Dissecting ADAM10 function in microglia-mediated synapse elimination

剖析 ADAM10 在小胶质细胞介导的突触消除中的功能

• 批准号: 10204719
• 负责人: Georgia Gunner
• 金额: $ 1.63万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-30 至 2021-08-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10204719
• 关键词: Affect; Alzheimer' s Disease; Alzheimer' s disease risk; Brain; Brain Mapping; Brain Stem; CX3CL1 gene; CX3CR1 gene; Cauterize; Cells; Cleaved cell; Collaborations; Data; Defect; Development; Disease; Excision; Foundations; Fractalkine; Functional disorder; Future; Genetic; Goals; Imaging Techniques; Immune; Immune signaling; Institution; Learning; Lesion; Ligands; Mediating; Membrane; Mentors; Mentorship; Messenger RNA; Metalloproteases; Microglia; Microscopy; Modeling; Molecular; Molecular Biology; Molecular Genetics; Mus; Nerve Degeneration; Neuraxis; Neurodegenerative Disorders; Neuroglia; Neurons; Peptide Hydrolases; Pharmacology; Phenocopy; Play; Population; Positioning Attribute; Post-Translational Protein Processing; Presynaptic Terminals; Process; Proteolysis; Research Personnel; Role; Sensory; Signal Pathway; Signal Transduction; Source; Specificity; Structure; Synapses; System; Testing; Thalamic structure; Therapeutic Intervention; Tissues; Training; Up-Regulation; Vibrissae; Work; barrel cortex; brain cell; brain remodeling; career; excitatory neuron; experience; experimental study; gamma secretase; genetic approach; in vivo; macrophage; molecular imaging; neonate; nervous system disorder; neural circuit; neuronal circuitry; receptor; relating to nervous system; response; risk variant; single-cell RNA sequencing; somatosensory; synaptic pruning; tool

7. Project Summary/Abstract The goal of this proposal is to dissect the molecular signaling between microglia and neurons that regulates synapse elimination in response to changes in sensory experience. Despite compelling evidence that microglia, the resident brain macrophages, play important roles in eliminating synapses in development and disease, the precise neuron-to-microglia molecular signaling that drives this process is poorly understood. I recently discovered a signaling pathway necessary for microglia-mediated synapse elimination by utilizing the well-described circuitry of the mouse barrel cortex circuit as a model to manipulate sensory experience and dampen neuronal activity. Here I found microglia robustly engulf synapses in the barrel cortex following either whisker lesioning or trimming, and that this engulfment is dependent on the microglial CX3CR1 receptor and its canonical neuronal ligand, CX3CL1, but not complement. Using single-cell RNAseq I also found that neuronal Cx3cl1 was not differentially regulated in the cortex following whisker removal, but the protease Adam10, known to cleave membrane-bound CX3CL1 into a soluble form, is increased following lesioning. Importantly, pharmacological inhibition of ADAM10 resulted in synapse elimination defects that phenocopied CX3CR1 and CX3CL1-deficient mice. These data suggest that post-translational modification of neuronal CX3CL1 by ADAM10 is required to regulate microglial synapse elimination in the cortex following whisker removal. Several exciting new questions have now arisen, which I will tackle in this proposal: 1) What is the cellular source of ADAM10 and is it localized to synapses (Aim 1)? 2) Do other subcortical synapses within the barrel circuit remodel via ADAM10-CX3CL1-CX3CR1 signaling and does this differ between whisker lesioning and trimming (Aim 2)? I hypothesize ADAM10 is derived from layer IV excitatory neurons to regulate microglia- mediated synapse remodeling and that ADAM10 signaling is specific for cortical synapse rewiring after whisker trimming and lesioning, but not for sub-cortical synapse remodeling. To test this hypothesis, I have acquired powerful in vivo molecular genetic tools to manipulate ADAM10 function in specific cells. I have also developed collaborations to learn and perform cutting-edge whole tissue clearing by iDISCO to assess structural remodeling of entire circuits. Finally, I have a strong mentoring team that includes my mentor Dr. Dorothy Schafer with expertise in microglial function within neural circuits, my co-mentor Dr. Andrew Tapper with expertise in structural and functional mapping of brain circuits, and collaborators with expertise in iDISCO. Together, I am in a strong position to molecularly dissect how ADAM10 modulates neuron-microglia signaling necessary for remodeling brain circuits. This could be highly relevant for neurodegenerative disease where microglial dysfunction, synapse loss, and ADAM10 have been implicated. In the process, I will receive training in a variety of microscopy and molecular genetic approaches that will provide a foundation for my future career as an independent principle investigator at an academic institution focused on dissecting functions for glial cells within neural circuits.

7. 项目总结/摘要 该提案的目标是剖析小胶质细胞和神经元之间的分子信号传导 它根据感官体验的变化调节突触消除。尽管令人信服 有证据表明，小胶质细胞（大脑中常驻的巨噬细胞）在消除突触方面发挥着重要作用 发育和疾病时，驱动这一过程的精确神经元到小胶质细胞的分子信号传导很差 明白了。我最近发现了小胶质细胞介导的突触消除所必需的信号通路 利用小鼠桶状皮层电路的良好描述的电路作为模型来操纵感觉 体验并抑制神经元活动。在这里，我发现小胶质细胞强有力地吞噬了桶状皮质中的突触 胡须损伤或修剪后，这种吞噬取决于小胶质细胞 CX3CR1 受体及其经典神经元配体 CX3CL1，但不是补体。使用单细胞 RNAseq 我还发现 胡须去除后，神经元 Cx3cl1 在皮层中没有受到差异调节，但蛋白酶 Adam10 已知可将膜结合的 CX3CL1 裂解成可溶形式，并在损伤后增加。 重要的是，ADAM10 的药理抑制会导致突触消除缺陷，从而导致表型复制 CX3CR1 和 CX3CL1 缺陷小鼠。这些数据表明神经元的翻译后修饰 ADAM10 的 CX3CL1 需要调节胡须后皮层中小胶质细胞突触的消除 移动。现在出现了几个令人兴奋的新问题，我将在本提案中解决这些问题：1）什么是 ADAM10 的细胞来源及其是否定位于突触（目标 1）？ 2）做其他皮层下突触 通过 ADAM10-CX3CL1-CX3CR1 信号传导进行桶状电路重塑，这在晶须损伤之间有何不同 和修剪（目标 2）？我假设 ADAM10 源自第四层兴奋性神经元来调节小胶质细胞 介导的突触重塑，并且 ADAM10 信号传导对于晶须后皮质突触重新布线具有特异性 修剪和损伤，但不适用于皮层下突触重塑。为了检验这个假设，我获得了 强大的体内分子遗传工具，可操纵特定细胞中的 ADAM10 功能。我也开发过 合作学习和执行 iDISCO 的尖端全组织透明化以评估结构重塑 整个电路。最后，我有一个强大的指导团队，其中包括我的导师 Dorothy Schafer 博士 我的合作导师 Andrew Tapper 博士拥有结构方面的专业知识 和大脑回路的功能图谱，以及具有 iDISCO 专业知识的合作者。在一起，我处于坚强的状态 从分子角度剖析 ADAM10 如何调节重塑所需的神经元-小胶质细胞信号传导 脑回路。这可能与神经退行性疾病高度相关，其中小胶质细胞功能障碍、突触 损失，ADAM10 受到牵连。在此过程中，我将接受各种显微镜和 分子遗传学方法将为我未来作为独立原则的职业生涯奠定基础 一家学术机构的研究员，专注于剖析神经回路内神经胶质细胞的功能。