Defining the interactions between microglia and synapses in brain development and disease

项目摘要/摘要 建立精确的突触连接对于正常的大脑功能至关重要。突触功能障碍可导致 神经元过度兴奋，导致包括癫痫在内的疾病。小胶质细胞是主要的免疫细胞。 并在突触发育、调节神经元兴奋性和吞噬等方面发挥多种作用 过度的兴奋性突触。然而，小胶质细胞撞击突触的机制主要是 用固定的组织学研究或用啮齿动物模型在成人大脑的有限区域进行研究。事实上, 在发育中的大脑中，还没有直接观察到小胶质细胞吞噬了整个突触。在这份提案中， 我将使用斑马鱼模型系统来研究完整发育的大脑中小胶质细胞和突触的相互作用。我的 最近发表的一项研究发现，斑马鱼中富含突触相关小胶质细胞(SAM) 并通过单细胞和区域批量测序确定其转录图谱。在这份提案中，我将 使用实时成像和候选基因缺失相结合的方法检查这一小胶质细胞亚群 在生理和过度兴奋的背景下。目标1将确定小胶质细胞是否吞噬突触 核心溶酶体蛋白水解酶的形成和免疫激活或缺失后的影响 组织蛋白B(Ctsba)--我转录工作中的最佳候选者。目标2将进一步评估这些表型 在化学诱导的过度兴奋的背景下，并使用惊吓行为记录来评估影响 关于神经回路的功能。最后，在目标3(R00阶段)，我将定义调控的分子机制 小胶质细胞吞噬过程中的溶酶体活性及神经元后小胶质细胞的转录水平 过度兴奋。总之，这些研究将开辟一个独特的方向，使用一种新的模型来识别分子 调节小胶质细胞-突触相互作用的通路与研究非神经元治疗的可能性 影响发展和癫痫等疾病状态的干预措施。

PROJECT SUMMARY/ ABSTRACT Establishing precise synaptic connections is critical for normal brain function. Synaptic dysfunction can lead to neuronal hyperexcitability, contributing to disorders including epilepsy. Microglia are the dominant immune cells in the brain and play multiple roles in synaptic development, modulating neuronal excitability, and engulfing excess excitatory synapses. However, the mechanisms by which microglia impact synapses have largely been investigated with fixed tissue histology or in limited regions of the adult brain using rodent models. In fact, microglial engulfment of whole synapses has not been directly observed in the developing brain. In this proposal, I will use a zebrafish model system to study microglial-synapse interactions in the intact developing brain. My recently published work identified a population of synapse-associated microglia (SAMs) enriched in the zebrafish hindbrain and defined its transcriptional profile by single-cell and regional bulk sequencing. In this proposal, I will examine this microglial subset using a combination of live imaging and candidate gene deletion in both physiology and in the context of hyperexcitability. Aim 1 will determine if microglia engulf synapses during development and the impact of immune activation or after deletion of a core lysosomal protease known as cathepsin b (ctsba) - a top candidate from my transcriptomic work. Aim 2 will further assess these phenotypes in the context of chemically induced hyperexcitability and use startle behavior recordings to assess the impact on neural circuit function. Finally, in Aim 3 (R00 phase), I will define the molecular mechanisms regulating lysosome activity during microglia phagocytosis and transcriptionally profile microglia following neuronal hyperexcitability. Together these studies will open a distinct direction using a new model to identify molecular pathways that regulate microglia-synaptic interactions with the potential to investigate non-neuronal therapeutic interventions that impact development and disease states such as epilepsy.