MYELINATION--ASSEMBLY OF LIPIDS AND PROTEINS

髓鞘形成——脂质和蛋白质的组装

• 批准号: 6647602
• 负责人: JOYCE A BENJAMINS
• 金额: $ 25.88万
• 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 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6647602
• 关键词: axon; biological models; calcium flux; calpain; cell death; cyclic AMP; disease /disorder model; endocytosis; flow cytometry; gene expression; immunocytochemistry; in situ hybridization; kainate; laboratory mouse; lipid biosynthesis; mixed tissue /cell culture; myelination; nerve /myelin protein; nitric oxide; northern blottings; oligodendroglia; protein biosynthesis; regulatory gene; thapsigargin; transcription factor

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）损伤会导致髓磷脂损失，髓鞘衰竭，轴突损伤和最终的细胞死亡。 我们已经表明，增加的细胞内Ca ++介导了成熟的膜薄板OL中的损伤。 我们假设细胞内Ca ++的持续时间，大小和来源是OL损伤性质和结果的主要决定因素。具体而言，我们将询问增加CA ++的给定模式是否导致膜片回缩，OL死亡或两者兼而有之。 我们将比较Thapsigargin引起的Ca ++ - 介导的损伤与NO供体SNAP和谷氨酸受体激动剂海藻酸盐所引起的损伤。 与对富集培养物的实验并行，我们将利用髓鞘的共培养系统来检查轴突对OLS对Ca ++介导的损伤的反应的影响。 我们的长期目标是确定保护OLS免受损害的策略，并优化与多发性硬化症或中风或创伤后的疾病有关的白质损伤中的髓磷脂修复。特定的目标1利用激光细胞仪来定义Thapsigargin，NO和Kainate在成熟OLS中引发的CA ++的变化。 将对它们对CA ++，膜片回收和OL生存力的影响进行比较。 具体目标2分析了伴随Ca ++介导的OL膜片回收的细胞骨架的变化和内吞作用的变化，重点是WGA受体和蛋白质蛋白质的内吞作用。具体目标3分析了CA ++在导致坏死或凋亡细胞死亡的途径中的作用。 每种途径的贡献将通过各种剂量的代理进行评估。 特定目标4分析了对细胞内Ca ++的反应伴随的基因表达的变化。 髓磷脂蛋白（MBP，PLP和DDM-20），相关转录因子（SCIP，GTX和CREB）的消息水平的变化以及核酸酶保护分析或Northern northern印刷作用将测量。 在特定的目标5中，将检查轴突对OL对OL Ca ++增加的响应的影响。 髓鞘的DRG-OL培养物将暴露于Thapsigargin，NO或Kainate。 培养物将在两个校区的系统中生长，以便可以将其应用于轴突膜蛋白 - 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生存力和轴突完整性的影响。 原位杂交和RT-PCR将评估消息水平的变化。在最后四个特定的目标中，将通过在不同时间评估Ca ++螯合的影响以及确定是否涉及CA ++螯合的影响来检查最终导致Ca ++介导的损伤的初始步骤。

项目成果

Effects of monensin on posttranslational processing of myelin proteins.

莫能菌素对髓磷脂蛋白翻译后加工的影响。

• DOI: 10.1111/j.1471-4159.1983.tb13575.x
• 发表时间: 1983
• 期刊: Journal of neurochemistry
• 影响因子: 4.7
• 作者: Townsend,LE;Benjamins,JA
• 通讯作者: Benjamins,JA

Redistribution and internalization of antibodies to galactocerebroside by oligodendroglia.

少突胶质细胞对半乳糖脑苷脂抗体的重新分布和内化。

• DOI: 10.1523/jneurosci.08-03-00883.1988
• 发表时间: 1988
• 期刊: The Journal of neuroscience : the official journal of the Society for Neuroscience
• 作者: Dyer,CA;Benjamins,JA
• 通讯作者: Benjamins,JA

Expression of P0 glycoprotein in CNS glia: effects of overexpression in N20.1 cells.

CNS 胶质细胞中 P0 糖蛋白的表达：N20.1 细胞中过度表达的影响。

• DOI: 10.1002/glia.20240
• 发表时间: 2005
• 期刊: Glia.
• 作者: Studzinski,DianeM;Benjamins,JoyceA
• 通讯作者: Benjamins,JoyceA

Recovery of proteolipid protein in mice heterozygous for the jimpy gene.

jimpy 基因杂合小鼠中蛋白脂质蛋白的回收。

• DOI: 10.1111/j.1471-4159.1989.tb07325.x
• 发表时间: 1989
• 期刊: Journal of neurochemistry
• 影响因子: 4.7
• 作者: Benjamins,JA;Studzinski,DM;Skoff,RP;Nedelkoska,L;Carrey,EA;Dyer,CA
• 通讯作者: Dyer,CA

Antibody to galactocerebroside alters organization of oligodendroglial membrane sheets in culture.

半乳糖脑苷脂抗体改变培养物中少突胶质细胞膜片的组织。

• DOI: 10.1523/jneurosci.08-11-04307.1988
• 发表时间: 1988
• 期刊: The Journal of neuroscience : the official journal of the Society for Neuroscience
• 作者: Dyer,CA;Benjamins,JA
• 通讯作者: Benjamins,JA