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

甲羟戊酸/胆固醇途径成功调节视神经再生

• 批准号: 10500994
• 负责人: Matthew B Veldman
• 金额: $ 40.38万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10500994
• 关键词: Acute; Affect; American; Animal Model; Animals; Axon; Biological; Blindness; Brain; Cell Survival; Cells; Chemicals; Cholesterol; Coenzyme Q10; Data; Data Set; Disease model; Drug Combinations; Exhibits; Failure; Financial Hardship; Future; Gene Expression; Gene Transfer; Genes; Genetic; Genetic Transcription; Glaucoma; Goals; Growth Cones; Human; Injury; Knowledge; Lipids; Low Density Lipoprotein Receptor; Mammals; Measures; Mediating; Mediator of activation protein; Medical; Metabolic Pathway; Modeling; Mus; Natural regeneration; Neuraxis; Optic Nerve; Optic Nerve Injuries; Organism; Pathology; Pathway interactions; Patients; Pharmacology; Pre-Clinical Model; Process; Protein Import; Protein Isoprenylation; Proteins; Recovery; Recycling; Regenerative Medicine; Regulation; Reporter; Retinal Ganglion Cells; Role; SET gene; Signal Pathway; Source; Synapses; System; Testing; Time; Tissue-Specific Gene Expression; Transgenic Organisms; Translating; Ubiquinone; United States; Up-Regulation; Vision; Visual; Visual system structure; Zebrafish; antagonist; axon injury; axon regeneration; cell injury; chemical genetics; cholesterol trafficking; cost; critical period; differential expression; experimental study; extracellular; gain of function; glycosylation; improved; in vivo; in vivo Model; innovation; knock-down; laser capture microdissection; loss of function; mevalonate; mouse model; negative affect; nerve injury; neuroprotection; novel; novel therapeutics; optic nerve disorder; optic nerve regeneration; overexpression; pre-clinical; prophylactic; receptor expression; relating to nervous system; response; sight restoration; success; teleost fish; tool; transcriptome; transcriptome sequencing; transcriptomics

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.

项目概要 在美国，青光眼等视神经病变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需要什么。相比之下，硬骨鱼， 例如斑马鱼，可以成功地再生视神经损伤并恢复失去的视力。我们正在使用这个 有机体研究 ON 成功再生的机制，希望转化这些发现 开发新疗法以改善哺乳动物疾病模型和患者的再生。在转录组中—— 通过对斑马鱼 ON 再生过程中视网膜神经节细胞 (RGC) 的广泛研究，我们发现了甲羟戊酸和 在此过程中胆固醇途径上调。我们的初步数据表明大师 这些途径的转录调节因子 srebf2 是 ON 成功再生所必需的。我们假设 srebf2 通过激活 RGC 内在甲羟戊酸和胆固醇合成来介导 ON 再生 途径和/或诱导细胞外胆固醇和脂质来源的受体表达。使用 由于斑马鱼系统拥有强大的遗传和化学工具，我们建议确定关键期 srebf2 活性及其功能的下游介体。目标 1 将确定 srebf2 何时起作用 RGC 对于 ON 再生至关重要，并使用 srebf2 活性的新型报告系来描述何时 发生转录活性。我们还将测试 srebf2 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）来识别差异基因 RGC 中 srebf2 功能的获得和丧失下的表达。目标 2 建议确定 RGC 内在或 srebf2 下游的外在途径介导其功能。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独立， 成功的 ON 再生相互依赖和/或补偿。取决于本研究的结果 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、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 表达是否足以提供神经保护并刺激轴突再生 在小鼠急性视神经损伤模型中。 LCM-seq 将用于鉴定诱导的基因表达变化 通过 Srebf2 检测小鼠 RGC 并与斑马鱼中鉴定的 RGC 进行比较，以提出成功的机制或 失败。这些实验将描绘有效 ON 所需的 srebf2 下游途径 再生并提出增强哺乳动物再生的前进道路。