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

项目摘要 在美国，由于视神经病变（如青光眼）导致的视力丧失是失明的常见原因。 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能力， 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。相反，硬骨鱼， 例如斑马鱼，可以成功地再生ON的损伤并恢复失去的视力。我们正在使用这 生物体研究成功的ON再生的机制，希望转化这些发现 用于改善哺乳动物疾病模型和患者再生的新疗法。在转录组中- 在斑马鱼ON再生期间对视网膜神经节细胞（RGC）的广泛研究中，我们鉴定了甲羟戊酸和 胆固醇途径在此过程中上调。我们的初步数据显示 这些途径的转录调节因子srebf 2是成功的ON再生所必需的。我们假设 srebf 2通过激活RGC内在甲羟戊酸和胆固醇合成介导ON再生 途径和/或诱导胆固醇和脂质的细胞外来源的受体的表达。使用 强大的遗传和化学工具可用于斑马鱼系统，我们建议确定关键时期 SREBF 2活性及其功能的下游介质。Aim 1将决定srebf 2何时在 RGC对于ON再生至关重要，并且使用srebf 2活性的新报告细胞系来描绘何时 转录活性发生。我们还将测试srebf 2活性的激活是否足以加速ON 再生最后，我们将使用激光捕获显微切割RNA-seq（LCM-seq）技术来鉴定差异基因 RGC中Srebf 2功能获得和丧失下的表达。目的2建议找出哪些研资局是内在或 SREBF 2下游的外源途径介导其功能。我们将使用药物和基因的组合 敲低以确定内在甲羟戊酸/胆固醇合成和外部供应是否是独立的， 相互依赖和/或补偿成功的ON再生。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 异戊二烯化和蛋白质N-糖基化或低密度脂蛋白受体及其下游加工。 目的3将测试Srebf 2表达是否足以提供神经保护和刺激轴突再生 在急性ON损伤的小鼠模型中。LCM-seq将用于鉴定诱导的基因表达变化， 通过Srebf 2检测小鼠RGCs，并与斑马鱼中鉴定的RGCs进行比较，以提示成功或 失败这些实验将描绘有效ON所必需的srebf 2下游途径。 再生，并提出了促进哺乳动物再生的途径。

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.