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.

概括 哺乳动物的视网膜在视网膜损伤后不会再生。相反，再生发生 在硬骨鱼中，通过米勒神经胶质细胞的增殖和重编程形成神经源性祖细胞 取代缺陷的视网膜细胞。因此，视网膜再生医学的目标是有效地指导 哺乳动物穆勒神经胶质细胞进入神经源性祖细胞状态。有几个障碍可以防止这种情况发生，其中包括 细胞周期再进入和增殖效率低下，缺乏去分化（神经胶质特性的丧失），以及 无法转变为神经源性状态。然而，最近的研究表明，这种类型 通过干预可以重新编程，并且需要继续研究来推动穆勒神经胶质细胞 基于重编程进入结构和功能再生领域。对于这款R21，我们将 确定小鼠米勒神经胶质细胞中 p27Kip1 失活是否会驱动这些细胞向神经源性祖细胞方向发展 状态。 p27Kip1 是一种细胞周期蛋白依赖性激酶抑制剂蛋白，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 p27Kip1 在没有损伤的情况下失活导致 Müller 胶质细胞增殖并迁移到外层 核层。我们现在有初步数据显示，老化的视网膜在老化后表现出强劲的增殖。 p27Kip1 失活，并且 p27Kip1 失活的缪勒神经胶质细胞的子集表达转录因子 与视网膜神经发生有关。在这个项目中，我们将确定表型变化 在存在或不存在两种类型诱导的情况下，老化视网膜中 p27Kip1 失活的米勒神经胶质细胞 视网膜损伤； N-甲基-D-天冬氨酸对视网膜内部神经元的兴奋毒性和热激光 损伤针对光感受器和 RPE。虽然初步研究将纳入非侵入性眼科治疗 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是 是单细胞RNA测序。将研究多个条件和时间点，并利用 现有技术的能力并降低成本，我们将优化视网膜细胞的方法 冷冻保存和样品复用。通过分析此处生成的数据集 通过整合现有数据集，我们将能够识别 p27Kip1 中的新细胞状态- 灭活米勒神经胶质细胞，确定它们是否去分化并获得神经源性特性，并识别 对不同类型伤害的选择性反应。这些研究将提供所需的解决方案 了解阻断 p27Kip1 功能如何影响 Müller 胶质细胞进入神经源性细胞的能力 祖状态。此外，视网膜细胞冻存及样本的开发成功 多路复用协议将增强视觉研究实验室扩展更复杂的能力 以具有成本效益的方式进行实验设计。