Role of Microglia in Synaptic Sculpting in the Healthy and Injured Adult Brain

描述(申请人提供)：虽然小胶质细胞作为大脑的免疫细胞而广为人知，但最近的证据表明，它们在塑造发育中大脑的突触格局方面发挥了作用，包括在修剪高峰期吞噬树突棘(1)。目前尚不清楚小胶质细胞是否在健康成人大脑的突触塑造中扮演类似的角色。此外，小胶质细胞迁移到神经元损伤的部位，也可以参与突触重塑，以帮助恢复-这一功能可能会与慢性神经炎性反应出错。然而，尽管我们知道小胶质细胞在发育过程中的作用，但目前还不清楚小胶质细胞是否在成人和受伤的大脑中具有这些能力，以及它们的功能后果。我已经开发了工具，使我能够第一次解决这些关键问题。我的团队发现，成人大脑中的小胶质细胞在生理上依赖于克隆刺激因子1受体(CSF1R)信号来生存，我们可以通过给予CSF1R抑制剂来利用这种依赖。这导致99%的小胶质细胞迅速消失，从而使我们能够以一种以前不可能的方式研究这些细胞的作用。此外，我一直在研究一种高度新颖的可诱导性神经元丢失的转基因模型，该模型在神经元中也少量表达EGFP，从而允许可视化、计数和分类树突棘。因此，通过结合使用这些技术，我处于一个独特的位置来回答小胶质细胞是否以及如何塑造成人突触景观，以及这种情况是如何随着神经元损伤而改变的。这些信息对于了解小胶质细胞在慢性神经退行性疾病期间或脑损伤后的激活如何塑造突触环境至关重要。为了回答这个问题，将在白喉毒素表达诱导下遭受前脑神经元损伤/丢失的CaM-Tet小鼠与在神经元亚群中表达EGFP的小鼠进行杂交(2，3)。一半诱导了神经元损伤的小鼠和一半没有诱导损伤的小鼠接受了CSF1R拮抗剂的治疗，这种拮抗剂可以在2个月内消除大脑中的小胶质细胞，从而使我能够观察慢性小胶质细胞消除的影响，以及慢性神经元损伤。我发现，与小胶质细胞完整的未受伤小鼠相比，小胶质细胞消失的未受伤小鼠的树突棘数量大大增加，这首次表明小胶质细胞参与了健康成人大脑中树突棘数量的调节。下一步，小胶质细胞对另外两组小鼠受损成年脑中突触景观的影响将通过分析海马区CA1区脊椎的数量和形态来确定。了解小胶质细胞对不同类型突触的独特贡献也很重要，因此将利用免疫组织化学和生物化学来确定谷氨酸、GABA和胆碱能信号是如何受到不同影响的。补体级联在小胶质细胞介导的突触建模中的参与也将被调查，因为最近的证据表明，小胶质细胞在发育过程中的修剪依赖于C3/C3R信号(4)。免疫组织化学和生化技术将用于确定C1q和C3在上述四组小鼠中的参与和表达，以及这些标志物与小胶质细胞形态和激活状态的相关性。事实上，我们最近在去除小胶质细胞的小鼠身上产生了qPCR数据，表明C3mRNA的表达减弱到未经治疗的小鼠的17%，支持了小胶质细胞是C3的主要生产者的观点。因此，我们将通过用载体或CSF1R拮抗剂治疗C3基因敲除小鼠和野生型小鼠来研究小胶质细胞对C3信号对树突棘修剪的依赖性。最后，长期激活的小胶质细胞会导致许多神经退行性疾病和脑损伤的长期炎症。因此，确定慢性小胶质细胞激活对突触结构的影响及其对认知的影响是很重要的。为了实现这一点，四组与最初实验中使用的小鼠相似的小鼠受到白喉毒素的表达，导致神经元丢失。一半未损毁的小鼠和一半的损毁小鼠在损伤期后而不是在损毁期间使用CSF1R拮抗剂治疗，将通过行为终点评估认知和运动能力。

DESCRIPTION (provided by applicant): While microglia are well known for their role as the immune cells of the brain, more recent evidence demonstrates their role in sculpting the synaptic landscape of the developing brain, including phagocytosing dendritic spines during peak pruning periods (1). It is unknown if microglia play similar roles in synaptic sculpting in th healthy adult brain. Additionally, microglia migrate to sites of neuronal injury, and could also participate in synaptic remodeling to aid in recovery - a function that could go awry with chronic neuroinflammatory responses. However, despite our knowledge of microglia in development, it is unknown if microglia possess these abilities in the adult and injured brain, and the functional consequences of them. I have developed tools that will allow me to address these key issues for the first time. My group has discovered that microglia in the adult brain are physiologically dependent upon colony-stimulating factor 1 receptor (CSF1R) signaling for their survival, and that we can take advantage of this dependency through the administration of CSF1R inhibitors. This results in the rapid elimination of >99% of all microglia, thus allowing us to study the role f these cells in a fashion not previously possible. Additionally, I have been working with a highly novel transgenic model of inducible neuronal loss, which also sparsely expresses EGFP in neurons, allowing for visualization, counting, and classification of dendritic spines. Thus, by using these technologies together, I am in a unique position to answer if and how microglia sculpt the adult synaptic landscape, and how this is altered with neuronal injury. Such information is critical to understand how microglia activation throughout a chronic neurodegenerative disease, or following a brain injury, shapes the synaptic environment. To answer this question, CaM-Tet mice that undergo forebrain neuronal injury/loss upon induction of diphtheria toxin expression were crossed to mice that express EGFP in neuronal subsets (2, 3). Half of the mice in which neuronal injury is induced and half of the mice in which it is not induced were treated with a CSF1R antagonist that eliminates microglia from the brain for 2 months, thus allowing me to look at the effects of chronic microglial elimination, as well as a chronic neuronal injury. I have discovered that dendritic spine number is greatly increased in uninjured mice with their microglia eliminated compared to uninjured mice with their microglia intact, showing for the first time that microglia are involved in modulating dendritic spine numbers in the healthy adult brain. Going forward, the impact of microglia on the synaptic landscape in the injured adult brain in other two groups of mice will be determined by analysis of the number and morphology of spines in the CA1 region of the hippocampus. It is also important to understand how microglia contribute uniquely to different types of synapses, so immunohistochemistry and biochemistry will be employed to determine how glutamatergic, GABAergic, and cholinergic signaling are differentially affected. The involvement of the complement cascade in microglia-mediated synaptic modeling will also be investigated, as recent evidence indicates that pruning during development by microglia is dependent on C3/C3R signaling (4). Immunohistochemical and biochemical techniques will be used to determine the involvement and expression of C1q and C3 in the four groups of mice previously described, and the correlation of these markers with microglial morphology and activation state. Indeed, we recently produced qPCR data from mice with their microglia eliminated demonstrating that C3 mRNA expression is attenuated to 17% of that in untreated mice, supporting the idea that microglia are the primary producers of C3. As a result, we will go forward by investigating the dependence of microglia on C3 signaling for dendritic spine pruning by treating both C3 knockout and wild type mice with either vehicle or CSF1R antagonist. Finally, chronically activated microglia contribute to long-term inflammation in many neurodegenerative disorders and brain injuries. Therefore, it is important to determine the consequences of chronic microglia activation, which extends beyond the initial insult period, on synapse structure and the resulting effects on cognition. To achieve this, four groups of mice comparable to those used in the initial experiments were subjected to expression of diphtheria toxin, resulting in neuronal loss. Half of unlesioned and half of lesioned mice were treated with the CSF1R antagonist following, but not during, the lesion period and cognition and motor abilities will be assessed via behavioral end points.