MOLECULAR SIGNALS INFLUENCING SPINAL CORD REGENERATION

影响脊髓再生的分子信号

• 批准号: 6805938
• 负责人: LAWRENCE F KROMER
• 金额: $ 28.71万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6805938
• 关键词: RNase protection assay; biological signal transduction; cell cell interaction; cell growth regulation; cytokine; developmental neurobiology; ephrins; extracellular matrix; glia; growth factor receptors; immunocytochemistry; in situ hybridization; laboratory mouse; laboratory rat; meninges; nervous system regeneration; neurogenesis; neuronal guidance; protein structure function; protein tyrosine kinase; receptor expression; spinal cord injury; western blottings

DESCRIPTION (provided by applicant): This proposal will investigate the hypothesis that ephrins and their associated Eph receptor tyrosine kinases regulate inhibitory cell-cell interactions that mediated segregation of glial cells and meningeal fibroblasts, induce glial scar formation, and inhibit axonal regeneration following spinal cord lesions. To address these questions, we will perform over-hemisection lesions of the thoracic spinal cord in adult rats and mice with gene deletions or mutations of specific ephrins or Eph receptors. Additional in vitro studies will evaluate the effects of cytokines/growth factors and proteins that block or stimulate signaling between ephrins and Eph receptors on glial cell and meningeal fibroblast functions. A variety of neuroanatomical, biochemical and molecular techniques will be used to address three specific aims. Aim I will test the hypothesis that changes in the levels and activity state of Eph/ephrins after spinal cord injury regulate cell intermixing, glial scar formation, and the reformation of the glial limitans. Cell types expressing specific Eph/ephrins will be identified and their location correlated with cellular changes in the glial scar and the distribution of inhibitory CSPGs. Aim II will use in vitro preparations to test the hypotheses that cytokineslgrowth factors regulate expression of Eph/ephrins in CNS glia and meningeal fibroblasts and that signaling through specific ephrins and Eph receptors mediates astrocyte-meningeal cell segregation and boundary formation. In these studies cytokines/trophic factors, ephrin/Eph-Fc proteins and antisense oligonucleotides will be used to regulate the production of individual ephrins/Eph receptors or alter their activation to identify their functions. Cultures of primary cells from mice with deletions and/or mutations of specific Eph/ephrins also will be used to identify the functions of specific molecules. Aim III will test the hypothesis that interactions between Eph receptors expressed on corticospinal axons and ephrins present on reactive glia in the lesioned spinal cord inhibit axonal regeneration. In these studies bilateral dorsal funiculus lesions in mice with deletions and/or mutations in ephrin-B3 or EphA4 and rats with fetal spinal cord transplants and intraspinal infusions of ephrin/Eph-Fc proteins will be used to study axonal regeneration from lesioned corticospinal axons. These experiments will permit us to investigate whether disrupting Eph-ephrin signaling enhances regeneration through the lesion/transplant interface and into the distal spinal cord. Thus, the proposed studies will expand our knowledge of the function of Eph/ephrins beyond current assumptions that they primarily regulate compartment formation and axonal guidance during nervous system development. It is anticipated that these studies also will provide important information for developing strategies to treat human spinal cord injuries.

描述（由申请人提供）：该提案将研究以下假设：ephrins及其相关的EPH受体酪氨酸激酶调节抑制性细胞细胞相互作用，该相互作用介导了神经胶质细胞和脑膜成纤维细胞的分离，诱导神经胶质疤痕形成，并诱导脊柱质结中轴突再生的轴突再生。为了解决这些问题，我们将对成年大鼠和小鼠的胸椎脊髓进行过度诊断病变，并具有基因缺失或特定ephrins或Eph受体的突变。额外的体外研究将评估细胞因子/生长因子和蛋白质的影响，这些因子和蛋白质刺激或刺激Ephrins和EPH受体之间的信号传导对神经胶质细胞和脑膜成纤维细胞功能的影响。各种神经解剖学，生化和分子技术将用于解决三个特定目标。目的我将检验以下假设：脊髓损伤后EPH/Ephrins的水平和活性状态变化调节细胞中断，神经胶质疤痕形成和神经胶质极限的改革。将确定表达特定EPH/Ephrins的细胞类型，其位置与胶质疤痕的细胞变化和抑制性CSPG的分布相关。 AIM II将使用体外制剂来测试CNS GliA和脑膜成纤维细胞中EPH/Ephrins的细胞因子生殖因子调节EPH/Ephrins的表达的假设，并且通过特定的Ephrins和EPH受体的信号传导介导星形胶质细胞膜细胞细胞分离和边界形成。在这些研究中，细胞因子/营养因子，Ephrin/Eph-FC蛋白和反义寡核苷酸将用于调节单个ephrins/eph受体的产生或改变其激活以识别其功能。来自缺失和/或特定EPH/Ephrins的突变的小鼠的原代细胞的培养也将用于识别特定分子的功能。 AIM III将检验以下假设：在病变的脊髓抑制轴突再生中，在反应性神经胶质上存在于皮质脊髓轴突和以ephrins表达的EPH受体之间的相互作用。在这些研究中，在埃弗林-B3或Epha4中具有缺失和/或突变的小鼠中双侧背侧曲并具有胎儿脊髓移植物和丝网膜内输注的大鼠，将用于研究从囊状皮质轴突的轴突再生研究的轴突再生。这些实验将使我们能够研究通过病变/移植界面并进入脊髓远端的EPH-磷信号传导是否会增强再生。因此，拟议的研究将扩大我们对EPH/Ephrins功能的了解，而不是当前的假设，即它们在神经系统发育过程中主要调节隔室形成和轴突指导。预计这些研究还将为制定治疗人脊髓损伤的策略提供重要信息。