Positive Feedback Loops in Growth Cone Symmetry Breaking and Guidance Signaling

描述（由申请人提供）：在中风、脊髓损伤和神经退行性疾病的有效治疗中，恢复损伤或疾病后神经系统内的功能连接是一项重大挑战。由于成人神经系统对神经回路的功能恢复具有相对的抵抗力，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促进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信号的微小梯度引导的。这些引导信号是如何在细长神经顶端的生长锥内局部放大的，这仍然是一个关键问题。许多模型系统显示出对细胞外信号趋化生长的证据表明，正反馈回路通常用于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性。本研究的目的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。基于其他研究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一个由脂质信号分子磷脂酰肌醇（PI） 3,4,5-三磷酸（PIP3）、脂质激酶PI-3激酶（PI3K）、Rho GTPase Rac和肌动蛋白动力学组成的正反馈回路不对称地放大。此外，我们提出Ca2+与PIP3在生长锥中的正反馈回路中起作用，以促进PIP3和Rac活性的局部积累。为了表征生长锥中这些假设的正反馈回路，我们将使用药理学和分子方法以及活神经元培养中的创新生物传感器来识别PIP3- pi3k - rac -actin动力学正反馈回路的成分和功能（目的1），以及细胞质Ca2+信号对不对称PIP3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，了解局部引导线索信号如何被放大，并有助于生长锥对称性破坏，这是转向和生长到适当的突触目标所必需的。

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.