Investigating the Role of Glia in Activity-Dependent Synapse Elimination

描述(申请人提供)：成熟神经回路的形成需要选择性地修剪不适当的突触并加强适当的突触连接。神经生物学中一个长期存在的问题是，是什么决定了哪些突触将被消除？虽然自发和经验驱动的突触活动在发育中的突触修剪中的作用是众所周知的，但令人惊讶的是，人们对将神经活动与特定突触的物理消除联系起来的分子和机制知之甚少。我们最近的研究表明，胶质细胞--小胶质细胞和星形胶质细胞--是发育中突触修剪的关键角色。我们发现，经典补体级联的起始蛋白C1q在发育中的神经元中被未成熟的星形胶质细胞显著上调。此外，C1q及其下游的C3都定位于突触，是视觉系统发育中消除突触所必需的；然而，补体介导突触修剪的机制完全不清楚。补体级联在先天免疫系统中的主要作用是调理或标记不需要的细胞或碎片，供吞噬细胞通过特定的补体受体清除。我们的初步研究支持一个模型，在这个模型中，发育中的大脑中不适当的突触类似地被补体标记，然后被小胶质细胞消除，小胶质细胞是中枢神经系统中的主要吞噬细胞。鉴于神经活动在突触修剪中的重要性，拟议中的研究的一个主要目标是确定补体系统是否以及如何与神经元活动合作，以产生精确的视觉回路连接。相邻轴突之间依赖活动的竞争被认为推动了弱突触输入的消除；然而，分子机制仍然难以捉摸。我们提出了一个模型，在这个模型中，活动和星形胶质细胞衍生因子(S)协同作用，上调发育中神经元的C1q，导致邻近弱突触的补体级联局部激活，小胶质细胞消除补体(C3)标记的突触。我们将使用小鼠的视黄醇生成系统作为一个模型来操纵体内特定突触的神经活动，以检测小胶质细胞(Aim 1)、C1q和C3(Aim 2)和星形胶质细胞(Aim 3)在活性依赖突触消除中的作用。具体地说，我们会问：1)补体是否专门标记弱突触以消除？2)小胶质细胞是否主动修剪突触或吞噬已被消除的突触？3)神经元活动是否调节补体级联，如果是，是如何调节的？这些问题的答案将有助于我们理解小胶质细胞和补体级联在发育中突触消除中的作用，并最终可能为我们提供新的见解，了解在正常的脑连接过程中，以及在涉及异常突触丢失和突触连接的疾病中，如癫痫和自闭症，中枢神经系统突触是如何被消除的。

DESCRIPTION (provided by applicant): The formation of mature neural circuits requires selective pruning of inappropriate synapses and strengthening of appropriate synaptic connections. A longstanding question in neurobiology is what determines which synapses will be eliminated? While the role of spontaneous and experience-driven synaptic activity in developmental synaptic pruning is well established, surprisingly little is known about the molecules and mechanisms that link neural activity with the physical elimination of specific synapses. Our recent studies reveal that glial cells-microglia and astrocytes--are key players in developmental synaptic pruning. We discovered that C1q, the initiating protein of the classical complement cascade, is significantly upregulated in developing neurons by immature astrocytes. Both C1q and downstream C3 are moreover localized to synapses and are required for developmental synapse elimination in the visual system; however the mechanisms by which complement mediates synaptic pruning are completely unknown. The primary role of the complement cascade in the innate immune system is to opsonize or tag unwanted cells or debris for removal by phagocytic macrophages via specific complement receptors. Our preliminary studies support a model in which inappropriate synapses in the developing brain are similarly tagged by complement and then eliminated by microglia, the primary phagocytic cells in CNS. Given the importance of neural activity in synaptic pruning, a major goal of the proposed research is to determine whether and how the complement system cooperates with neuronal activity to give rise to precise visual circuit wiring. Activity-dependent competition between neighboring axons is thought to drive the elimination of weak synaptic inputs; however, the molecular mechanisms remain elusive. We propose a model in which activity and astrocyte-derived factor(s) act cooperatively to upregulate C1q in developing neurons which leads to local activation of the complement cascade at neighboring weak synapses and the elimination of complement (C3) tagged synapses by microglia. We will use the mouse retinogeniculate system as a model to manipulate neural activity at specific synapses in vivo to examine the role of microglia (Aim 1), C1q and C3 (Aim 2) and astrocytes (Aim 3) in activity-dependent synapse elimination. Specifically, we will ask: 1) Does complement specifically tag weak synapses for elimination? 2) Do microglia actively prune synapses or engulf synapses already undergoing elimination? 3) Does neuronal activity regulate the complement cascade, and if so, how? The answers to these questions will add to our understanding of the role of microglia and the complement cascade in developmental synapse elimination, and may ultimately provide new insight into how CNS synapses are eliminated during normal brain wiring, and possibly in diseases involving aberrant synapse loss and synaptic connectivity, such as epilepsy and autism.