Investigating the Role of Microglia in Developmental CNS Synaptic Remodeling

描述（由申请人提供）：在发育过程中，神经回路经历重塑过程，其中多余的突触被消除或修剪，剩余的突触被加强。虽然发育性突触重塑是一个活性依赖性过程，其中较弱或较不活跃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组分（C1 q和C3）对于发育和患病的中枢神经系统（CNS）中的突触消除是必需的（Stevens等人，2007年）。此外，C1 q蛋白在神经元中特异性表达，并定位于与活跃的突触修剪一致的年龄和脑区域的突触。从这些发现中产生的一个主要问题是补体通过什么机制促进突触的去除。补体级联反应传统上与先天免疫系统相关，其中补体组分包被或调理碎片（例如病原体、细胞尸体等）。用于移除。补体调理的碎片清除的典型途径是通过表达补体蛋白受体的细胞的吞噬作用。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，以标记弱突触，以通过活化的小胶质细胞（CNS中的主要吞噬细胞）清除。为了表征小胶质细胞在突触修剪中的作用（目标1），我们将使用各种成像技术（例如，体内活体成像等）测试小胶质细胞吞噬突触末梢的能力（子目标1a）。此外，我们将通过评估补体受体KO小鼠中的修剪缺陷和C3和C1 q KO小鼠中的小胶质细胞生理学来确定小胶质细胞介导的突触清除是否是补体依赖性过程（子目标1b）。本建议的最后一部分（目的2）是测试小胶质细胞介导的突触重塑在整个发育中的CNS中的普遍性。先前和拟议的工作已经在视网膜神经系统的发育中完成;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补体依赖性过程（子目标2b）。除了正常发育之外，这种修剪过程中的缺陷还涉及广泛的发育（例如自闭症）和精神病（例如精神分裂症）障碍（Innocenti等人，2003; Pardo等人，2005; Vargas等人，2005; Woo and Crowell，2005）.此外，有证据表明，突触移除发生在神经退行性疾病（例如阿尔茨海默氏症）的早期阶段（Selkoe，2002）。因此，研究正常发育的基本机制提供了对生物学机制的基本理解，也为研究CNS疾病的机制和治疗策略的开发提供了框架。 公共卫生相关性：出生后，在一个被称为突触修剪的过程中，神经元与其神经系统目标之间的许多连接被移除，留下一个高度精细的电路。这种修剪过程中的异常与发育（例如自闭症）和精神病（例如精神分裂症）障碍以及神经退行性疾病（例如阿尔茨海默病）的早期阶段有关。因此，除了阐明神经系统发育的基本机制之外，理解突触修剪对于开发靶向治疗和/或预防神经系统疾病的治疗策略具有意义。.

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. .