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

信号蛋白信号传导在树突损伤神经元恢复中的作用：体外和体内比较案例研究

• 批准号: 10358797
• 负责人: Gal Haspel
• 金额: $ 45.43万
• 依托单位: NEW JERSEY INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10358797
• 关键词: Affect; Axon; Behavior; Biological Assay; Biological Models; Caenorhabditis elegans; Case Study; Cells; Communities; Curiosities; Custom; Dendrites; Development; Doctor of Philosophy; Exhibits; Future; Genes; Genome; Goals; Immobilization; In Vitro; Individual; Injury; Institutes; Invertebrates; Knock-out; Lasers; Ligands; Locomotor Recovery; Membrane; Mentors; Methods; Microfluidic Microchips; Microsurgery; Molecular; Morphogenesis; Morphology; Motor Neurons; Mus; Natural regeneration; Nematoda; Nerve Regeneration; Nervous system structure; Neurons; Neuropilin-1; Neuropilins; New Jersey; Output; Pathway interactions; Pattern; Physiologic pulse; Process; Proteins; Recombinants; Recovery; Recovery of Function; Research; Research Personnel; Role; Scientist; Semaphorin-3; Semaphorin-3A; Semaphorins; Signal Pathway; Signal Transduction; Signaling Molecule; Small Interfering RNA; Spinal cord injury; Students; Synapses; Talents; Technology; Testing; Therapeutic; Time; Training; Transgenic Organisms; Traumatic Brain Injury; Vertebrates; axon guidance; axon regeneration; base; career; comparative; doctoral student; graduate student; high school; improved; in vivo; in vivo regeneration; injury recovery; member; mutant; neural circuit; neural network; overexpression; plexin; receptor; receptor function; recruit; relating to nervous system; response; response to injury; skills; synaptogenesis; undergraduate student

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.

创伤性脑损伤和脊髓损伤期间轴突和树突都会受损，导致神经元丢失。 突触连接和神经网络崩溃。然而，大多数神经再生的研究集中在轴突， 使得树突状细胞对损伤的反应还未被广泛研究。树突是否以及如何重建尚不清楚 影响轴突再生，甚至促进回路重新连接。轴突的正确模式 发育和再生过程中的树突，在很大程度上依赖于这些细胞的精确形态发生。 这反过来又依赖于引导分子信号传导。在这里，我们重点关注信号素及其 神经丛蛋白和神经纤毛蛋白。在进化上保守，脑信号蛋白信号通路是至关重要的 从无脊椎动物到脊椎动物的神经回路的建立， 对损伤的反应是未知的。 我们的长期目标是确定网络，细胞和分子机制，影响恢复， 损伤我们的中心假设是可溶性脑信号蛋白促进损伤后树突的增殖，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 以允许树突增殖以及突触形成。在这里，我们开始确定共同的基本原则， 树突状细胞对损伤的反应机制，通过利用无脊椎动物C.优雅， 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的神经元的单个树突， 线虫和小鼠皮层神经元的原代培养物中。对于每一个这些免费的模型系统 我们采用了一种微流体装置，该装置将改善配体应用的控制和存活性。然后我们将测试 利用C.研究不同信号蛋白和丛蛋白在树突状细胞横断反应中的作用。elegans基因敲除 菌株的基因，以及转基因过表达和siRNA来调节蛋白质水平，和 合成的Sema3A配体和siRNA在小鼠神经元上的应用。我们将分析 形态学变化、突触发生、单个细胞中的神经元活动和相应的回路， 以及连接的功能恢复。 这些目标将共同研究体外和体内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细胞和网络机制，共享一个分子 信号通路拟议的研究将由一位才华横溢的博士候选人和几位导师进行 新泽西理工学院的本科生。我们将招募积极的，多样化的， 有能力的学生，并鼓励他们的好奇心和支持他们的职业目标。技能和方法， 参与的年轻科学家将获得的知识将增加他们在职业生涯中下一步的可取性。