Cellular and Molecular Dynamics of Retinal Microglial in the Context of Photoreceptor Degeneration

感光器变性背景下视网膜小胶质细胞的细胞和分子动力学

• 批准号: 10223314
• 负责人: Daniel Raphael Saban
• 金额: $ 44.31万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10223314
• 关键词: Address; Age; Anaphylatoxins; Blindness; Bone Marrow; Bone Marrow Purging; C3AR1 gene; Cell Death; Cells; Chemotaxis; Chimera organism; Clinical; Complex; Cytoprotection; Data; Data Set; Defect; Development; Disease; Environment; Etiology; Excision; Explosion; Eye; Galectin 3; Genetic; Genetic Transcription; Immune; Immune response; In Situ; Interruption; Knock-in Mouse; Knock-out; Knockout Mice; Knowledge; Lead; Left; Letters; Life; Ligands; Light; Mediating; Methods; Microglia; Modeling; Molecular; Molecular Profiling; Mus; Myeloid Cells; Neuraxis; Neurons; Outcome; Pathology; Patients; Phagocytes; Phagocytosis; Pharmacology; Phenocopy; Photoreceptors; Physiological; Physiology; Play; Population; Process; Property; Protein Tyrosine Kinase; Reagent; Reporter; Retina; Retinal Degeneration; Retinal Diseases; Rhodopsin; Role; Series; Signal Transduction; Stargardt' s disease; Structure of retinal pigment epithelium; TREM2 gene; Techniques; Testing; Time; Tissues; Transgenic Organisms; Validation; Vision; experimental study; gain of function; imaging modality; in vivo; loss of function; macrophage; migration; molecular dynamics; monocyte; new therapeutic target; novel; novel therapeutic intervention; novel therapeutics; patient population; photoreceptor degeneration; prenatal; preservation; programs; receptor; recruit; response; single-cell RNA sequencing; small molecule; tool

SUMMARY Many, if not most, forms of retinal degeneration involve ectopic accumulation of subretinal macrophages. However, the contribution of bona fide microglia to this immune response and their independent role in disease is poorly understood. It is now widely appreciated that macrophages in degenerating neuronal tissues are not only comprised of microglia but may also include monocyte-derived macrophages. The former are endogenous prenatal-derived cells maintained locally throughout life, whereas the latter represent transiently recruited passengers in disease states. Hence, these two distinct lineages have nonredundant activities in the disease process and should thereby be studied as distinct entities. However, distinguishing microglia is technically challenging since standard techniques such as immunolabeling, conventional reporter mice, or myeloablation bone marrow chimeras are insufficient to study these two populations. In fact, the only method to achieve such separation is through recently established Cx3cr1-CreER microglia lineage tracing mice. Yet, few studies have employed this approach, which has resulted in a knowledge gap in the field. As it is also now known that microglia are essential in establishing and preserving neuronal activity in physiological conditions, determining microglia-specific activities in retinal disease is now imperative. Using the lineage tracing approach and single- cell RNA sequencing (scRNA-seq), our lab recently identified a novel population of cytoprotective retinal microglia in photoreceptor degeneration models. We now wish to build upon these findings in our current proposal by applying novel tools to unravel these cells mechanistically, as well as to determine the significance of their cytoprotective program across etiologically distinct retinal degenerative diseases. We begin in Aim 1 by leveraging our scRNA-seq dataset to inhibit microglial chemotaxis that will allow us to determine whether cytoprotection is a subretinal-specific response. Aim 2 takes advantage of our scRNA-seq dataset as well for targeted conditional and global knockouts to establish the molecular underpinnings of microglia-mediated protection. Lastly, in Aim 3 we will apply loss- and gain-of-function studies to examine whether this cytoprotective microglial program is operative in both primary photoreceptor degeneration and retinal pigment epithelial pathology-related degeneration. In summary, our proposal is not only timely, but is also poised to unravel the innerworkings of this novel microglial population across etiologically distinct retinal degeneration models and perhaps help uncover novel therapeutic targets that can bolster their activities for vision preservation in photoreceptor degeneration.

总结 许多（如果不是大多数）形式的视网膜变性涉及视网膜下巨噬细胞的异位积聚。 然而，真正的小胶质细胞对这种免疫反应的贡献及其在疾病中的独立作用， 是很难理解的。现在广泛认识到，变性神经元组织中的巨噬细胞并不 仅由小胶质细胞组成，但也可包括单核细胞衍生的巨噬细胞。前者是内生的 出生前衍生的细胞在整个生命过程中保持在局部，而后者代表短暂招募 旅客在疾病状态下因此，这两个不同的谱系在疾病中具有非冗余的活动 这一过程，因此应该作为不同的实体进行研究。然而，从技术上讲， 具有挑战性，因为标准技术如免疫标记、常规报告小鼠或骨髓消融 骨髓嵌合体不足以研究这两个群体。事实上，唯一的方法来实现这样的 分离是通过最近建立的Cx 3cr 1-CreER小胶质细胞谱系追踪小鼠进行的。然而，很少有研究 采用了这种方法，这导致了该领域的知识差距。正如现在所知， 小胶质细胞在生理条件下建立和保持神经元活性是必不可少的， 视网膜疾病中的小胶质细胞特异性活动现在是必要的。使用血统追踪方法和单一- 细胞RNA测序（scRNA-seq），我们实验室最近发现了一种新型细胞保护性视网膜病变群体 光感受器变性模型中的小胶质细胞。我们现在希望在我们目前的调查结果的基础上， 通过应用新的工具来解开这些细胞的机制，以及确定的意义， 他们的细胞保护计划在病因不同的视网膜变性疾病。我们开始目标1， 利用我们的scRNA-seq数据集来抑制小胶质细胞的趋化性，这将使我们能够确定 细胞保护是视网膜下特异性反应。Aim 2也利用了我们的scRNA-seq数据集， 有针对性的条件和整体敲除，以建立小胶质细胞介导的 保护最后，在目标3中，我们将应用功能丧失和获得研究来检查这是否 细胞保护性小胶质细胞程序在原发性光感受器变性和视网膜色素变性中均起作用 上皮病理相关变性。总之，我们的建议不仅是及时的，而且是准备好的， 揭示这种新的小胶质细胞群体在病因学上不同的视网膜变性中的内部运作， 模型，也许有助于发现可以增强他们视力活动的新治疗靶点 光感受器变性中的保存。