How do astrocytes remodel neural circuits?

项目摘要 需要修剪神经元连接并消除多余的神经元以生成优化的神经元连接。 成熟大脑中的回路尽管已经确定神经元和神经胶质协调神经元的精细化， 神经回路，这一过程的分子机制仍然不清楚。此K99/R 00提案 将帮助我推进我的职业生涯，因为我调查的细胞机制，胶质细胞帮助完善神经元 开发过程中的电路为了深入了解神经元重塑的分子机制，我将使用 果蝇幼虫的神经系统在变态过程中经历了广泛的神经元重塑。在 在初步筛选研究体内神经元重塑的新模型细胞时，我发现了几个新标记物 表现出新型重塑事件的细胞。此外，我进行了两次大规模筛选， 新的神经胶质通路，有助于修剪和消除神经元碎片。首先，使用单个神经元 谱系，我进行了体内RNAi筛选神经胶质细胞修剪神经元所需的神经胶质基因。二我 在修剪过程中对神经胶质细胞进行转录谱分析，鉴定神经胶质细胞中上调的基因，并对它们进行筛选， 神经元重塑的调节剂。这些基因将作为我定义神经胶质细胞如何 在开发过程中完善神经网络。在指导奖励阶段，我将建立一个系统， 我监测动态细胞生物学变化，发生在胶质细胞吞噬，并将使用这一新的 系统来测试新分子的角色重塑。在第一个目标中，我将研究胶质细胞的具体过程， 使用遗传编码的细胞标记物和外植体活体成像观察吞噬作用。这些试验将作为 这是理解我发现的分子如何在神经胶质吞噬作用中发挥作用的基础。在目标2中，我将 利用这个新系统来了解Tweek，一个高度保守的分子，在神经胶质吞噬过程中的功能 修剪的神经元。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基因组工程在果蝇中产生人类疾病相关突变这可能会 让我了解特维克的人类同源物KIAA 1109的突变如何导致罕见的常染色体 神经系统疾病最后，在目标3中，我将使用我构建的工具， 在这项研究新发现的吞噬细胞受体如何驱动神经元重塑的建议中， 突触，神经突，以及多种类型的谱系。我概述了一系列的研究和职业发展 这些里程碑将在这个奖项中实现，并让我成长为一名科学家。加强所需领域 对于一个成功的研究生涯，目标是结合神经科学课程和培训领域 例如科学写作、指导和项目设计。我有一个由一群优秀的 科学家们致力于我的成功，并渴望在我的专业发展中帮助我。我的长- 作为一名独立的研究者，我的职业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如何与神经发育障碍相关。

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.