质和蛋白质的组装

• 批准号: 3395115
• 负责人: JOYCE A BENJAMINS
• 金额: $ 14.73万
• 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/3395115
• 关键词: Golgi apparatus; cell membrane; developmental neurobiology; galactolipids; histochemistry /cytochemistry; immunochemistry; immunologic techniques; intracellular transport; 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.

该提案的目的是定义机制 调节细胞内脂质和蛋白质的运输 髓磷脂。 一组实验表征了 蛋白脂蛋白 (PLP) 和硫苷脂的位点 合成到组装成髓磷脂或少突胶质细胞 (OL) 血浆 膜。 PLP 和脑硫脂共同转运的假设 将通过比较分布和代谢来研究 PLP 与细胞内池中的硫苷脂。 硫脂将会 与 PLP 相比，关于 (1) 积累位点 运输被离子载体或破坏剂阻断 细胞骨架：（2）细胞内囊泡和OL的定向 质膜； (3) 髓磷脂池外的长期周转。 这些研究将利用体内和体外标记 (3H)亮氨酸和(35S)硫酸盐，以及亚细胞分级分离 从大鼠脑干中分离出整个组织和 OL。 OL 及其膜片的免疫细胞化学染色 培养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 半乳糖脑苷脂：培养的 OL 和随后的抗体复合物 对 OL 膜和代谢的影响。 GalC Ab 对的影响 OL 将与 Ab 对其他糖脂的影响进行比较 凝集素花生凝集素。 在第二个存在的情况下抗体到 GalC Ab 导致 OL 膜上的 GalC 修补，并且 GalC:Ab 复合物迅速内化。 这些事件的敏感性 代谢抑制剂、离子载体和破坏剂 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 由 GalC:Ab 复合物的内化刺激。 的接触 OL 与 GalC Ab 反应 2-24 小时，导致可逆缩合 膜片；细胞骨架在这种变化中的作用是 通过免疫细胞化学和生化方法进行研究。 这 持续暴露于 GalC Ab 会改变增殖的假设 并通过比较来调查 OL 的成熟度 (3H)胸苷的掺入和GalC的出现时间， 处理和对照培养物中的 2'3'-CNP、碱性蛋白和 PLP。 这些研究有助于进一步理解和最终 治疗涉及髓磷脂的疾病。 表征 控制髓磷脂组装和 OL 功能的机制将提供 有关正常髓鞘形成调节的重要信息 损伤后的发育和髓鞘再生。