Identification of Cellular, Molecular and Genetic Factors Regulating RGC Regeneration

项目摘要 硬骨鱼具有再生失去的视网膜神经元的天然能力。这是由于激活内源性 视网膜干细胞，Müller Glia（MG），经历重编程并响应于 损伤相比之下，哺乳动物MG对视网膜损伤有反应，不分裂和替换视网膜中丢失的细胞。 缺乏外部刺激。先前的研究已经成功地确定了诸如Achaete-scute 同源物1（ASCL 1）和Lin-28同源物A（LIN 28 A）作为MG再生潜力的关键调节剂。 有趣的是，通过诱导ASCL 1的表达变化和治疗，可以刺激小鼠MG分裂。 与组蛋白脱乙酰酶一起使用，表明再生潜力完好无损。这些研究几乎完全 在研究再生潜力之前诱导广泛的视网膜损伤。关于视网膜的功能 再生在具有明确疾病相关性的离散细胞类型的损失之后受到调节。 选择性视网膜神经节细胞（RGC）变性与几种与视力相关的人类疾病有关 损失青光眼是由视神经损伤引起的疾病的一个例子，是导致不可逆性青光眼的主要原因。 世界上的盲人为了研究RGC再生，我们建立了一种新的转基因模型， 斑马鱼的选择性RGC消融。这些鱼共表达细菌酶硝基还原酶（NTR）和 RGCs中的黄色荧光蛋白（YFP）报告基因。NTR将前药如甲硝唑（MTZ）转化为 DNA损伤诱导剂，导致RGCs的快速靶向消融。最近，我们使用了NTR- 前体药物消融系统研究视杆细胞再生。我们发现了免疫细胞的一个关键作用 在视杆细胞再生和神经保护药物筛选结论。使用我们的新模型，我们建议 鉴定调节斑马鱼RGC再生新因子并比较其在再生缺陷中的功能 小鼠模型。我推测，在斑马鱼中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，这些因素的一个子集将刺激再生 小鼠的反应。这些见解可能会导致RGC变性疾病的变革性治疗。 除了它们的再生能力，斑马鱼还适合高通量筛选（HTS）， 体内成像和快速基因组操作。我们将利用这些优势， 再生模型和确定对RGC死亡的关键免疫细胞应答者（目的1），筛选药物 增强再生或保护RGCs免于细胞死亡，并在互补小鼠中测试命中药物 RGC退化模型（Aim 2），并破坏新发现的“再生相关”基因在RGC退化中的作用。 RGC再生（目标3）。这些目标及其背后的综合研究计划与以下目标保持一致： 国家眼科研究所（NEI）强调的重点领域：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.