GSK-3B/APC in developmental & regenerative axon growth

描述(申请人提供)：调节神经元形态的神经营养因子信号和神经元细胞骨架之间的联系仍然难以捉摸。在体外，我们已经确定了NGF下游的一条途径，涉及空间定位的PI3K和GSK-3b信号以及腺瘤性息肉病结肠蛋白(APC)与生长锥处的微管末端的结合。我们推测，在NGF诱导的轴突生长过程中，这一途径参与了微管的组装。APC和GSK-3b也显著地定位于体外“预适应损伤”的DRG神经元快速生长的轴突的远端轴突尖端和生长锥体。因此，我们推测GSK-3b/APC通路也参与了再生轴突生长过程中的微管组装。在体内，GSK-3b和APC与神经系统发育相关的功能尚未被探索，因为在基因靶向的小鼠中，GSK-3b和APC具有胚胎致死性，而且它们都有相关的家族成员，在哺乳动物的神经系统中也大量表达，可能具有“代偿”功能。我们计划在目前的提案中解决这些蛋白质在体内的作用。我们将为GSK-3a、GSK-3b、APC、APC-L和上游激酶ILK产生可诱导的、DRG特异性的敲除基因。利用DRG特异性轴突报告小鼠，我们将确定GSK-3b/APC通路在周围和脊髓DRG投射发育以及周围神经挤压通常诱导的轴突再生中的作用。如果在体内轴突再生过程中发现微管的组装需要GSK-3b和APC，并且如果能够确定关键的上游调控因子，我们的研究将为促进损伤后的轴突再生提供新的药理学途径。

DESCRIPTION (provided by applicant): Links between neurotrophin signals that regulate neuronal morphology and the neuronal cytoskeleton have remained elusive. In vitro, we have identified a pathway downstream of NGF involving spatially localized PI3K and GSK-3b signaling and binding of the Adenomatous Polyposis Coli protein (APC) to microtubule + ends at the growth cone. We hypothesize that this pathway mediates microtubule assembly during NGF induced axon growth. Both APC and GSK-3b are also strikingly localized to the distal axon tips and growth cones of rapidly growing axons of "precondition lesioned" DRG neurons in vitro. Thus we hypothesize the GSK-3b/APC pathway mediates microtubule assembly in regenerative axon growth as well. In vivo functions of GSK-3b and APC related to nervous system development have not yet been explored because of embryonic lethality in gene targeted mice and because each has a related family member also heavily expressed in the mammalian nervous system that may have a "compensatory" function. We plan to address in vivo roles of these proteins in the current proposal. We will generate inducible, DRG-specific knock-outs for GSK-3a, GSK-3b, APC, APC-L, and an upstream kinase, ILK. Using DRG specific axonal reporter mice, we will determine the roles of the GSK-3b/APC pathway in the development of peripheral and spinal cord DRG projections and in the axon regeneration normally induced by a peripheral nerve crush. If GSK-3b and APC are found to be required for assembly of microtubules during axon regeneration in vivo, and if key upstream regulators can be identified, our studies would provide a new pharmacological approach to enhancing axon regeneration after injur