NSF-BSF: Uncovering the specific mechanisms of spine and axonal pruning mediated by Semaphorin-Plexin signaling

大脑回路首先在神经细胞（神经元）之间形成过多的连接，然后对这些多余的连接进行精确的细化，以产生功能齐全的大脑网络。通过修剪细胞连接的神经元结构，即树突棘（位于接收端）和轴突末梢（位于发送端），连接得以完善。正确修剪神经元连接对于神经系统的正常组织和功能至关重要；修剪中断会导致大脑连接功能失调和生物体行为异常。连接的发育移除是如何被调节的，这是大脑未解之谜之一。这个联合项目的目的是揭示一个叫做信号蛋白的分子家族是如何指导消除大脑区域中多余的连接的，该区域对啮齿动物和人类的记忆形成和空间定向/识别行为（海马体）都很重要。为了实现研究目标，该项目将使用培养箱中培养的分离小鼠神经元，以及以特定方式进行基因修饰的小鼠大脑内完整神经元，以检查信号蛋白在大脑发育过程中轴突和树突修剪中所起的作用。此外，该项目将向大量本科生介绍尖端的三维脑图像分析技术，并扩大在当地一所少数民族入学率为98%的学校（其中87%的学生经济状况不理想）开展的一项成功的高中生科学推广计划，并为本科生提供深入的研究培训和参与。这项研究将为神经元如何建立正常连接提供新的机制见解，并将为长期存在的特定分子如何调节神经元回路优化的复杂过程提供答案。在大脑发育过程中，引导线索主要通过激活响应神经元上的受体来控制树突棘（突触后）和轴突末端（突触前）所经历的复杂修剪过程。具体的细胞内信号传导机制如何引导线索诱导这些反应是知之甚少。有趣的是，相同的信号素/丛蛋白信号系统（配体-受体对）似乎同时作用于小鼠海马颗粒细胞的突触后侧和突触前侧，分别诱导脊柱和轴突修剪。在这个项目中，新的转基因小鼠品系（使用CRISPR/cas9方法生成）将与各种体外和体内方法相结合，以了解信号素/丛状蛋白信号通路如何在三个不同的水平上调节突触修剪。具体目标将涉及：1)负责脊柱和轴突修剪的配体/受体对的身份，2)丛蛋白受体中特定细胞质基序的作用，以及3)涉及每个过程的新型丛蛋白下游信号元件。本研究汇集了分子生物学、细胞生物学、生物化学和小鼠遗传学方面的专业知识，以协同合作的方式研究发育生物体中控制树突脊柱和轴突修剪的具体机制。这些预期的发现将使人们对控制哺乳动物大脑线路的分子机制的理解达到一个新的水平。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Brain circuits wire themselves by first forming too many connections between nerve cells (neurons), followed by a precise refinement of these excess connections to produce fully functional brain networks. Connections are refined by pruning the neuronal structures that cells connect with, called dendritic spines (on the receiving end) and axonal terminals (on the sending end). Proper pruning of neuronal connections is essential for the proper organization and function of the nervous system; disrupted pruning results in dysfunctional brain connections and abnormal behavior in organisms. The ways in which the developmental removal of connections are regulated is one of the big unsolved mysteries of the brain. The aim of this joint project is to uncover how a family of molecules called Semaphorins instructs the elimination of excess connections in a brain region important for memory formation and spatial orientation/recognition behavior in both rodents and humans (the hippocampus). To address the research objectives, the project will use isolated mouse neurons grown in an incubator, as well as intact neurons inside mouse brains that have been genetically modified in specific ways to examine the role that Semaphorins play in axonal and dendritic pruning during brain development. In addition, the project will introduce large numbers of undergraduate students to cutting-edge 3-dimensional brain image analysis techniques, and expand a successful science outreach program for high school students conducted at a local school with a 98% minority enrollment (and where 87% of the students are economically disadvantaged), as well as providing in-depth research training and participation to undergraduate students. This study will provide new mechanistic insights into how neurons establish their normal connections, and will provide answers to the long-standing question of how specific molecules regulate the complex process of neuronal circuit refinement. During brain development, guidance cues control the complex pruning processes that dendritic spines (postsynaptic) and axonal terminals (presynaptic) undergo, mainly through the activation of receptors on the responding neurons. The specific intracellular signaling mechanisms underlying how guidance cues induce these responses are poorly understood. Interestingly, the same Semaphorin/Plexin-signaling system (ligand-receptor pair) appears to operate on both the postsynaptic and presynaptic side of mouse hippocampal granule cells to induce spine and axonal pruning, respectively. In this project, novel genetically-modified mouse lines (generated using CRISPR/cas9 methodology) will be combined with a variety of in-vitro and in-vivo approaches to ask how the Semaphorin/Plexin-signaling pathway(s) regulate synaptic pruning at three different levels. The specific aims will address: 1) the identity of the ligand/receptor pair(s) responsible for spine versus axon pruning, 2) the role of specific cytoplasmic motifs within the Plexin receptors and, 3) the novel Plexin downstream signaling elements involved with each process. This study brings together expertise in molecular, cellular biology, biochemistry, and mouse genetics, in a synergistic and collaborative effort to investigate the specific mechanisms that control dendritic spine and axonal pruning in the developing organism. The anticipated findings will provide a new level of understanding about the molecular mechanisms that govern the wiring of the mammalian brain.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.