Guidance molecules in spinal cord regeneration

描述（由申请人提供）：在海七鳃鳗脊髓横断后，轴突在正确的路径上选择性地再生。一些属于信号蛋白/崩溃蛋白和网络蛋白家族的分子被认为在胚胎发育过程中帮助引导轴突，也可能在引导受伤的成熟中枢神经系统的再生中发挥作用。在以往的研究中，netrin和信号素受体的表达仅发生在再生不良的神经元中。因此，这些引导分子可能作为化学补给剂，阻止轴突再生到脊髓中的目标。目的1。我们将确定抑制netrin和信号素受体的表达是否会增强网状脊髓神经元的再生能力。在脊髓横断部位注射荧光素标记的反义morpholino寡核苷酸，逆行转运可抑制这些神经元中的表达。通过轴突内注射示踪剂，并与注射随机寡核苷酸的对照动物进行比较，确定对再生距离和方向的影响。目标2。由于化学排斥可以通过操纵环鸟苷-单磷酸（cGMP）和环腺苷-单磷酸（cAMP）信号通路转化为吸引，我们将通过细胞外注射膜渗透性类似物来确定改变细胞内cAMP和/或cGMP浓度对网状脊髓神经元再生能力和/或途径特异性的影响。目标3。我们将继续寻找在再生良好的神经元中优先表达的轴突引导分子。我们将确定ephrin及其受体Eph、新的信号蛋白受体CD72、intergins和RTPR是否在正确的路径上引导再生轴突。利用已经克隆的部分序列引物和其他引物的退化引物，我们将通过RTPCR克隆出七鳃鳗cd72、ephrins及其相应受体的同源物，并通过半定量原位杂交确定其mrna的解剖分布。脊髓横断对其表达的影响将与鉴定的网状脊髓神经元的再生能力相关。通过阐明中枢神经系统轴突的再生机制，这些实验可能会改善脊髓损伤、头部创伤或中风后人类患者诱导再生和功能恢复的治疗方法。

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.