Reprogramming adult murine Müller glia via L-Myc expression

通过 L-Myc 表达对成年小鼠 Müller 胶质细胞进行重编程

• 批准号: 10751295
• 负责人: Megan Stone
• 金额: $ 3.3万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10751295
• 关键词: ATAC-seq; Adult; Affect; Alleles; BHLH Protein; Binding; Biological Process; Blindness; Bromodeoxyuridine; Cell Cycle; Cell Reprogramming; Cell division; Cells; Chromatin; Color; Competence; DNA; Data; Disease; Electroporation; Euchromatin; Family; Fibroblasts; Fishes; Fluorescence; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Heritability; Histone H3; Histones; Hour; In Vitro; Injury; Label; MYCL1 gene; Mammals; Measures; Modeling; Muller' s cell; Multiomic Data; Mus; Natural regeneration; Neuroglia; Neurons; Ontology; Orthologous Gene; Pattern; Plasmids; Play; Population; Process; Proliferating; Proteins; Regenerative capacity; Retina; Role; Signal Transduction; Somatic Cell; Sorting; Source; Stains; System; Testing; Time; Tissue-Specific Gene Expression; Tissues; Vertebrates; Vision; Visual; Visual impairment; Zebrafish; bioinformatics network; cell growth; cell type; data integration; design; fluorophore; functional restoration; gene regulatory network; gene therapy; induced pluripotent stem cell; insight; mouse model; overexpression; prevent; progenitor; programs; response; response to injury; restoration; retinal neuron; retinal progenitor cell; retinal regeneration; selective expression; sight restoration; stem cells; teleost fish; tissue regeneration; transcriptome sequencing; transcriptomics; vector

PROJECT SUMMARY In response to damage from injury or disease, lost neurons in the adult mammalian retina are irreplaceable which can lead to visual impairment including blindness. Some non-mammalian vertebrates, such as teleost fish, can regenerate the retina in response to damage via the Müller glia. In teleost fish, Müller glia will respond to signals from damaged neurons by asymmetrically dividing to produce retinal progenitor cells. These progenitor cells then differentiate into neuronal cell types and functionally restore tissue. Mammalian Müller glia are quiescent and do not display regenerative capacity in response to damage. However, mammalian Müller glia demonstrate some cell state plasticity and can be reprogrammed into induced pluripotent stem cells (iPSC) in vitro. Mammalian Müller glia likely have the capacity for reprogramming but lack the cell intrinsic- or extrinsic-signaling necessary to do so. I have found that overexpression of L-Myc, a robust reprogramming factor used to reprogram many somatic cell types to iPSCs, leads to cell cycle re-entry in adult mammalian Müller glia in ex vivo retinal tissue. Using murine retinal explant tissue as an injury model, I will characterize the effects of L-Myc overexpression on proliferation and dedifferentiation in mammalian Müller glia. I hypothesize that L-Myc overexpression in Müller glia leads to direct and indirect changes in gene expression and chromatin accessibility that reprogram Müller glia toward a progenitor-like state. To test this hypothesis, I will first characterize the effects of L-Myc expression on proliferation. I will do so by defining the interval of proliferation as it relates to L-Myc expression and determining if Müller glia or their descendants are able to divide repeatedly following L-Myc overexpression. Next, I will characterize the effect of L-Myc overexpression in Müller glia on cell identity and gene regulation. Using data integration of RNA-sequencing, ATAC-sequencing, and CUT&Tag of FACS sorted L-Myc overexpressing Müller glia, I will identify gene regulatory networks affected directly or indirectly by L-Myc overexpression and identify changes in cell state. Combining Müller glia, a highly promising population within the mammalian retina for reprogramming, and L-Myc, a potent reprogramming factor to promote proliferation and dedifferentiation, creates an ideal scenario to reveal targets to promote regeneration in the mammalian retina through the analysis of gene regulatory networks activated or inhibited by L-Myc overexpression. Regardless of the cell state changes that occur in response to L-Myc overexpression in Müller glia, this project will reveal what signaling barriers exist that prevent tissue regeneration via Müller glia in the mammalian retina. This study is designed to identify future targets for genetic therapies to promote restoration of tissue and visual function after injury in the mammalian retina, which one day could lead to treatments for currently untreatable visual diseases.

项目摘要 在对损伤或疾病造成的损害的反应中，成年哺乳动物视网膜中丢失的神经元被修复。 不可替代，可能导致视力障碍，包括失明。一些非哺乳类脊椎动物， 例如硬骨鱼，可以通过缪勒神经胶质再生视网膜以应对损伤。在硬骨鱼中， 神经胶质将对来自受损神经元的信号作出反应，通过不对称分裂产生视网膜祖细胞 细胞这些祖细胞然后分化成神经元细胞类型并在功能上恢复组织。 哺乳动物的缪勒神经胶质是静止的，对损伤不显示再生能力。 然而，哺乳动物Müller神经胶质细胞表现出一定的细胞状态可塑性，并且可以被重新编程为 体外诱导多能干细胞（iPSC）。哺乳动物的缪勒神经胶质细胞可能有能力 这些细胞可以重编程，但缺乏这样做所必需的细胞内在或外在信号传导。 我发现L-Myc的过度表达，L-Myc是一种强大的重编程因子， 体细胞类型转化为iPSC，导致离体视网膜组织中成年哺乳动物Müller神经胶质细胞的细胞周期重新进入。 使用小鼠视网膜外植体组织作为损伤模型，我将描述L-Myc过表达的影响， 对哺乳动物Müller胶质细胞增殖和去分化的影响。我推测L-Myc的过度表达可能与 Müller胶质细胞导致基因表达和染色质可及性的直接和间接变化， Müller胶质细胞向祖细胞样状态发展。为了验证这一假设，我将首先描述L-Myc的作用， 增殖的表达。我将通过定义与L-Myc表达相关的增殖间隔来做到这一点 并确定Müller胶质细胞或其后代是否能够在L-Myc后重复分裂 过度表达接下来，我将描述Müller胶质细胞中L-Myc过表达对细胞身份的影响， 基因调控使用RNA测序、ATAC测序和FACS分选的CUT&Tag的数据整合 L-Myc过表达的Müller胶质细胞，我将鉴定直接或间接受L-Myc影响的基因调控网络 过表达和识别细胞状态的变化。 结合Müller神经胶质，哺乳动物视网膜内一个非常有前途的重新编程群体， 和L-Myc，一种促进增殖和去分化的有效重编程因子， 通过基因分析揭示促进哺乳动物视网膜再生的靶点 L-Myc过表达激活或抑制的调控网络。不管细胞状态如何变化， Müller胶质细胞中L-Myc过表达的反应，该项目将揭示存在什么样的信号障碍 通过哺乳动物视网膜中的米勒神经胶质阻止组织再生。这项研究旨在确定 基因治疗的未来目标，以促进损伤后的组织和视觉功能的恢复， 哺乳动物视网膜，有一天可能导致治疗目前无法治愈的视觉疾病。