MYELINATION--ASSEMBLY OF LIPIDS AND PROTEINS

髓鞘形成——脂质和蛋白质的组装

基本信息

批准号：
2037014
负责人：
JOYCE A BENJAMINS
金额：
$ 22.3万
依托单位：
WAYNE STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1977
资助国家：
美国
起止时间：
1977-04-01 至 1998-11-30
项目状态：
已结题

项目摘要

DESCRIPTION: (Applicant's Abstract) Changes in the ionic and cellular environment in and around multiple sclerosis plaques affect the ability of mature OLs to maintain myelin sheaths and reform myelin after injury. Understanding the regulation of myelin assembly and maintenance in mature OLs will lead to strategies for blocking demyelination or enhancing remyelination in demyelinated areas. Previous studies from this laboratory have shown that a sustained increase in intracellular Ca++ levels in cultured OLs leads to a reversible retraction of membrane sheets. To further understand the relationship between Ca++ levels and maintenance of membrane sheets, they will examine the capacity of mature OLs to regulate calcium when exposed to ionophores, depolarization, inhibition or activation of voltage-gated Ca++ channels, or release of intracellular calcium. Morphology and maintenance of membrane sheets will be assessed by immunocytochemical staining and quantitative morphometry. Cell viability will be evaluated by visualization of nuclear lysis with propidium iodide and assay of mitochondrial function. Changes in Ca++ levels will be measured with Indo-1 and lase cytometry imaging. Agents identified as altering Ca++ levels, damaging mature OLs, or disrupting membrane sheets will be examined for their effects on the pattern of IEG and myelin protein gene expression. The IEGs to be examined are c-fos, c-jun, jun b, HLH 462 and zif 268, selected because of their known activity as transcription factors and their responsiveness to injury in OLs or other cell types. Myelin protein genes are likely to be downstream targets of IEG changes. In situ hybridization, immunocytochemistry, and Western and Northern blotting will be used to examine expression of the genes. The pattern of changes will be correlated with the ability of OLs to recover and maintain or reform membrane sheets. Another series of experiments will examine a critical end point in myelin assembly, the transport of intracellular vesicles containing PLP/DM-20 into and out of the plasma membrane of OL and associated membrane sheets. The balance between insertion and removal of PLp/DM-20 in the plasma membrane of OL cell bodies and membrane sheets will be examined by exposing cells to inhibitors of endocytosis. Effects of co-culture with neurons from dorsal root ganglia and the effects of upregulation of PLP/DM-20 synthesis with cyclic AMP will be assessed using immunocytochemistry and iodination of surface PLP. Related studies of insertion of galactocerebroside (GalC) and sulfatide into the extracellular face of the plasma membrane will be carried out in the immortalized OL cell line N20.1 which synthesizes the galactolipids but does not express them on the surface of the cell.

描述：（申请人的摘要）离子和细胞的变化多发性硬化斑块内及其周围的环境影响能力成熟的 OL 可以维持髓鞘并在损伤后重塑髓鞘。了解成熟期髓磷脂组装和维护的调节 OLs 将导致阻止脱髓鞘或增强脱髓鞘作用的策略脱髓鞘区域的髓鞘再生。以前的研究从这里实验室表明，细胞内 Ca++ 持续增加培养的 OL 水平会导致膜可逆性收缩床单。进一步了解Ca++水平与膜片的维护，他们会检查成熟的能力当暴露于离子载体、去极化、抑制或激活电压门控 Ca++ 通道，或释放细胞内钙。膜片的形态和维护将通过免疫细胞化学染色和定量进行评估形态测量。细胞活力将通过可视化进行评估用碘化丙啶进行核裂解并测定线粒体功能。 Ca++ 水平的变化将通过 Indo-1 和激光细胞术测量成像。被确定为改变 Ca++ 水平、损害成熟 OL 的药物，或破坏膜片将检查其对 IEG 和髓磷脂蛋白基因表达模式。 IEG 将检查的是 c-fos、c-jun、jun b、HLH 462 和 zif 268，选择是因为它们作为转录因子的已知活性及其 OL 或其他细胞类型对损伤的反应。髓磷脂蛋白基因可能是 IEG 变化的下游目标。原位杂交、免疫细胞化学、Western 和 Northern 印迹将用于检查基因的表达。变化的模式将与 OL 恢复和维持或改革膜片。另一系列的实验将检验髓鞘质组装、细胞内转运的关键终点含有 PLP/DM-20 的囊泡进出 OL 的质膜以及相关的膜片。插入和插入之间的平衡去除 OL 细胞体质膜中的 PLp/DM-20 和将通过将细胞暴露于抑制剂来检查膜片内吞作用。与背根神经元共培养的影响神经节和 PLP/DM-20 合成上调的影响环磷酸腺苷将使用免疫细胞化学和碘化进行评估表面PLP。半乳糖脑苷脂(GalC)插入的相关研究硫苷脂进入质膜的细胞外表面将在合成的永生化OL细胞系N20.1中进行半乳糖脂，但不在细胞表面表达。