Guidance molecules in spinal cord regeneration

描述（由申请人提供）：在海七鳃鳗脊髓横切后，轴突选择性地在正确的路径上再生。属于semaphorin/sapsin和netrin家族的几种分子被认为有助于在胚胎发育期间引导轴突，并且还可能在引导损伤的成熟CNS中的再生中发挥作用。在以前的研究中，netrin和semaphorin受体的表达只发生在再生不良的神经元中。因此，这些引导分子可以作为化学排斥剂，防止轴突再生到脊髓中的目标。目标1。我们将确定抑制netrin和semaphorin受体的表达是否会增强已鉴定的网状脊髓神经元的再生能力。在这些神经元中的表达将通过在脊髓横断部位注射荧光素标记的反义吗啉代寡核苷酸的逆行运输来抑制。通过轴突内注射示踪剂确定对再生距离和方向的影响，并与注射随机寡核苷酸的对照动物进行比较。目标2.由于化学排斥可以通过操纵环鸟苷一磷酸（cGMP）和环腺苷一磷酸（cAMP）信号通路转换为吸引力，我们将确定通过细胞外注射膜渗透性类似物改变细胞内cAMP和/或cGMP浓度对再生能力和/或识别的网状脊髓神经元的通路特异性的影响。目标3.我们将继续寻找轴突导向分子，这些分子优先在再生良好的神经元中表达。我们将确定ephrins及其受体Eph，新的脑信号蛋白受体CD 72，intergins和RTPR是否指导再生轴突的正确路径。使用已克隆的部分序列的引物和其他人的简并引物，我们将通过RTPCR克隆七鳃鳗同源物的CD 72，ephrin，及其相应的受体，并确定其mRNA的解剖分布的半定量原位杂交。脊髓横断对其表达的影响将与已鉴定的网状脊髓神经元的再生能力相关。通过阐明CNS轴突再生的机制，这些实验可能导致改善的治疗方法，以诱导脊髓损伤、头部创伤或中风后的人类患者的再生和功能恢复。

DESCRIPTION (provided by applicant): Following spinal cord transection in the sea lamprey, axons regenerate selectively in their correct paths. Several molecules belonging to the semaphorin/collapsin and netrin families are thought to help guide axons during embryonic development and may also play a role in guiding regeneration in the injured, mature CNS. In previous studies, expression of netrin and semaphorin receptors occurred only in bad-regenerating neurons. Thus these guidance molecules may act as chemorepellents preventing axons from regenerating to their targets in the spinal cord. Aim 1. We will determine whether inhibiting expression of netrin and semaphorin receptors will enhance the regenerative capacities of identified reticulospinal neurons. Expression in these neurons will be inhibited by retrograde transport of fluoroscein-tagged antisense morpholino oligonucleotides injected at the site of spinal cord transection. The effect on distance and direction of regeneration will be determined by intra-axonal injection of tracer and compared with those in control animals injected with random oligonucleotides. Aim 2. Because chemorepulsion can be converted to attraction by manipulation of the cyclic guanosine-monophosphate(cGMP) and cyclic adenosine monophosphate (cAMP) signaling pathways, we will determine the effect of altering intracellular cAMP and/or cGMP concentrations by extracellular injections of membrane-permeable analogs on the regenerative capacities and/or pathway specificities of identified reticulo-spinal neurons. Aim 3. We will continue our search for axonal guidance molecules that are expessed preferentially in good-regenerating neurons. We will determine whether ephrins and their receptors Eph, the new semaphorin receptor CD72, intergins, and RTPR guide regenerating axons in their correct paths. Using primers from partial sequences already cloned and degenerate primers for others, we will clone by RTPCR the lamprey homologs of CD 72, ephrins, and their corresponding receptors, and determine the anatomical distribution of their mRNAs by semiquantitative in situ hybridization. The effect of spinal transection on their expression will be correlated in identified reticulospinal neurons with their regenerative capacities. By elucidating the mechanisms of regeneration in CNS axons, these experiments may lead to improved therapeutic approaches to inducing regeneration and functional recovery in human patients following spinal cord injury, head trauma or stroke.