GUIDANCE MOLECULES IN SPINAL CORD REGENERATION

描述：(改编自调查人员摘要)：这是一项建议，以 确定脊髓轴突的再生是被引导还是被调节 通过Netrins和信号素的作用，引导轴突的分子 并存在于成熟脊椎动物的中枢神经系统中。跟随脊椎 脊髓横断，哺乳动物的轴突通常不会再生，这 使再生的分子机制研究复杂化。相反， 七鳃鳗从完全脊髓横断中恢复，轴突再生 有选择地走上正确的道路。七鳗鱼的其他优点是 再生研究包括：1)鉴定出巨大的网状脊髓神经元 它们的再生能力不同，它们的再生能力 先前确定的。2)这些神经元在活体和中枢神经系统中均可见。 全额的。3)分子表达模式可以与 单个神经元的再生能力和已确定的神经元类型。4)英寸 携带基因枪的神经元体内转基因可获得长效转基因 表情。我们已经有了两个信号素的部分序列，一个是netrin 和两种七鳃鳗网状蛋白受体。我们有三个本地化的表达 它们通过原位杂交，并有证据表明它们的表达是 受脊髓横断的影响。我们现在建议确定是否 网织蛋白和信号素受体的高表达或低表达 网状脊髓神经元改变脑出血的概率和/或通路特异性 他们的再生。我们将完成对建议的 引导分子及其受体，并将其表达定位于 未损伤和脊髓横断的动物。受体的表达将 然后通过基因枪增强或抑制网状脊髓神经元 用正义或反义(或者，截断显性 网织蛋白和信号素受体的c DNA。与重组病毒共转染法 记者绿色荧光蛋白将区分转染自 未转染细胞。再生的概率将由以下因素决定 HRP的逆行运输，而再生的方向性 将通过细胞内注射HRP和观察 脊髓全层再生轴突。如果发展性指导 影响中枢神经系统轴突再生效果的分子，其 手法可以形成治疗促进再生的基础 脊髓损伤、创伤性脑损伤患者轴突中断 受伤和中风。

DESCRIPTION: (Adapted from the Investigator's Abstract): This is a proposal to determine if regeneration of spinal cord axons is guided or otherwise modulated by the actions of netrins and semaphorins, molecules that guide axons during development and are present in the CNS of mature vertebrates. Following spinal cord transection, mammalian axons ordinarily do not regenerate, which complicates the study of molecular mechanisms of regeneration. By contrast, lampreys recover from complete spinal transection and axons regenerate selectively in their correct paths. Other advantages of the lamprey for regeneration research include: 1) Identified giant reticulospinal neurons differ from one another in their regenerative abilities, which have been determined previously. 2) These neurons can be visualized in vivo and in CNS wholemounts. 3) Molecular expression patterns can be correlated with regenerative abilities in individual neurons and identified neuron types. 4) In vivo transfection of neurons with Gene Gun results in long-lasting transgene expression. We already have partial sequences for two semaphorins, one netrin and two netrin receptors in lamprey. We have localized expression of three of these by in situ hybridization and have evidence that their expression is modulated by spinal cord transection. We now propose to determine whether overexpression or underexpression of netrin and semaphorin receptors in reticulospinal neurons alters the probability and/or pathway specificity of their regeneration. We will complete the full length cloning of the proposed guidance molecules and their receptors, and localize their expression in uninjured and spinal cord transected animals. The expression of receptors will then be enhanced or inhibited in reticulospinal neurons by Gene Gun transfection with the sense or antisense (alternatively, a truncated dominant negative) cDNA for netrin and semaphorin receptors. Cotransfection with the reporter Green Fluorescent Protein will distinguish transfected from untransfected cells. The probability of regeneration will be determined by retrograde transport of HRP, while the directional specificity of regeneration will be determined by intracellular injection of HRP and observation of the regenerated axons in spinal cord wholemounts. If developmental guidance molecules influence the effectiveness of axon regeneration in CNS, their manipulation could form the basis for therapies to improve regeneration of interrupted axons in patients with spinal cord injuries, traumatic brain injuries and stroke.