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.

项目总结 对于损伤或疾病造成的损害，成年哺乳动物视网膜中丢失的神经元是 不可替代，会导致包括失明在内的视力损害。一些非哺乳类脊椎动物， 例如硬骨鱼，可以通过Müler神经胶质细胞对损伤做出反应来再生视网膜。在硬骨鱼中，米勒 神经胶质细胞会对受损神经元发出的信号做出反应，不对称分裂产生视网膜前体细胞 细胞。然后，这些祖细胞分化为神经细胞类型，并在功能上恢复组织。 哺乳动物的Müler神经胶质细胞处于静止状态，对损伤没有表现出再生能力。 然而，哺乳动物的Müler胶质细胞表现出一些细胞状态的可塑性，并可以重新编程为 体外诱导多能干细胞(IPSC)。哺乳动物的Müler胶质细胞可能有能力 重新编程，但缺乏这样做所需的细胞内在或外在信号。 我发现L-Myc的过度表达，一种用于重新编程的强大的重新编程因子 体细胞类型到ipSCs，导致成年哺乳动物视网膜组织中Müler胶质细胞周期的重新进入。 以小鼠视网膜外植体组织为损伤模型，研究L-MYC过表达对损伤的影响 哺乳动物Müler胶质细胞增殖和去分化的研究我推测L-Myc在 Müler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，从而重新编程 穆勒正在走向一种类似祖先的状态。为了验证这一假设，我将首先描述L-Myc的效果 在增殖上的表达。我将通过定义增殖间隔来实现这一点，因为它与L-MYC的表达有关 并确定米勒胶质细胞或其后代是否能够在L-Myc之后重复分裂 过度表达。接下来，我将表征L-Myc在Müler胶质细胞中过表达对细胞鉴定和 基因调控。利用流式细胞仪的RNA测序、ATAC测序和切割标签的数据集成 L-Myc过表达穆勒胶质细胞，我将确定L-Myc直接或间接影响的基因调控网络 过度表达并识别细胞状态的变化。 穆勒胶质细胞是哺乳动物视网膜内一个非常有希望进行重新编程的群体， 而L-Myc，一种促进增殖和去分化的强有力的重新编程因子，创造了一个理想的 通过基因分析揭示促进哺乳动物视网膜再生的靶点的情景 L-Myc过表达激活或抑制调控网络。无论单元状态如何变化， 由于L-Myc在Müler胶质细胞中过表达，该项目将揭示存在哪些信号障碍 这阻止了哺乳动物视网膜中通过Müler胶质细胞的组织再生。这项研究旨在确定 基因治疗的未来目标是促进损伤后组织和视觉功能的恢复 哺乳动物的视网膜，有一天可能会导致目前无法治愈的视觉疾病的治疗。