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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.

总结