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

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

概括哺乳动物视网膜在视网膜损伤后不会再生，相反，会发生再生。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因此，视网膜再生医学的目标是有效地引导。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阻止这一过程，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），以及然而，最近的研究表明这种类型的。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对于这个 R21，我们将基于重编程进入结构和功能再生领域。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 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，我们实验室之前的工作表明，分化、细胞迁移、代谢和基因表达。 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在这个项目中，我们将确定与视网膜神经发生相关的表型变化。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性毒性和热激光初步研究将包括非侵入性眼部病变。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通过分析此处生成的数据集进行冷冻保存和样本复用。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，并识别这些研究将为不同类型的伤害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此外，视网膜细胞冷冻保存和样本的开发成功。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。