Neurogenic potential of murine Müller glia following retinal injury and conditional inactivation of p27Kip1

视网膜损伤和 p27Kip1 条件失活后小鼠 Müller 胶质细胞的神经源性潜力

• 批准号: 10541894
• 负责人: EDWARD M LEVINE
• 金额: $ 21.63万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10541894
• 关键词: Address; Adult; Alleles; Animal Model; Animals; Atlases; Bar Codes; Behavior; Cell Cycle; Cell Cycle Regulation; Cell Differentiation process; Cells; Complex; Cryopreserved Cell; Cyclin-Dependent Kinase Inhibitor; Data; Data Analyses; Data Collection; Data Set; Development; Disease; Down-Regulation; Exhibits; Experimental Designs; Functional Regeneration; Future; Gene Expression; Gene Silencing; Genetic; Genomics; Gliosis; Goals; Human; Injury; Intervention; Label; Lasers; Lesion; Metabolism; Methods; Modeling; Muller' s cell; Mus; N-Methylaspartate; Natural regeneration; Neuroglia; Neuronal Differentiation; Nuclear; Ophthalmoscopy; Optical Coherence Tomography; Phenotype; Photoreceptors; Proliferating; Property; Proteins; Protocols documentation; Regenerative Medicine; Regenerative research; Research; Resolution; Retina; Sampling; Scanning; Spatial Distribution; Stimulus; Structure; Sum; Technology; Testing; Time; Tissues; Up-Regulation; Variant; Vesicle; Vision research; Work; aged; cell motility; cost; cost effective; cyclin-dependent kinase inhibitor 1B; excitotoxicity; injured; migration; mouse model; neurogenesis; non-genetic; ocular imaging; particle; pharmacologic; prevent; progenitor; protein kinase inhibitor; response; retinal damage; retinal neuron; retinal regeneration; selective expression; serial imaging; single-cell RNA sequencing; stem cells; teleost fish; tissue fixing; transcription factor; transcriptomics

Summary The mammalian retina does not regenerate following retinal damage. In contrast, regeneration occurs in teleost fish through the proliferation and reprogramming of Müller glia into neurogenic progenitor cells that replace the retinal cells in deficit. Thus, a goal of retinal regenerative medicine is to efficiently direct mammalian Müller glia into a neurogenic progenitor state. Several barriers prevent this and they include inefficient cell cycle reentry and proliferation, the lack of dedifferentiation (loss of glial properties), and inability to transition into a neurogenic state. However, recent studies show that this type of reprogramming is possible with intervention, and continued research is needed to push Müller glia- based reprogramming into the realm of structural and functional regeneration. For this R21, we will determine if p27Kip1 inactivation in mouse Müller glia drives these cells toward a neurogenic progenitor state. p27Kip1 is a cyclin-dependent kinase inhibitor protein that has multiple functions in the cell cycle, differentiation, cell migration, metabolism, and gene expression. Prior work from our lab showed that p27Kip1 inactivation in the absence of injury caused Müller glia proliferation and migration into the outer nuclear layer. We now have preliminary data showing that aged retinas exhibit robust proliferation after p27Kip1 inactivation, and that a subset of p27Kip1-inactivated Müller glia express a transcription factor associated with retinal neurogenesis. In this project, we will determine the phenotypic changes of p27Kip1-inactivated Müller glia in aged retinas in the presence or absence of two types of induced retinal damage; N-methyl-D-aspartate excitotoxicity to target inner retinal neurons, and thermal laser lesions to target photoreceptors and RPE. While initial studies will incorporate noninvasive ocular imaging in live animals and immunohistology of fixed tissue, the primary method of data collection will be single cell RNA sequencing. Multiple conditions and timepoints will be studied, and to leverage the capabilities of existing technology and reduce costs, we will optimize methods for retinal cell cryopreservation and sample multiplexing. Through analysis of the datasets generated here accompanied by integration of existing datasets, we will be able to identify new cell states in p27Kip1- inactivated Müller glia, determine if they dedifferentiate and acquire neurogenic properties, and identify selective responses to different types of injury. These studies will provide the needed resolution to understand how blocking p27Kip1 function impacts the ability of Müller glia to enter into a neurogenic progenitor state. Additionally, the successful development of retinal cell cryopreservation and sample multiplexing protocols will enhance the ability of vision research labs to expand upon more complex experimental designs in a cost effective manner.

总结 哺乳动物视网膜损伤后不会再生。相反， 通过Müller胶质细胞增殖和重编程为神经源性祖细胞， 来替代受损的视网膜细胞因此，视网膜再生医学的目标是有效地引导 将哺乳动物的米勒神经胶质转化为神经原性祖细胞状态。有几个障碍阻止了这一点，它们包括 无效的细胞周期再进入和增殖，缺乏去分化（神经胶质性质的丧失），以及 无法过渡到神经原性状态。然而，最近的研究表明，这种类型的 通过干预，重新编程是可能的，需要继续研究以推动Müller胶质细胞- 基于重编程的结构和功能再生领域。对于R21，我们将 确定小鼠Müller胶质细胞中p27 Kip 1失活是否驱动这些细胞向神经原性祖细胞方向发展 状态p27 Kip 1是一种细胞周期蛋白依赖性激酶抑制蛋白，在细胞周期中具有多种功能， 分化、细胞迁移、代谢和基因表达。我们实验室之前的工作表明， 在无损伤的情况下，p27 Kip 1失活导致Müller胶质细胞增殖并迁移到外膜。 核层我们现在有初步的数据表明，老年视网膜在经过 p27 Kip 1失活，并且p27 Kip 1失活的Müller胶质细胞亚群表达转录因子 与视网膜神经发生有关。在本项目中，我们将确定 在存在或不存在两种类型的诱导的情况下，老化视网膜中p27 Kip 1失活的Müller胶质细胞 视网膜损伤; N-甲基-D-天冬氨酸对靶视网膜内神经元的兴奋性毒性，以及热激光 靶向光感受器和RPE的损伤。虽然最初的研究将包括非侵入性眼 活体动物成像和固定组织的免疫组织学，数据收集的主要方法将 是单细胞RNA测序。将研究多个条件和时间点，并利用 现有技术的能力和降低成本，我们将优化视网膜细胞的方法， 冷冻保存和样品多路复用。通过分析这里生成的数据集 伴随着现有数据集的整合，我们将能够识别p27 Kip 1中的新细胞状态。 灭活的Müller神经胶质，确定它们是否去分化并获得神经原性特性，并鉴定 对不同类型损伤的选择性反应。这些研究将提供必要的解决办法， 了解阻断p27 Kip 1功能如何影响Müller胶质细胞进入神经源性 祖国此外，视网膜细胞冷冻保存和样品的成功开发 多路复用协议将增强视觉研究实验室的能力， 以成本效益的方式进行实验设计。