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: 10354817
• 负责人: EDWARD M LEVINE
• 金额: $ 25.95万
• 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 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10354817
• 关键词: Address; Adult; Alleles; Animal Model; Animals; Atlases; Bar Codes; 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; 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; Pharmacology; Phenotype; Photoreceptors; 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; base; cell motility; cost; cost effective; excitotoxicity; injured; migration; mouse model; neurogenesis; non-genetic; ocular imaging; particle; prevent; progenitor; protein kinase inhibitor; response; retinal damage; retinal neuron; retinal regeneration; selective expression; serial imaging; single-cell RNA sequencing; stem cells; teleost fish; 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.

摘要 哺乳动物的视网膜在视网膜损伤后不会再生。相反，再生发生了 在硬骨鱼中，通过Müler神经胶质细胞的增殖和重新编程成为神经源性祖细胞 来替代缺失的视网膜细胞。因此，视网膜再生医学的一个目标是有效地引导 哺乳动物Müler神经胶质细胞进入神经源性祖细胞状态。有几个障碍阻止了这一点，其中包括 细胞周期重新进入和增殖效率低下，缺乏去分化(丧失胶质细胞特性)，以及 不能过渡到神经性状态。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，还需要继续研究，以推动Müler glia- 基于重新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Müler神经胶质细胞中p27Kip1的失活是否促使这些细胞向神经源性祖细胞方向发展 州政府。P27Kip1是一种细胞周期蛋白依赖性的蛋白，在细胞周期中具有多种功能， 分化、细胞迁移、新陈代谢和基因表达。我们实验室之前的工作表明 P27Kip1在无损伤的情况下失活导致Müler胶质细胞增殖和向外迁移 核层。我们现在有初步数据显示，老化的视网膜在 P27Kip1失活，p27Kip1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。在这个项目中，我们将确定表型变化 P27Kip1-存在或不存在两种诱导因素时老化视网膜Müler神经胶质细胞的失活 视网膜损伤；N-甲基-D-天冬氨酸对视网膜内神经元的兴奋毒性，以及热激光 损伤以光感受器和RPE为靶点。虽然最初的研究将包括非侵入性眼球 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 进行单细胞RNA测序。将研究多个条件和时间点，并利用 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 超低温保存和样品多路传输。通过对这里生成的数据集的分析 伴随着现有数据集的整合，我们将能够识别p27Kip1中的新细胞状态- 灭活的Müler胶质细胞，确定它们是否去分化并获得神经源性特性，并鉴定 对不同类型伤害的选择性反应。这些研究将提供必要的解决办法，以 了解阻断p27Kip1功能如何影响Müler神经胶质细胞进入神经源性 始祖国家。此外，成功研制了视网膜细胞冷冻保存和标本 多路传输协议将增强视觉研究实验室扩展更复杂内容的能力 以具有成本效益的方式进行实验设计。