Identification of Cellular, Molecular and Genetic Factors Regulating RGC Regeneration

鉴定调节 RGC 再生的细胞、分子和遗传因素

基本信息

批准号：
10519102
负责人：
Kevin Emmerich
金额：
$ 4.77万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-12-12 至 2024-12-11
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10519102
关键词：
Ablation Acute Address Apoptosis Area Articulation Blindness Brain CRISPR/Cas technology Candidate Disease Gene Cell Death Cell Survival Cells Chemicals Chronic Clinical Collaborations Competence Computer Analysis DNA Damage Data Development Disease Drug Screening Enzymes Fishes Genes Genetic Genome Genomics Glaucoma Goals Histone Deacetylase Homologous Gene Human Immune Immune system Individual Inflammatory Injury Interneurons Kinetics Knowledge Larva Leber&apos s Hereditary Optic Neuropathy Light Link Mammals Measurement Methods Metronidazole Microglia Microscopy Modeling Molecular Muller&apos s cell Mus N-Methylaspartate National Eye Institute Natural regeneration Necrosis Nerve Crush Neurodegenerative Disorders Neuroprotective Agents Nitroreductases Optic Nerve Outcome Pathway interactions Pharmaceutical Preparations Play Process Prodrugs Proteins Regenerative capacity Regenerative research Regenerative response Reporter Research Resolution Retina Retinal Degeneration Retinal Ganglion Cells Role Signal Transduction Specificity Stimulus System Techniques Testing Therapeutic Transforming Growth Factor beta Transgenic Model Vertebrates Vision Visual Work Zebrafish adaptive optics axon regeneration behavior test cell injury cell regeneration cell type confocal imaging cytokine design gene function high throughput screening human disease in vivo in vivo imaging insight knockout gene light transmission model design mouse model mutant nerve damage neuroprotection novel optical lattices protective effect regeneration model regeneration potential regenerative response response to injury retinal damage retinal ganglion cell degeneration retinal ganglion cell regeneration retinal neuron retinal progenitor cell retinal regeneration retinal rods screening single-cell RNA sequencing stem cells teleost fish transcriptomics

项目摘要

Project Summary Teleost fish have a natural capacity to regenerate lost retinal neurons. This is due to activation of endogenous retinal stem cells, Müller Glia (MG), that undergo reprogramming and divide asymmetrically in response to injury. In contrast, mammalian MG are reactive to retinal injury do not divide and replace lost cells in the absence of exogenous stimulation. Prior research has successfully identified factors such as Achaete-scute homolog 1 (ASCL1) and Lin-28 homologue A (LIN28A) as critical regulators of MG regenerative potential. Intriguingly, mouse MG can be stimulated to divide by inducing expression changes in ASCL1 and treatment with histone deacetylases, demonstrating that regenerative potential is intact. These studies almost exclusively induce broad retinal damage prior to investigating regenerative potential. Much less is known about how retinal regeneration is regulated following the loss of discrete cell-types that have clear disease relevance. Selective retinal ganglion cell (RGC) degeneration is implicated in several human diseases linked to vision loss. Glaucoma, one example of disease caused by optic nerve damage, is the leading causing of irreversible blindness in the world. To investigate RGC regeneration, we created a novel transgenic model enabling selective RGC ablation in zebrafish. These fish co-express a bacterial enzyme Nitroreductase (NTR) and a yellow fluorescent protein (YFP) reporter in RGCs. NTR converts prodrugs such as metronidazole (MTZ) into DNA damage inducing agents, resulting in rapid targeted ablation of RGCs. Recently, we used the NTR- prodrug ablation system to study rod photoreceptor regeneration. We identified a critical role for immune cells in rod cell regeneration and concluded a neuroprotective drug screen. Using our new model, we propose to identify novel factors regulating zebrafish RGC regeneration and compare function in regeneration-deficient mouse models. I hypothesize that large-scale discovery in zebrafish will reveal novel cellular, molecular, and/or genetic factors that regulate RGC regeneration, and that a subset of these factors will stimulate regenerative responses in mice. Such insights may lead to transformative therapeutics for RGC degeneration diseases. In addition to their regenerative competence, zebrafish are amenable to high-throughput screening (HTS), in vivo imaging, and rapid genomic manipulation. We will take advantage of these strengths by characterizing our regeneration model and determining key immune cell responders to RGC death (Aim 1), screening for drugs that enhance regeneration or protect RGCs from cell death and testing hit drugs in complementary mouse RGC degeneration models (Aim 2), and disrupting newly identified “regeneration-associated” genes for roles in RGC regeneration (Aim 3). These aims, and the comprehensive research plan behind them, are aligned with areas of emphasis articulated by the National Eye Institute (NEI): the emerging field of regeneration, the immune system’s role in visual disease, and connecting disease-associated genes to mechanisms.

项目摘要硬骨鱼自然而然地再生视网膜神经元。视网膜干细胞Müller神经胶质（MG），其重新编程和不对称地分裂。相比缺乏外源刺激。同源物1（ASCL1）和LIN-28同源物A（LIN28A）作为MG再生潜力的关键调节剂。有趣的是，可以通过诱导ASCL1的表达变化和处理来刺激小鼠MG分裂使用组蛋白的deacelasses，证明再生潜力是完整的。在研究再生潜力之前会引起广泛的视网膜损害。在失去离散细胞类型的明确疾病相关性后，定期定期。选择性视网膜神经节细胞（RGC）变性与几种与视觉相关的人类疾病损失。世界上的失明。斑马鱼中的选择性RGC消融。 RGC中的黄色荧光蛋白（YFP）记者。 DNA损伤诱导剂，重新占用RGC的快速靶向。研究杆光感受器再生的前瞻性消融系统。在杆细胞再生中，结论了神经保护药物筛查。确定调节斑马鱼RGC再生的新因素，并比较在再生缺陷中的功能鼠标模型。调节RGC再生的遗传因素，这些因素的子集将刺激再生小鼠的反应。除了它们的再生能力外，斑马鱼还适合高脚齿筛选（HTS）体内成像和快速的基因组操作。再生模型并确定对RGC死亡的关键免疫细胞反应（AIM 1），筛查药物这增强了再生或保护RGC免受细胞死亡和测试击中编译小鼠中的药物 RGC变性模型（AIM 2），并破坏了新鉴定的“与再生相关”基因 RGC再生（目标3）。由国家眼科研究所（NEI）提出的强调领域：新兴领域，Thee 免疫系统在视觉疾病中的作用，并将与疾病相关的基因与机制联系起来。