Dissecting ADAM10 function in microglia-mediated synapse elimination

7.项目摘要/摘要 这项提议的目标是剖析小胶质细胞和神经元之间的分子信号。 它调节突触的消除，以回应感觉体验的变化。尽管令人信服 有证据表明，驻留在脑内的巨噬细胞小胶质细胞在消除突触方面发挥重要作用 发展和疾病，驱动这一过程的精确的神经元到小胶质细胞分子信号转导很差 明白了。我最近发现了一条小胶质细胞介导的突触消除所必需的信号通路 利用描述得很好的小鼠桶状皮质回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烈地吞噬桶状皮质中的突触。 在胡须丢失或修剪之后，这种吞噬依赖于小胶质细胞CX3CR1 受体及其典型的神经元配体CX3CL1，但不是补体。利用单细胞RNAseq我还发现 在去除胡须后，神经元Cx3cl1在皮质中没有差异调节，而是蛋白酶 ADAM10已知能将膜结合的CX3CL1裂解成可溶性形式，在损伤后增加。 重要的是，ADAM10的药理抑制导致突触消除缺陷，从而导致表观复制 CX3CR1和CX3CL1缺陷小鼠。这些数据表明，神经元的翻译后修饰 需要ADAM10的CX3CL1来调节胡须后皮质小胶质突触的消除 移走。现在出现了几个令人兴奋的新问题，我将在这个提案中解决这些问题：1)什么是 ADAM10的细胞来源以及它是否定位于突触(目标1)？2)在大脑皮质下的其他突触 通过ADAM10-CX3CL1-CX3CR1信号进行桶状电路重建，这在晶须剥离之间是否有区别 和修剪(目标2)？I假设ADAM10来自第四层兴奋性神经元，以调节小胶质细胞- 介导的突触重塑和ADAM10信号对胡须后皮质突触重新布线的特异性 修剪和损伤，但不是皮质下突触重塑。为了验证这一假设，我获得了 强大的体内分子遗传学工具，可在特定细胞中操纵ADAM10的功能。我还开发出了 合作学习和执行iDISCO的尖端全组织清理，以评估结构重建 整个赛道。最后，我有一个强大的指导团队，其中包括我的导师多萝西·谢弗博士 在神经回路中的小胶质细胞功能方面的专业知识，我的同事安德鲁·塔珀博士在结构方面的专业知识 和大脑回路的功能图，以及在iDISCO方面具有专业知识的合作者。在一起，我正处于一个强大的 定位分子解剖ADAM10如何调节重塑所必需的神经元-小胶质细胞信号 大脑回路。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 Lost和ADAM10已被牵连。在这个过程中，我将接受各种显微镜和 分子遗传学方法，这将为我未来的职业生涯提供一个独立的原则 一家学术机构的研究人员专注于解剖神经回路中神经胶质细胞的功能。

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.