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：
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
项目类别：
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&apos 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

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.

概括视网膜损伤后，哺乳动物视网膜不会再生。相反，再生发生通过将müller神经胶质扩散和重编程到神经源细胞中，在硬骨鱼中在赤字中取代了视网膜细胞。那是视网膜再生医学的目标是有效地指导哺乳动物müller神经胶质进入神经源性祖细胞状态。几个障碍阻止了这一点，它们包括无效的细胞周期再入和增殖，缺乏去分化（神经胶质特性的丧失）和无法过渡到神经源。但是，最近的研究表明，这种类型的通过干预是可能的重新编程，并且需要继续研究以推动müllerGlia- 基于结构和功能再生领域的重新编程。对于此R21，我们将确定小鼠müller神经胶质中的p27KIP1是否将这些细胞驱动到神经源性祖细胞状态。 P27KIP1是一种依赖细胞周期蛋白的激酶抑制剂蛋白，在细胞周期中具有多个功能，分化，细胞迁移，代谢和基因表达。我们实验室的先前工作表明 p27KIP1在没有损伤的情况下失活导致müller神经胶质增殖并迁移到外部核层。现在，我们有初步数据表明，老化的视网膜在 p27KIP1失活，以及p27KIP1灭活的müller胶质的子集表达转录因子与残留神经发生有关。在这个项目中，我们将确定在存在或不存在两种类型的诱导的情况下视网膜损害； N-甲基-D-天冬氨酸兴奋性靶向内部残留神经元和热激光器靶向感光体和RPE的病变。初始研究将融合非侵入性眼现场动物的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。多个条件和时间点将进行研究，并利用现有技术的功能并降低成本，我们将优化永久单元的方法冷冻保存和样品多路复用。通过分析此处生成的数据集伴随着现有数据集的集成，我们将能够在P27KIP1-中识别新的单元格状态灭活的müller神经胶质，确定它们是否去分化并获得神经源特性，并识别对不同类型的伤害的选择性反应。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MüllerGlia进入神经源的能力祖细胞状态。此外，残留细胞冷冻保存和样品的成功发展多路复用协议将增强视力研究实验室扩展更复杂的能力实验设计以具有成本效益的方式。