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

小胶质细胞在健康和受伤成人大脑突触塑造中的作用

• 批准号: 8717468
• 负责人: Rachel Rice
• 金额: $ 3.11万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8717468
• 关键词: AMPA Receptors; Acute; Address; Adult; Affect; Agaricales; Age; Alzheimer' s Disease; Animals; Antibodies; Apical; Attenuated; Behavioral; Behavioral Assay; Biochemical; Biochemistry; Brain; Brain Injuries; Categories; Cells; Chronic; Classification; Cognition; Cognitive; Complement; Complement 1q; Computer software; Control Animal; Control Groups; DLG4 gene; Data; Dendrites; Dendritic Spines; Dependence; Dependency; Development; Diphtheria; Diphtheria Toxin; Disease; Environment; Freezing; Glutamates; Hippocampus (Brain); Image; Image Analysis; Imagery; Immune; Immunohistochemistry; Inflammation; Inflammatory; Injury; Knock-out; Knockout Mice; Knowledge; Length; Lesion; Life; Macrophage Colony-Stimulating Factor Receptor; Maintenance; Mediating; Microglia; Microscope; Modeling; Molecular Profiling; Morphology; Motor; Mus; Muscarinic Acetylcholine Receptor; N-Methyl-D-Aspartate Receptors; Neurodegenerative Disorders; Neuronal Injury; Neurons; Outcome; Phagocytosis; Play; Positioning Attribute; Process; Prosencephalon; Receptor Signaling; Recovery; Role; Shapes; Signal Transduction; Site; Stress; Structure; Synapses; Synaptophysin; Techniques; Technology; Tetanus Helper Peptide; Time; Tissue Banking; Tissue Banks; Tissues; Transgenic Model; Traumatic Brain Injury; Up-Regulation; Vertebral column; Wild Type Mouse; Work; brain cell; cholinergic; cohort; complement system; density; functional outcomes; inhibitor/antagonist; injured; insight; mRNA Expression; neuron loss; novel; public health relevance; receptor; relating to nervous system; research study; response; retinal rods; tool; treatment duration

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.

描述（由申请人提供）：虽然小胶质细胞以其作为大脑免疫细胞的作用而闻名，但最近的证据表明它们在塑造发育中大脑的突触景观方面的作用，包括在高峰修剪期吞噬树突棘 (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发挥类似的作用。此外，小胶质细胞迁移到神经元损伤部位，还可以参与突触重塑以帮助恢复——这种功能可能会因慢性神经炎症反应而出错。然而，尽管我们了解小胶质细胞的发育情况，但尚不清楚小胶质细胞在成人和受伤的大脑中是否具有这些能力，以及它们的功能后果。我开发了一些工具，使我能够首次解决这些关键问题。我的小组发现，成人大脑中的小胶质细胞在生理上依赖于集落刺激因子 1 受体 (CSF1R) 信号传导来维持其生存，并且我们可以通过施用 CSF1R 抑制剂来利用这种依赖性。这导致 99% 以上的小胶质细胞被快速消除，从而使我们能够以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诱导性神经元丢失转基因模型，该模型也在神经元中稀疏表达 EGFP，从而可以对树突棘进行可视化、计数和分类。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，以及它如何随着神经元损伤而改变。这些信息对于了解慢性神经退行性疾病期间或脑损伤后的小胶质细胞激活如何塑造突触环境至关重要。为了回答这个问题，将在诱导白喉毒素表达后经历前脑神经元损伤/损失的 CaM-Tet 小鼠与在神经元亚群中表达 EGFP 的小鼠杂交 (2, 3)。一半诱导神经元损伤的小鼠和一半未诱导神经元损伤的小鼠接受 CSF1R 拮抗剂治疗，该拮抗剂可消除大脑中的小胶质细胞，持续 2 个月，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，小胶质细胞被消除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调节健康成人大脑中的树突棘数量。展望未来，小胶质细胞对其他两组小鼠受伤成年大脑突触景观的影响将通过分析海马 CA1 区域棘的数量和形态来确定。了解小胶质细胞如何对不同类型的突触做出独特的贡献也很重要，因此将采用免疫组织化学和生物化学来确定谷氨酸能、GABA 能和胆碱能信号传导如何受到不同的影响。还将研究补体级联在小胶质细胞介导的突触建模中的参与，因为最近的证据表明小胶质细胞发育过程中的修剪依赖于 C3/C3R 信号传导 (4)。将使用免疫组织化学和生化技术来确定先前描述的四组小鼠中C1q和C3的参与和表达，以及这些标记物与小胶质细胞形态和激活状态的相关性。事实上，我们最近从小胶质细胞被消除的小鼠中获得了 qPCR 数据，表明 C3 mRNA 表达减弱至未治疗小鼠的 17%，支持了小胶质细胞是 C3 的主要产生者的观点。因此，我们将通过用载体或 CSF1R 拮抗剂治疗 C3 敲除小鼠和野生型小鼠，研究小胶质细胞对树突棘修剪的 C3 信号传导的依赖性。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，确定小胶质细胞慢性激活（其持续超出初始损伤期）对突触结构的影响及其对认知的影响非常重要。为了实现这一目标，与最初实验中使用的小鼠相当的四组小鼠被表达白喉毒素，导致神经元损失。一半未损伤小鼠和一半损伤小鼠在损伤期后（但不是损伤期间）接受 CSF1R 拮抗剂治疗，并且将通过行为终点评估认知和运动能力。