Regulation of successful optic nerve regeneration by the mevalonate/cholesterol pathway

项目总结 在美国，像青光眼这样的视神经疾病导致的视力丧失是导致失明的常见原因。 不幸的是，这些情况通常是永久性的，因为中枢神经系统缺乏 再生受损的轴突。视神经(ON)损伤的哺乳动物模型概括了在 患者很难理解成功再生所需的条件。相比之下，硬骨鱼， 例如斑马鱼，可以成功地再生受损的ON和恢复失去的视力。我们正在使用这个 有机体研究成功再生的机制，希望将这些发现转化为 进入新的治疗方法，以改善哺乳动物疾病模型和患者的再生。在转录组中- 斑马鱼再生过程中视网膜神经节细胞(RGCs)的广泛研究 胆固醇途径在这一过程中被上调。我们的初步数据显示 这些途径的转录调控因子srebf2是成功再生所必需的。我们假设 Srebf2通过激活RGC固有的甲氧丙戊酸和胆固醇合成来调节再生。 细胞外胆固醇和脂类来源的受体的途径和/或诱导表达。使用 对于斑马鱼系统可用的强大的遗传和化学工具，我们建议识别关键时期 Srebf2活性及其功能的下游介体(S)。目标1将确定srebf2何时在 RGCs对于ON再生和使用srebf2活性的新报告系来描绘何时 转录活动就会发生。我们还将测试srebf2活性的激活是否足以加速 再生。最后，我们将使用激光捕获显微切割rna-seq(lcm-seq)来鉴定差异基因。 视网膜神经节细胞中srebf2增益性和损失性功能的表达。目标2建议确定哪个RGC内在或 Srebf2下游的外在通路参与其功能的调节。我们将使用药物和基因的组合 敲除以确定甲氧戊酸/胆固醇的内在合成和外部供应是否独立， 相互依赖的，和/或对成功再生的补偿。根据这项研究的结果 我们将进一步研究胆固醇、泛醌、蛋白质的下游内在合成途径。 以及蛋白质N-糖基化或低密度脂蛋白受体及其下游加工。 目标3将测试Srebf2表达的充分性，以提供神经保护和刺激轴突再生 在急性损伤的小鼠模型上。LCM-SEQ将被用来识别诱导的基因表达变化 并与斑马鱼中发现的RGCs进行比较，以提示成功或 失败了。这些实验将描绘出srebf2下游有效发挥作用所必需的通路。 并提出了促进哺乳动物再生的前进道路。

PROJECT SUMMARY Loss of vision due to optic neuropathies, like glaucoma, is a common cause of blindness in the United States. Unfortunately, these conditions are usually permanent because the central nervous system lacks the ability to regenerate damaged axons. Mammalian models of optic nerve (ON) injury recapitulate the pathology seen in patients making it difficult to understand what is needed for successful regeneration. In contrast, teleost fish, such as the zebrafish, can successfully regenerate damage to the ON and recover lost vision. We are using this organism to study the mechanisms of successful ON regeneration with the hopes of translating these findings into novel therapeutics to improve regeneration in mammalian disease models and patients. In a transcriptome- wide study of retinal ganglion cells (RGCs) during zebrafish ON regeneration we identified the mevalonate and cholesterol pathways as up regulated during this process. Our preliminary data suggests the master transcriptional regulator of these pathways, srebf2, is necessary for successful ON regeneration. We hypothesize that srebf2 mediates ON regeneration by activating the RGC intrinsic mevalonate and cholesterol synthesis pathway and/or inducing expression of receptors for extracellular sources of cholesterol and lipids. Using the powerful genetic and chemical tools available for the zebrafish system, we propose to identify the critical period of srebf2 activity and the downstream mediator(s) of its function. Aim 1 will determine when srebf2 function in RGCs is critical for ON regeneration and using novel reporter lines of srebf2 activity to delineate when transcriptional activity occurs. We will also test if activation of srebf2 activity is sufficient to accelerate ON regeneration. Lastly, we will use laser capture microdissection RNA-seq (LCM-seq) to identify differential gene expression under gain- and loss-of-srebf2 function in RGCs. Aim 2 proposes to identify which RGC intrinsic or extrinsic pathways downstream of srebf2 mediate its function. We will use combinations of drugs and gene knockdown to determine if intrinsic mevalonate/cholesterol synthesis and external supplies are independent, interdependent, and/or compensatory for successful ON regeneration. Depending upon the results of this study we will further examine the downstream intrinsic synthesis pathways for cholesterol, ubiquinone, protein prenylation, and protein N-glycosylation or low-density lipoprotein receptors and their downstream processing. Aim 3 will test the sufficiency of Srebf2 expression to provide neuroprotection and stimulate axon regeneration in a mouse model of acute ON injury. LCM-seq will be used to identify gene expression changes induced in mouse RGCs by Srebf2 and compared to those identified in zebrafish to suggest mechanisms for success or failure. These experiments will delineate the pathways downstream of srebf2 necessary for efficient ON regeneration and suggest paths forward to enhance mammalian regeneration.