How do astrocytes remodel neural circuits?

项目总结 修剪神经元连接和消除多余神经元是产生优化神经元所必需的 成熟大脑中的回路。虽然已经确定神经元和神经胶质细胞协调精炼 神经回路，这一过程背后的分子机制仍然不清楚。此K99/R00建议书 将帮助我推进我的事业，因为我研究了神经胶质细胞帮助提炼神经元的细胞机制 开发过程中的电路。为了加深对神经元重塑的分子理解，我将使用 果蝇幼虫神经系统，在变态过程中经历广泛的神经元重塑。在……里面 初步筛选研究活体神经元重塑的新模型细胞，我发现了几个新的标志物 对于表现出新类型重塑事件的细胞。此外，我还进行了两次大规模筛查，以确定 帮助修剪和清除神经元碎片的新的神经胶质通路。首先，使用单个神经元 我在体内进行了一次RNAi筛选，寻找神经胶质修剪所需的神经胶质基因。第二，我 在修剪过程中对胶质细胞进行转录分析，识别胶质细胞中上调的基因，并对它们进行筛选 神经元重塑的调节因子。这些基因将成为我定义神经胶质细胞的分子切入点 在开发过程中完善神经网络。在指导颁奖阶段，我将建立一个系统，使 我将监测胶质细胞吞噬过程中发生的动态细胞生物学变化，并将使用这一新的 系统测试新分子的角色重塑。在Aim1中，我将研究神经胶质细胞的具体过程 使用遗传编码细胞标记和外植体活体成像的吞噬作用。这些化验将作为 这是了解我确定的分子如何在胶质细胞吞噬中发挥作用的基础。在《目标2》中，我会 利用这个新系统来了解高度保守的分子TWeek在神经胶质细胞吞噬过程中的功能 修剪过的神经元。令人惊讶的是，TWeek没有已知的蛋白质结构域或分子功能。我会用 CRISPR/Cas9基因组工程在苍蝇中创造与人类疾病相关的突变。这可能会 请允许我了解特威克人类同源基因KIAA1109的突变是如何导致一种罕见的常染色体 神经系统疾病。最后，在将主要在我自己的实验室中执行的Aim 3中，我将使用我构建的工具 在这项研究一组新发现的吞噬细胞受体如何驱动神经元重塑的提案中 突触，神经突起，以及多种血统。我概述了一系列的研究和职业发展 在这个奖项期间将达到的里程碑，并使我作为一名科学家成长。加强需要的领域 对于成功的研究生涯，目标是结合神经科学课程和领域的培训。 例如科学写作、指导和项目设计。我有一个委员会，由一群优秀的 致力于我的成功并渴望在我的职业发展中帮助我的科学家。我的龙- 作为一名独立研究员的职业生涯目标是了解神经元的分子机制 重塑以及这一过程中的异常与神经发育障碍的关系。

PROJECT SUMMARY Pruning neuronal connections and eliminating superfluous neurons is required to generate optimized neuronal circuits in the mature brain. Although it is well established that neurons and glia coordinate the refinement of neural circuits, the molecular mechanisms underlying this process remain poorly defined. This K99/R00 proposal will help me advance my career as I investigate the cellular mechanisms by which glia help refine neuronal circuits during development. To generate a deep molecular understanding of neuronal remodeling, I will use the Drosophila larval nervous system, which goes through extensive neuronal remodeling during metamorphosis. In a preliminary screen for new model cells to study neuronal remodeling in vivo, I discovered several new markers for cells that exhibit novel types of remodeling events. In addition, I conducted two large-scale screens to identify new glial pathways that assist in pruning and the elimination of neuronal debris. First, using a single neuronal lineage, I performed an in vivo RNAi screen for glial genes required for glial pruning of neurons. Second, I transcriptionally profiled glia during pruning, identifying upregulated genes in glia, and also screened them for regulators of neuronal remodeling. These genes will serve as a molecular entry point for me to define how glia refine neural networks during development. During the mentored award phase, I will build a system that will allow me to monitor the dynamic cell biological changes that occur during glial phagocytosis and will use this new system to test novel molecules for their role remodeling. In Aim1, I will study the specific process of glial phagocytosis using genetically encoded cellular markers and explant live imaging. These assays will serve as the foundation for understanding how the molecules I identified play a role in glial phagocytosis. In Aim 2, I will use this new system to understand how Tweek, a highly conserved molecule, functions during glial phagocytosis of pruned neurons. Surprisingly, Tweek has no known protein domains or molecular function. I will use CRISPR/Cas9 genomic engineering to create human disease-associated mutations in the fly. This will potentially allow me to understand how mutations in Tweek's human homolog KIAA1109 cause a rare autosomal neurological disease. Finally, in Aim 3, which will be mostly carried out in my own lab, I will use the tools I build in this proposal to examine how a collection of newly identified phagocytic receptors drive neuronal remodeling of synapses, neurites, and in multiple types of lineages. I outlined a series of research and career development milestones that will be met during this award and allow me to grow as a scientist. To strengthen areas needed for a successful research career, the aims are combined with neuroscience coursework and training in areas such as scientific writing, mentoring, and project design. I have a committee made up of an excellent group of scientists who are dedicated to my success and eager to assist me in my professional development. My long- term career goal as an independent investigator is to understand the molecular mechanisms of neuronal remodeling and how abnormalities in this process are associated with neurodevelopmental disorders.