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：
2902328
负责人：
JOYCE A BENJAMINS
金额：
$ 23.18万
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 2004-07-31
项目状态：
已结题

项目摘要

Oligodendrocyte (OL) injury results in loss of myelin, failure of myelination, axonal damage and ultimately cell death. We have shown that increased intracellular Ca++ mediates damage in mature membrane sheet-bearing OLs. We hypothesize that the duration, magnitude and source of increased intracellular Ca++ are major determinants of the nature and outcome of OL damage. Specifically, we will ask whether a given mode of increasing Ca++ leads to membrane sheet retraction, OL death or both. We will compare Ca++-mediated injury elicited by thapsigargin to that elicited by the NO donor SNAP and by the glutamate receptor agonist kainate. In parallel with experiments on enriched OL cultures, we will utilize a myelinating co-culture system to examine the effects of axons on the responses of OLs to Ca++-mediated injury. Our long term objective is to identify strategies for protecting OLs from damage and optimizing myelin repair in white matter injury associated with diseases such as multiple sclerosis or with the sequelae of stroke or trauma. Specific Aim 1 utilizes laser cytometry to define the changes in Ca++ initiated in mature OLs by thapsigargin, NO and kainate. A range of doses will be compared for their effects on Ca++, membrane sheet retraction, and OL viability. Specific Aim 2 analyzes changes in cytoskeleton and rates of endocytosis which accompany Ca++-mediated retraction of OL membrane sheets, with focus on endocytosis of WGA receptors and proteolipid protein. Specific Aim 3 analyzes the role of increased Ca++ in the pathways leading to necrotic or apoptotic cell death. The contribution of each pathway will be assessed with various doses of the agents. Specific Aim 4 analyzes changes in gene expression accompanying OL responses to increased intracellular Ca++. Changes in message levels for myelin proteins (MBP, PLP, and DDM-20), relevant transcription factors (SCIP, GtX, and CREB) and immediate early genes (zif , c-fos, and c-jun) will be measured by nuclease protection assay or Northern blotting. In Specific Aim 5, the effects of axons on OL responses to increased OL Ca++ will be examined. Myelinated DRG-OL cultures will be exposed to thapsigargin, NO, or kainate. Cultures will be grown in a two-compartment system, so that agents can be applied to the axon-myelin-glia compartment, but not the neuronal cell bodies. Effects on myelin, OL viability, and axonal integrity will be evaluated by immunocytochemical staining. In situ hybridization and RT-PCR will assess changes in message levels. In the last four specific aims, the initial steps in signaling pathways that ultimately lead to Ca++-mediated injury will be examined by assessing the effects of Ca++ chelation at various times, and by determining whether cyclic AMP or calpains are involved.

少突胶质细胞 (OL) 损伤会导致髓磷脂缺失、髓鞘形成失败、轴突损伤并最终导致细胞死亡。我们已经表明，细胞内 Ca++ 的增加会介导成熟膜片承载 OL 的损伤。我们假设细胞内 Ca++ 增加的持续时间、程度和来源是 OL 损伤的性质和结果的主要决定因素。具体来说，我们将询问增加 Ca++ 的给定模式是否会导致膜片收缩、OL 死亡或两者兼而有之。我们将比较毒胡萝卜素引起的 Ca++ 介导的损伤与 NO 供体 SNAP 和谷氨酸受体激动剂红藻氨酸引起的损伤。在对丰富的 OL 培养物进行实验的同时，我们将利用髓鞘共培养系统来检查轴突对 OL 对 Ca++ 介导的损伤反应的影响。我们的长期目标是确定保护 OL 免受损伤并优化与多发性硬化症或中风或创伤后遗症相关的白质损伤的髓磷脂修复的策略。具体目标 1 利用激光细胞术来确定毒胡萝卜素、NO 和红藻氨酸在成熟 OL 中引发的 Ca++ 变化。将比较一系列剂量对 Ca++、膜片收缩和 OL 活力的影响。具体目标 2 分析伴随 Ca++ 介导的 OL 膜片收缩的细胞骨架和内吞速率的变化，重点关注 WGA 受体和蛋白脂质蛋白的内吞作用。具体目标 3 分析了 Ca++ 增加在导致细胞坏死或凋亡的途径中的作用。将使用不同剂量的药物来评估每个途径的贡献。具体目标 4 分析伴随 OL 对细胞内 Ca++ 增加的反应而发生的基因表达变化。将通过核酸酶测量髓鞘蛋白（MBP、PLP 和 DDM-20）、相关转录因子（SCIP、GtX 和 CREB）和即早期基因（zif、c-fos 和 c-jun）信息水平的变化保护测定或Northern印迹。在具体目标 5 中，将检查轴突对 OL 对 OL Ca++ 增加的反应的影响。有髓鞘的 DRG-OL 培养物将暴露于毒胡萝卜素、NO 或红藻氨酸。培养物将在两室系统中生长，因此药剂可以应用于轴突-髓磷脂-神经胶质室，但不能应用于神经元细胞体。通过免疫细胞化学染色评估对髓磷脂、OL 活力和轴突完整性的影响。原位杂交和 RT-PCR 将评估信息水平的变化。在最后四个具体目标中，将通过评估不同时间 Ca++ 螯合的影响，并确定是否涉及环 AMP 或钙蛋白酶，来检查最终导致 Ca++ 介导的损伤的信号传导途径的初始步骤。