Elucidating and bypassing molecular mechanisms that suppress Muller glia-dependent regeneration of cones in two zebrafish models of chronic retinal damage

阐明和绕过抑制两种慢性视网膜损伤斑马鱼模型中穆勒胶质细胞依赖性视锥细胞再生的分子机制

• 批准号: 10567836
• 负责人: Seth Blackshaw
• 金额: $ 59.18万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10567836
• 关键词: ATAC-seq; Acute; Agonist; Attention; Behavior; Bioinformatics; Blindness; Bypass; CRISPR/Cas technology; Candidate Disease Gene; Cells; Central Nervous System; Chromatin; Chronic; Competence; Cone; DNA Sequence Alteration; Data Set; Development; Exhibits; Fishes; Future; Gene Expression; Gene Expression Profiling; Generations; Genes; Gliosis; Growth Factor; Hour; Human; Immune; Inflammation; Injections; Injury; Knowledge; Ligands; Light; Mammals; Medical; Methods; Microglia; Modeling; Molecular; Molecular Analysis; Muller' s cell; Mus; N-Methylaspartate; Natural regeneration; Neurons; Nuclear Pore Complex; Nuclear RNA; Paper; Persons; Play; Population; Process; Proliferating; Proteins; Regenerative capacity; Regenerative response; Reproducibility; Retina; Retinal Cone; Retinal Degeneration; Role; Science; Signal Pathway; Signal Transduction; Societies; Source; Stimulus; Testing; Translations; United States; Vertebrate Photoreceptors; Visual; Visual impairment; Work; Zebrafish; antagonist; blind; comparative; cytokine; disability; early onset; epigenomics; gamma secretase; inhibitor; knock-down; multiple omics; mutant; nerve stem cell; neuron regeneration; novel therapeutics; overexpression; photoreceptor degeneration; prevent; programs; receptor; regenerative; regenerative therapy; response; retinal damage; retinal neuron; retinal regeneration; sight restoration; single-cell RNA sequencing; small molecule; species difference; transcription factor; transcriptome sequencing

Project Summary: Retinal degenerative diseases are a major medical issue for society. One potentially exciting approach to restore vision is the regeneration of lost retinal neurons from an endogenous population of retinal cells, the Müller glia. We are studying this process in zebrafish, which unlike mammals, exhibits a natural Müller glia- dependent retinal regeneration response. However, there are two major gaps in our understanding of this retinal regeneration response. The first is why rapid acute damage exhibits a regeneration response and a slow chronic damage, which is what is often observed in human retinal degenerative diseases, does not induce a regeneration response in zebrafish. The second gap in our understanding is the role of the microglia, the immune cells of the central nervous system, which are the major source of inflammation resulting from damage and a known regulator of the Müller glia-dependent retinal regeneration. We will address these two gaps in three Specific Aims. Aim 1 will determine the potential of two different chronic zebrafish retinal degeneration mutants (gosh, an early onset rapid cone photoreceptor degeneration mutant and cep290, a late onset slow cone degeneration mutant) to induce Müller glia proliferation and regenerate lost cones using different stimuli. We will determine to what extent either a secondary acute damage or the introduction of molecules that stimulate Müller glia proliferation can induce cone regeneration in chronically damaged fish and how complete the regeneration process is. In Aim 2, we will conduct a comprehensive and unbiased, comparative analysis of gene expression and chromatin accessibility in Müller glia and microglia using a multiomic single-nuclear RNA- Seq and ATAC-Seq analysis in these two chronic degeneration mutants, along with two mouse chronic retinal degeneration mutants. We will determine the similarities and differences in gene expression and chromatin accessibility in the Müller glia and microglia between the acutely and chronically damaged retinas. These bioinformatic analyses will reveal transcription factors and signaling (cytokine, growth factors, ligand/ receptor pairs) molecules that are essential for regeneration following acute damage and blocking regeneration in the chronically damaged zebrafish retina. We will also determine the differences and similarities between the chronically damaged zebrafish and mouse retinas to determine how similar these regulatory components are between the zebrafish and mouse. Aim 3 will then functionally test the roles of the candidate regulators previously identified in our scRNA-Seq datasets or in Aim 2 by either modifying their expression or their activity in the chronically and acutely damaged zebrafish retina. This work will be the first molecular analysis of how retinal regeneration is regulated in the chronically damaged zebrafish retina and will be critical in the translation of Müller glial-dependent retinal regenerative therapies into human retinal degenerative diseases.

项目概要： 视网膜退行性疾病是社会的主要医学问题。一个潜在的令人兴奋的方法， 恢复视力是从内源性视网膜细胞群再生丢失的视网膜神经元， 穆勒胶。我们正在研究斑马鱼的这一过程，与哺乳动物不同，斑马鱼表现出天然的米勒神经胶质- 依赖性视网膜再生反应然而，我们对此的认识存在两大空白 视网膜再生反应第一个是为什么快速的急性损伤会表现出再生反应， 在人类视网膜变性疾病中经常观察到的缓慢慢性损伤， 斑马鱼的再生反应。我们理解的第二个差距是小胶质细胞的作用， 中枢神经系统的免疫细胞，是损伤导致炎症的主要来源 和一种已知的米勒神经胶质依赖性视网膜再生调节剂。我们将解决这两个差距， 三个具体目标。目的1将确定两种不同的慢性斑马鱼视网膜变性的潜力 突变体（gosh，早发性快速锥光感受器变性突变体和cep 290，迟发性缓慢锥光感受器变性突变体）。 视锥变性突变体）诱导Müller胶质细胞增殖并使用不同刺激再生丢失的视锥。 我们将确定继发性急性损伤或引入分子的程度， 刺激Müller胶质细胞增殖可诱导慢性损伤鱼类视锥细胞再生， 再生过程是。在目标2中，我们将进行全面、公正的比较分析 Müller胶质细胞和小胶质细胞中的基因表达和染色质可及性，使用多组单核RNA- 在这两种慢性变性突变体中的Seq和ATAC-Seq分析，沿着两种小鼠慢性视网膜变性突变。 退化突变体我们将确定基因表达和染色质的异同 急性和慢性损伤视网膜之间的Müller胶质细胞和小胶质细胞的可及性。这些 生物信息学分析将揭示转录因子和信号传导（细胞因子、生长因子、配体/受体 对）的分子，对急性损伤后的再生至关重要，并阻止再生， 长期受损的斑马鱼视网膜我们还将确定之间的差异和相似之处， 慢性损伤的斑马鱼和小鼠视网膜，以确定这些调节成分有多相似 在斑马鱼和老鼠之间。然后，目标3将对候选监管机构的作用进行功能测试 先前在我们的scRNA-Seq数据集或Aim 2中通过修饰它们的表达或它们的活性而鉴定 在慢性和急性损伤的斑马鱼视网膜中。这项工作将是第一个分子分析如何 视网膜再生在慢性损伤的斑马鱼视网膜中受到调节， Müller神经胶质依赖性视网膜再生疗法用于人类视网膜退行性疾病。