MOLECULAR SIGNALS INFLUENCING SPINAL CORD REGENERATION

描述(由申请人提供)：该提案将研究一种假设，即肾上腺素及其相关的Eph受体酪氨酸激酶调节抑制性细胞-细胞相互作用，该细胞-细胞相互作用介导胶质细胞和脑膜成纤维细胞的分离，诱导胶质瘢痕形成，并抑制脊髓损伤后的轴突再生。为了解决这些问题，我们将对成年大鼠和小鼠的胸髓进行过半切损伤，这些大鼠和小鼠的基因缺失或特定的肾上腺素或Eph受体突变。此外，体外研究还将评估细胞因子/生长因子以及阻断或刺激肾上腺素和Eph受体之间的信号传递的蛋白质对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、生化和分子技术来解决三个具体目标。目的验证脊髓损伤后Eph/Ephins水平和活性状态的变化调节细胞混杂、胶质瘢痕形成和神经胶质细胞界限层重建的假说。将确定表达特定Eph/eparins的细胞类型，并将它们的位置与胶质瘢痕中的细胞变化和抑制性CSPG的分布相关联。目的利用体外实验研究细胞分裂素样生长因子调节中枢神经系统神经胶质细胞和脑膜成纤维细胞中Eph/Ephins的表达，以及通过特定的Ephin和Eph受体介导星形胶质细胞-脑膜细胞分离和边界形成的假设。在这些研究中，将使用细胞因子/营养因子、Ephin/Eph-Fc蛋白和反义寡核苷酸来调节单个Eparins/Eph受体的产生或改变其激活以确定其功能。具有特定Eph/Eparin缺失和/或突变的小鼠的原代细胞培养也将用于鉴定特定分子的功能。目的III将验证这一假说，即皮质脊髓轴突上表达的Eph受体与受损脊髓内反应性胶质细胞上存在的肾上腺素之间的相互作用抑制了轴突再生。在这些研究中，我们将利用双侧脊髓背索损伤的小鼠和胚胎脊髓移植大鼠的双侧背索损伤来研究受损皮质脊髓轴突的再生。这些实验将使我们能够研究干扰Eph-ephin信号是否促进了通过病变/移植界面和远端脊髓的再生。因此，拟议的研究将扩大我们对Eph/Ephins功能的了解，而不是目前的假设，即它们主要调节神经系统发育过程中的脑室形成和轴突引导。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。

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.