MOLECULAR SIGNALS INFLUENCING SPINAL CORD REGENERATION

影响脊髓再生的分子信号

• 批准号: 7081302
• 负责人: LAWRENCE F KROMER
• 金额: $ 28.04万
• 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 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7081302
• 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）：本提案将研究以下假设：肝配蛋白及其相关的 Eph 受体酪氨酸激酶调节抑制性细胞间相互作用，介导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，并抑制脊髓损伤后的轴突再生。为了解决这些问题，我们将对具有特定肝配蛋白或 Eph 受体基因缺失或突变的成年大鼠和小鼠进行胸脊髓过度半切损伤。其他体外研究将评估阻断或刺激肝配蛋白和 Eph 受体之间信号传导的细胞因子/生长因子和蛋白质对神经胶质细胞和脑膜成纤维细胞功能的影响。将使用各种神经解剖学、生化和分子技术来实现三个具体目标。目的 我将检验以下假设：脊髓损伤后 Eph/ephrins 水平和活性状态的变化调节细胞混合、神经胶质疤痕形成和神经胶质界限的重建。将鉴定表达特定 Eph/ephrins 的细胞类型，并将其位置与神经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以下假设：细胞因子生长因子调节 CNS 胶质细胞和脑膜成纤维细胞中 Eph/ephrins 的表达，以及通过特定 ephrins 和 Eph 受体的信号传导介导星形胶质细胞-脑膜细胞分离和边界形成。在这些研究中，细胞因子/营养因子、肝配蛋白/Eph-Fc 蛋白和反义寡核苷酸将用于调节单个肝配蛋白/Eph 受体的产生或改变其激活以鉴定其功能。来自具有特定 Eph/ephrins 缺失和/或突变的小鼠的原代细胞培养物也将用于鉴定特定分子的功能。目标 III 将检验以下假设：皮质脊髓轴突上表达的 Eph 受体与受损脊髓中反应性神经胶质细胞上存在的肝配蛋白之间的相互作用会抑制轴突再生。在这些研究中，肝配蛋白 B3 或 EphA4 缺失和/或突变的小鼠以及胎儿脊髓移植和肝配蛋白/Eph-Fc 蛋白脊柱内输注的大鼠的双侧背索损伤将用于研究受损皮质脊髓轴突的轴突再生。这些实验将使我们能够研究破坏 Eph-ephrin 信号传导是否会增强通过病变/移植界面并进入远端脊髓的再生。因此，拟议的研究将扩展我们对 Eph/ephrins 功能的了解，超越目前的假设，即它们主要调节神经系统发育过程中的室形成和轴突引导。预计这些研究还将为制定治疗人类脊髓损伤的策略提供重要信息。