Identification of Cellular, Molecular and Genetic Factors Regulating RGC Regeneration

项目摘要 硬骨鱼具有再生丢失的视网膜神经元的天然能力。这是由于内源性激素的激活。 视网膜干细胞，Müler Glia(MG)，经历重新编程并不对称分裂，以响应 受伤。相比之下，哺乳动物MG对视网膜损伤有反应，不会分裂和取代视网膜中丢失的细胞 缺乏外源刺激。先前的研究已经成功地确定了诸如Achaete-Scut等因素 同系物1(ASCL1)和LIN-28同源物A(LIN28a)是MG再生潜力的关键调节因子。 有趣的是，可以通过诱导ASCL1的表达变化和治疗来刺激小鼠MG分裂 与组蛋白脱乙酰酶，表明再生潜力是完整的。这些研究几乎是独一无二的 在研究再生潜力之前，诱导广泛的视网膜损伤。更少人知道视网膜如何 再生是在失去与明确疾病相关的离散细胞类型后受到调控的。 选择性视网膜神经节细胞(RGC)变性与几种与视力有关的人类疾病有关 损失。青光眼是由视神经损伤引起的疾病之一，是不可逆转的主要原因。 世界上的盲人。为了研究RGC的再生，我们创建了一个新的转基因模型，使 斑马鱼选择性RGC消融。这些鱼共同表达细菌酶硝基还原酶(NTR)和 黄色荧光蛋白(YFP)在视网膜节细胞中表达。NTR将甲硝唑(MTZ)等前体药物转化为 DNA损伤诱导剂，导致快速靶向消融视网膜节细胞。最近，我们使用了正常贸易关系-- 前药消融系统研究视杆感光细胞再生。我们确定了免疫细胞的关键作用 在视杆细胞再生和总结神经保护药物筛选。使用我们的新模型，我们建议 寻找调控斑马鱼RGC再生的新因子并比较其在再生缺陷中的作用 老鼠模型。我假设在斑马鱼上的大规模发现将揭示新的细胞、分子和/或 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。这些见解可能会导致对RGC退行性疾病的变革性治疗。 除了再生能力外，斑马鱼还可以进行高通量筛选(HTS)， 活体成像和快速基因组操作。我们将利用这些优势，通过将我们的 再生模型和确定RGC死亡的关键免疫细胞反应因子(目标1)，药物筛选 促进再生或保护视网膜节细胞免于细胞死亡，并在互补小鼠中测试热门药物 RGC退变模型(AIM 2)，以及扰乱新发现的与再生相关的基因在 RGC再生(目标3)。这些目标及其背后的全面研究计划与 国家眼科研究所(NEI)阐述的重点领域：新兴的再生领域， 免疫系统在视觉疾病中的作用，以及将疾病相关基因与机制联系起来。

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.