Investigating the Role of Microglia in Developmental CNS Synaptic Remodeling

描述（由申请人提供）：在发育过程中，神经回路经历一个重塑过程，在这个过程中，多余的突触被消除或修剪，剩余的突触得到加强。虽然发育突触重塑是一个活动依赖的过程，其中较弱或活性较低的突触被选择性地消除，但精确的分子机制尚未阐明。最近，我们的实验室发现经典补体级联的成分（C1q和C3）是发育中和患病中枢神经系统（CNS）突触消除所必需的（Stevens et al., 2007）。此外，C1q蛋白在神经元中特异性表达，并定位于与活跃突触修剪一致的年龄和大脑区域的突触。从这些发现中产生的一个主要问题是补体促进突触移除的机制是什么。补体级联传统上与先天免疫系统有关，其中补体成分包裹或调理碎片（例如病原体，细胞尸体等）以去除。补体活化碎片清除的典型途径是通过表达补体蛋白受体的细胞吞噬，与先天免疫系统类似，我们认为补体在发育中的神经系统中起作用，标记弱突触，由激活的小胶质细胞（中枢神经系统的主要吞噬细胞）清除。为了描述小胶质细胞在突触修剪中的作用（目的1），我们将使用各种成像技术（例如体内实时成像等）测试小胶质细胞吞噬突触末梢的能力（目的1a）。此外，我们将通过评估补体受体KO小鼠的修剪缺陷和C3和C1q KO小鼠的小胶质细胞生理，来确定小胶质细胞介导的突触去除是否是补体依赖的过程（subaim 1b）。本提案的最后一个组成部分（目的2）是测试小胶质细胞介导的突触重构在整个发育中的中枢神经系统的普遍性。以前和建议的工作已经在发展视网膜新生系统；因此，我将测试小胶质细胞介导的突触重塑是否也发生在另一个大脑区域，小脑（subaim 2a）。此外，我将确定这是否是一个互补依赖的过程（第2b条）。除了正常发育外，这种修剪过程的缺陷还涉及广泛的发育（如自闭症）和精神（如精神分裂症）疾病（Innocenti等人，2003；Pardo等人，2005；Vargas等人，2005；Woo和Crowell， 2005）。此外，有证据表明突触移除发生在神经退行性疾病（如阿尔茨海默氏症）的早期阶段（Selkoe, 2002）。因此，研究正常发育的基本机制不仅提供了对生物学机制的基本认识，而且为研究中枢神经系统疾病的机制和制定治疗策略提供了框架。

DESCRIPTION (provided by applicant): During development, neural circuitry undergoes a remodeling process in which excess synapses are eliminated or pruned and the remaining synapses are strengthened. While it is clear that developmental synaptic remodeling is an activity-dependent process whereby weaker or less-active synapses are selectively eliminated, the precise molecular mechanisms have not been elucidated. Recently, our laboratory discovered that components of the classical complement cascade (C1q and C3) were necessary for synapse elimination in the developing and diseased central nervous system (CNS)(Stevens et al., 2007). In addition, C1q protein was specifically expressed in neurons and localized to synapses at ages and brain regions consistent with active synaptic pruning. One of the major questions arising from these findings is by what mechanism is complement facilitating synapse removal. The complement cascade is traditionally associated with the innate immune system in which complement components coat or opsonizes debris (e.g. pathogens, cell corpses, etc.) for removal. A canonical pathway for complement-opsonized debris removal is through phagocytosis by cells that express receptors for complement proteins Similar to the innate immune system, we suggest that complement is acting in the developing nervous system to tag weak synapses for removal by activated microglia, the primary phagocytic cell in the CNS. To characterize the role of microglia in synaptic pruning (Aim 1), we will test the capacity of microglia to phagocytose synaptic endings (subaim 1a) using various imaging techniques (e.g in vivo live imaging, etc.). In addition, we will deterimine if microglia-mediated synaptic removal is a complement-dependent process (subaim 1b) by assessing pruning deficits in complement receptor KO mice and microglia physiology in C3 and C1q KO mice. The final component of this proposal (Aim 2) is to test the pervasive nature of microglia-mediated synapse remodeling throughout the developing CNS. Previous and proposed work has been done in the developing retinogeniculate system; therefore, I will test whether microglia-mediated synaptic remodeling also occurs in another brain region, the cerebellum (subaim 2a). Furthermore, I will determine if this is a complement-dependent process (subaim 2b). In addition to normal development, deficits in this pruning process have been implicated in a broad range of developmental (e.g. autism) and psychiatric (e.g. schizophrenia) disorders (Innocenti et al., 2003; Pardo et al., 2005; Vargas et al., 2005; Woo and Crowell, 2005). Furthermore, there is evidence that synapse removal occurs during early stages of neurodegenerative disease (e.g. Alzheimer's)(Selkoe, 2002). Therefore, studying basic mechanisms of normal development provides both a basic understanding of a biological mechanism but also a framework for studying mechanisms of CNS disease and development of therapeutic strategies. PUBLIC HEALTH RELEVANCE: Following birth, in a process called synaptic pruning, many connections between neurons and their nervous system targets are removed leaving behind a highly refined circuitry. Abnormalities in this pruning process are associated with developmental (e.g. autism) and psychiatric (e.g. schizophrenia) disorders as well as early stages of neurodegenerative disease (e.g. Alzheimer's disease). Thus, in addition to elucidating basic mechanisms of nervous system development, understanding synaptic pruning has implications for development of therapeutic strategies targeted toward treatment and/or prevention of nervous system diseases. .