GUIDANCE MOLECULES IN SPINAL CORD REGENERATION

描述：（改编自研究者摘要）：这是一项提案， 确定脊髓轴突的再生是否被引导或以其他方式调节 通过netrins和semaphorins的作用， 发育和存在于成熟脊椎动物的中枢神经系统。继脊髓 脊髓横断，哺乳动物的轴突通常不会再生， 使再生的分子机制的研究复杂化。相比之下， 七鳃鳗从完全脊髓横断中恢复且轴突再生 选择正确的道路。七鳃鳗的其他优点 再生研究包括：1）鉴定巨大网状脊髓神经元 它们的再生能力各不相同， 以前确定的。2)这些神经元可以在体内和中枢神经系统中可视化 全山3)分子表达模式可以与 单个神经元的再生能力和识别的神经元类型。4)在 用基因枪体内转染神经元导致持久的转基因 表情我们已经有了两个信号素的部分序列，一个netrin 和两个netrin受体。我们有三种局部表达， 这些通过原位杂交，并有证据表明，他们的表达是 通过脊髓横切来调节。我们现在建议确定 netrin和semaphorin受体的过表达或低表达， 网状脊髓神经元改变神经元的可能性和/或通路特异性 他们的再生。我们将完成全长克隆的建议 引导分子及其受体，并将其表达定位于 未受伤和脊髓横断的动物。受体的表达将 在网状脊髓神经元中， 用有义或反义（或者，截短的显性基因）转染 阴性）netrin和semaphorin受体的cDNA。共转染 报告基因绿色荧光蛋白将区分转染的 未转染细胞再生的概率将由以下因素决定： HRP的逆行运输，而再生的方向特异性 将通过细胞内注射HRP并观察 再生轴突在脊髓整体。如果发展指导 分子影响CNS中轴突再生的有效性，它们 操作可以形成治疗的基础，以改善再生的 脊髓损伤、脑外伤患者的轴突中断 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.