Role of semaphorin signaling in neuronal recovery from dendritic injury: a comparative case study in-vitro and in-vivo

轴突和树突在创伤性脑损伤和脊髓损伤期间都会受损，导致神经元丧失 突触连接和神经网络崩溃。然而，大多数神经再生研究都集中在轴突上， 树突对损伤的反应尚未被探索。目前尚不清楚树突是否以及如何重建 本身，影响轴突再生，甚至促进回路重新连接。轴突的正确模式 树突在发育和再生过程中很大程度上依赖于这些树突的精确形态发生 过程，而这又依赖于引导分子信号传导。这里我们重点关注信号素及其 受体、丛蛋白和神经毡蛋白。进化保守，信号蛋白信号通路至关重要 发育过程中从无脊椎动物到脊椎动物的神经回路的建立，但它们的参与 对受伤的反应尚不清楚。 我们的长期目标是确定影响恢复的网络、细胞和分子机制 受伤。我们的中心假设是可溶性信号蛋白在损伤后促进树突增殖，类似于 它们在发育过程中的作用，而膜结合信号蛋白是需要克服的限制因素 允许树突增殖以及突触形成。在这里，我们着手确定共同的基本原则 通过利用无脊椎动物秀丽隐杆线虫的体内方法来研究树突对损伤的反应机制， 采用基于小鼠原代神经元培养的体外方法。 我们将使用飞秒脉冲激光精确地断开活体、行为神经元的单个树突 线虫和小鼠皮质神经元的原代培养物中。对于每个免费模型系统 我们采用了微流体装置，该装置将改善配体应用的控制和生存能力。然后我们将测试 使用线虫敲除不同信号蛋白和丛蛋白对树突横断反应的作用 菌株的基因，以及转基因过表达和 siRNA 来调节蛋白质水平，以及 合成的 Sema3A 配体和 siRNA 在小鼠神经元上的应用。我们将分析的时间进程 形态变化、突触发生、单个细胞的神经元活动以及相应的回路，如 以及连接的功能恢复。 这些目标将共同在体外和体内检查信号蛋白信号传导在神经元反应中的作用 树突损伤后。我们的预期结果将揭示共享分子的细胞和网络机制 信号通路。拟议的研究将由一名才华横溢的博士生和几名受指导的人进行 新泽西理工学院的本科生。我们将招募积极进取、多元化、 有能力的学生，鼓励他们的好奇心并支持他们的职业目标。所掌握的技巧和方法 参与的年轻科学家将获得的知识将增加他们对职业生涯下一步的渴望。

Both axons and dendrites are damaged during traumatic brain injury and Spinal Cord Injury, causing a loss of synaptic connectivity and neural network breakdown. Yet, most studies of neural regeneration focus on axons, leaving dendritic responses to injury vastly unexplored. It is unknown whether and how dendrites reestablish themselves, influence axonal regeneration, or even promote circuit reconnection. The proper patterning of axons and dendrites during development, and regeneration, rely heavily on the precise morphogenesis of these processes, which in turn relies on guidance molecule signaling. Here we focus on semaphorins and their receptors, the plexins and neuropilins. Evolutionary conserved, the semaphorin signaling pathways are crucial to the establishment of neural circuits from invertebrates to vertebrates during development but their involvement in response from injury is unknown. Our long-term goal is to determine the network, cellular, and molecular mechanisms that affect recovery from injury. Our central hypotheses are that soluble semaphorins promote dendritic proliferation after injury, similar to their role during development, while membrane-bound semaphorins are limiting factors that need to be overcome to allow dendritic proliferation as well as synaptic formation. Here we set out to determine the shared fundamental mechanisms in dendritic response to injury by leveraging the in-vivo approach using the invertebrate C. elegans, with in-vitro methods based on mouse primary neuronal culture. We will use a femtosecond-pulse laser to precisely disconnect individual dendrites of neurons in live, behaving nematode and in a primary culture of mouse cortical neurons. For each of these complimentary model systems we adapted a microfluidic device that will improve control of ligand application and survivability. We will then test the roles of different semaphorins and plexins in response to dendritic transection by using C. elegans knockout strains for their genes, as well as transgenic overexpression and siRNA to regulate the protein levels, and application of synthetic Sema3A ligand and siRNA on mouse neurons. We will assay the time course of morphological changes, synaptogenesis, neuronal activity in individual cells and the corresponding circuit, as well as functional recovery of connections. Together, these aims will examine, both in-vitro and in-vivo, roles of semaphorin signaling in neuronal response after dendritic injury. Our anticipated results will uncover cellular and network mechanisms that share a molecular signaling pathway. The proposed research will be carried out by a talented PhD candidate and several mentored undergraduate students at the New Jersey Institute of Technology. We will recruit motivated, diverse, and capable students, and encourage their curiosity and supporting their career goals. The skills and methods that the participating young scientists will acquire will increase their desirability for the next steps in their career.