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的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具有相对抗性，因此认为在这些回路的初始形成期间使用的重演程序对于鼓励损伤或疾病后的神经再生具有重要的希望。在胚胎发育期间，神经生长由细胞外引导线索的微小梯度引导。这些引导信号如何在伸长神经尖端的生长锥内局部放大仍然是一个关键问题。从一些模型系统，表现出对细胞外信号的趋化性生长的证据表明，正反馈回路通常用于局部放大细胞外的线索，并产生所需的细胞不对称性引导细胞迁移。本提案的目标是确定在神经元生长锥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。基于其他小组和我们自己的工作，我们假设细胞外引导信号在生长锥内通过由脂质信号分子磷脂酰肌醇（PI）3，4，5-三磷酸（PIP 3）、脂质激酶PI-3激酶（PI 3 K）、Rho GT3 Rac和肌动蛋白动力学组成的正反馈环不对称地放大。此外，我们提出，Ca 2+功能的正反馈回路与生长锥中的PIP 3，以促进PIP 3和Rac活性的本地化积累。为了表征生长锥中这些假定的正反馈回路，我们将在活神经元培养物中使用药理学和分子方法沿着创新的生物传感器来鉴定PIP 3-PI 3 K-Rac-肌动蛋白动力学正反馈回路的组分和功能（目的1），以及胞质Ca 2+信号传导对不对称PIP 3信号放大和生长锥转向的贡献（目的2）。总的来说，本提案中描述的工作将提供新的见解，以了解局部引导信号是如何被放大的，并有助于生长锥对称性破坏所需的转向和生长到适当的突触目标。 公共卫生相关性：成人神经系统在中风、损伤或神经退行性疾病后对显著的功能再生具有相对抗性。人们普遍认为，能够更接近地再现成人神经系统中的发育过程的治疗干预具有恢复受损或患病的神经回路的最大潜力。该提案的目标是了解细胞外引导信号如何在发育或再生神经内局部放大，以确保神经系统的正确布线。

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. PUBLIC HEALTH RELEVANCE: The adult nervous system is relatively resistant to significant functional regeneration following stroke, injury, or neurodegenerative disease. It is widely believed that therapeutic interventions which can more closely recapitulate developmental processes in the adult nervous system have the greatest potential for restoring damaged or diseased neural circuits. The goal of this proposal is to understand how extracellular guidance cue signals are locally amplified within developing or regenerating nerves to ensure the proper wiring of the nervous system.