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

在创伤性脑损伤和脊髓损伤中，轴突和树突都受到破坏，导致 突触连接和神经网络崩溃。然而，大多数关于神经再生的研究都集中在轴突上， 树突状细胞对损伤的反应极大地未被研究。目前尚不清楚树突是否以及如何重建。 影响轴突再生，甚至促进回路重新连接。轴突的正确构型 在发育和再生过程中，树突在很大程度上依赖于它们的精确形态发生 过程，而这又依赖于引导分子信号。在这里，我们将重点放在信号量及其 受体，神经丛蛋白和神经粘连蛋白。进化保守，信号素信号通路至关重要 从无脊椎动物到脊椎动物在发育过程中神经回路的建立，但它们参与了 受伤后的反应尚不清楚。 我们的长期目标是确定影响康复的网络、细胞和分子机制。 受伤。我们的中心假设是，可溶性信号素促进损伤后树突状细胞的增殖，类似于 它们在发育过程中的作用，而膜结合的信号素是需要克服的限制因素 以允许树突的增殖和突触的形成。在这里，我们开始确定共享的基本原理 利用无脊椎动物线虫体内方法研究树突状细胞对伤害反应的机制， 以小鼠神经元原代培养为基础的体外培养方法。 我们将使用飞秒脉冲激光来精确地断开活的、行为的 线虫和小鼠皮质神经元的原代培养。这些免费的模型系统中的每一个 我们采用了一种微流控装置，这将改善对配体应用的控制和生存能力。然后我们将测试 不同信号素和丛蛋白在线虫基因敲除树突状横断反应中的作用 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。我们将分析其时间进程 单个细胞的形态变化、突触发生、神经元活动和相应的电路，如 以及连接的功能恢复。 这些目标将在体外和体内检验信号素信号在神经元反应中的作用。 树突状细胞损伤后。我们的预期结果将揭示共享一个分子的细胞和网络机制 信号通路。拟议的研究将由一位有才华的博士生和几位导师进行 新泽西理工学院的本科生。我们将招聘有动力的、多样化的和 鼓励有能力的学生，鼓励他们的好奇心，支持他们的职业目标。具备以下技能和方法 将获得的参与的年轻科学家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.