Positive Feedback Loops in Growth Cone Symmetry Breaking and Guidance Signaling

描述(由申请人提供)：在损伤或疾病后恢复神经系统内的功能连接是开发中风、脊髓损伤和神经退行性疾病有效治疗方法的重大挑战。由于成人神经系统对神经回路的功能恢复相对抵抗，因此人们认为，在这些回路的初始形成过程中使用的重述程序对于鼓励损伤或疾病后的神经再生具有重要的前景。在胚胎发育期间，神经生长是由细胞外引导线索的微小梯度来指导的。这些引导信号如何在伸长神经末端的生长锥体内被局部放大仍然是一个关键问题。来自一些模型系统的证据表明，正反馈环经常被用来局部放大细胞外信号，并产生引导细胞迁移所需的细胞不对称。这项建议的目的是确定神经元生长锥体中是否存在类似的机制，并确定这些信号通路的组成和功能。基于其他研究小组和我们自己的工作，我们假设细胞外的指导信号在生长锥内被一个正反馈环不对称地放大，该正反馈环由脂质信号分子磷脂酰肌醇(PI)3，4，5-三磷酸(PIP3)、脂蛋白激酶PI-3激酶(PI3K)、Rho GTP酶Rac和肌动蛋白动力学组成。此外，我们认为，在生长锥体中，钙离子与PIP3处于正反馈环中，促进PIP3和RAC活性的局部积累。为了确定生长锥中这些假定的正反馈环的特征，我们将在活的神经元培养中使用药理学和分子方法以及创新的生物传感器来确定PIP3-PI3K-RAC-肌动蛋白动力学正反馈环(AIM 1)的组成和功能，以及细胞质Ca~(2+)信号在不对称PIP3信号放大和生长锥转向(AIM 2)中的作用。总的来说，这项建议中描述的工作将为局部指导线索信号如何被放大提供新的见解，并有助于打破生长锥对称，从而转向和生长到适当的突触靶点。

DESCRIPTION (provided by applicant): Restoring functional connections within the nervous system following injury or disease represents a significant challenge in the development of effective treatments for stroke, spinal cord injury, and neurodegenerative conditions. Because the adult nervous system is relatively resistant to the functional restoration of neural circuits, t is believed that recapitulating programs used during the initial formation of these circuits holds significant promise for encouraging nerve regeneration following injury or disease. During embryonic development, nerve growth is guided by minute gradients of extracellular guidance cues. How these guidance cue signals are locally amplified within growth cones at the tip of elongating nerves remains a critical question. Evidence from a number of model systems that exhibit chemotactic growth towards extracellular signals indicates that positive feedback loops are often used to locally amplify extracellular cues and generate the cellular asymmetry required for guided cell migration. The goal of this proposal is to ascertain whether similar mechanisms are at work in neuronal growth cones, and to define the components and function of these signaling pathways. Based on work by other groups and our own, WE HYPOTHESIZE that extracellular guidance cue signals are asymmetrically amplified within the growth cone by a positive feedback loop comprised of the lipid signaling molecule phosphatidylinositol (PI) 3,4,5-triphosphate (PIP3), the lipid kinase PI-3 kinase (PI3K), the Rho GTPase Rac, and actin dynamics. Furthermore, we propose that Ca2+ functions in a positive feedback loop with PIP3 in growth cones to promote the localized accumulation of PIP3 and Rac activity. In order to characterize these putative positive feedback loops in growth cones, we will use pharmacological and molecular approaches along with innovative biosensors in live neuronal cultures to identify the components and function of a PIP3-PI3K-Rac-actin dynamics positive feedback loop (aim 1), as well as the contribution of cytoplasmic Ca2+ signaling to the amplification of asymmetric PIP3 signals and growth cone turning (aim 2). Collectively, the work described in this proposal will provide novel insight into how local guidance cue signals are amplified and contribute to the growth cone symmetry breaking required for turning and outgrowth to proper synaptic targets.