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

摘要 许多（如果不是大多数）形式的视网膜变性都涉及视网膜下巨噬细胞的异位积累。 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 人们对此知之甚少。现在人们普遍认识到，退化神经组织中的巨噬细胞并不 仅由小胶质细胞组成，但也可能包括单核细胞衍生的巨噬细胞。前者是内生的 产前衍生的细胞在整个生命过程中局部维持，而后者代表短暂招募 处于疾病状态的乘客。因此，这两个不同的谱系在疾病中具有非冗余的活性 过程，因此应将其作为不同的实体进行研究。然而，区分小胶质细胞在技术上是 由于免疫标记、传统报告小鼠或清髓等标准技术具有挑战性 骨髓嵌合体不足以研究这两个群体。事实上，实现这一目标的唯一方法 分离是通过最近建立的 Cx3cr1-CreER 小胶质细胞谱系追踪小鼠进行的。然而，很少有研究 采用这种方法，导致了该领域的知识差距。现在也知道 小胶质细胞对于在生理条件下建立和维持神经元活动至关重要，决定 小胶质细胞在视网膜疾病中的特异性活性现在势在必行。使用谱系追踪方法和单 细胞 RNA 测序 (scRNA-seq)，我们的实验室最近发现了一种新的细胞保护性视网膜群体 光感受器变性模型中的小胶质细胞。我们现在希望以这些发现为基础 通过应用新颖的工具从机械上解开这些细胞并确定其重要性来提出建议 他们针对不同病因的视网膜退行性疾病的细胞保护计划。我们从目标 1 开始： 利用我们的 scRNA-seq 数据集抑制小胶质细胞趋化性，这将使我们能够确定是否 细胞保护是视网膜下特异性反应。目标 2 也利用了我们的 scRNA-seq 数据集 有针对性的条件和全局基因敲除，以建立小胶质细胞介导的分子基础 保护。最后，在目标 3 中，我们将应用功能丧失和获得功能研究来检验这是否 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中均起作用 上皮病理相关的变性。综上所述，我们的建议不仅是及时的，而且是有准备的。 揭示这种新型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机制 模型，或许有助于发现新的治疗靶点，从而增强他们的视觉活动 光感受器变性中的保存。

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.