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

大脑电路首先在神经细胞(神经元)之间形成太多的连接，然后对这些多余的连接进行精确的细化，以产生完全功能的大脑网络。通过修剪细胞连接的神经元结构，称为树突棘(在接收端)和轴突终末(在发送端)，来精炼连接。神经元连接的适当修剪对于神经系统的正确组织和功能是必不可少的；修剪中断会导致大脑连接功能障碍和生物体的异常行为。发育过程中移除连接的调控方式是大脑中一大未解之谜。这个联合项目的目的是揭示一种名为信号素的分子家族如何指示消除大脑区域中的过度连接，该区域对啮齿类动物和人类(海马体)的记忆形成和空间定位/识别行为至关重要。为了达到研究目标，该项目将使用培养箱中培养的孤立小鼠神经元，以及小鼠大脑中以特定方式进行基因改造的完整神经元，以检查信号素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%(87%的学生处于经济困难的地区)的学校为高中生开展的成功的科学外展计划，以及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，并将为特定分子如何调节神经元电路精化的复杂过程这个长期存在的问题提供答案。在大脑发育过程中，引导线索主要通过激活反应神经元上的受体来控制树突棘(突触后)和轴突终末(突触前)经历的复杂修剪过程。指导线索如何引发这些反应的具体细胞内信号机制尚不清楚。有趣的是，相同的Semaphorin/Plexin信号系统(配体-受体对)似乎分别作用于小鼠海马颗粒细胞的突触后和突触前侧，以诱导脊髓和轴突修剪。在这个项目中，新的转基因小鼠品系(使用CRISPR/CAS9方法产生)将结合各种体外和体内方法来研究信号转导/丛连蛋白信号通路(S)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)负责脊椎和轴突修剪的配体/受体对(S)的身份，2)特定细胞质基序在丛蛋白受体中的作用，3)参与每个过程的新的丛状蛋白下游信号元件。这项研究汇集了分子、细胞生物学、生物化学和小鼠遗传学方面的专业知识，协同合作，研究在发育中的有机体中控制树突棘和轴突修剪的具体机制。这些预期的发现将提供对支配哺乳动物大脑连接的分子机制的新水平的理解。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.