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

描述（由申请人提供）：调节神经元形态的神经营养因子信号与神经元细胞骨架之间的联系仍然难以捉摸。在体外，我们已经确定了一个途径下游的NGF涉及空间定位PI 3 K和GSK-3b信号和结合的腺瘤性息肉病大肠杆菌蛋白（APC）微管+结束在生长锥。我们推测，这一途径介导的微管组装在神经生长因子诱导的轴突生长。APC和GSK-3b在体外也明显定位于“预处理损伤”DRG神经元的快速生长轴突的远端轴突尖端和生长锥。因此，我们假设GSK-3b/APC途径也介导再生轴突生长中的微管组装。GSK-3b和APC与神经系统发育相关的体内功能尚未被探索，因为基因靶向小鼠的胚胎致死性，并且因为各自具有相关的家族成员，其也在哺乳动物神经系统中大量表达，可能具有“补偿”功能。我们计划在目前的提案中解决这些蛋白质的体内作用。我们将产生GSK-3a、GSK-3b、APC、APC-L和上游激酶ILK的可诱导的DRG特异性敲除。使用DRG特异性轴突报告小鼠，我们将确定GSK-3b/APC通路在外周和脊髓DRG投射的发展中的作用，以及在外周神经挤压正常诱导的轴突再生中的作用。如果GSK-3b和APC被发现是轴突再生过程中微管组装所必需的，并且如果关键的上游调控因子能够被识别，我们的研究将为促进损伤后轴突再生提供一种新的药理学方法。

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