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

摘要 许多(如果不是大多数)视网膜变性涉及视网膜下巨噬细胞的异位聚集。 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，退化神经元组织中的巨噬细胞不是 仅由小胶质细胞组成，但也可能包括单核细胞来源的巨噬细胞。前者是内生性的 出生前衍生的细胞在整个生命过程中保持在局部，而后者代表瞬时招募 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多余的活动。 因此，应将其作为不同的实体加以研究。然而，从技术上讲，区分小胶质细胞 具有挑战性的标准技术，如免疫标记、常规报告小鼠或骨髓清除术 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，很少有研究表明 采用了这一方法，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存神经元活动是必不可少的，决定 视网膜疾病中的小胶质细胞特异性活动现在势在必行。使用血统追踪方法和单一的- 细胞RNA测序(scRNA-seq)，我们的实验室最近发现了一个新的细胞保护性视网膜种群 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研究成果的基础上 通过应用新的工具来机械地解开这些细胞并确定其意义 他们的细胞保护计划跨越不同的病因视网膜退行性疾病。我们从目标1开始 利用我们的scRNA-seq数据集来抑制小胶质细胞的趋化作用，这将使我们能够确定 细胞保护是一种视网膜下特异性反应。Aim 2也利用了我们的scRNA-seq数据集 有针对性的条件性和全局性基因敲除建立小胶质细胞介导的分子基础 保护。最后，在目标3中，我们将应用功能损失和功能增益研究来检验这一点 细胞保护性小胶质细胞程序对初级光感受器变性和视网膜色素均有效 上皮病理相关变性。总而言之，我们的建议不仅是及时的，而且也准备好 解开这种新的小胶质细胞群在病因上不同的视网膜变性中的内部结构 模型，也许有助于发现新的治疗靶点，可以增强他们的视力活动 光感受器退行性变的保存。

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.