Novel Role for Sirtuin Signaling Mechanisms and Downstream Targets in Depression

项目摘要 表观遗传因素可能在揭示遗传性疾病的分子机制中起关键作用。 发病机制的几个神经精神疾病，包括抑郁症和药物成瘾。基因似乎 仅解释了发展这些疾病的部分风险因素。NIH独立之路奖 (K99/R 00）将大大促进候选人的能力，开始他的职业生涯作为一个独立的科学家， 这使他能够研究表观遗传机制，这些机制是在基因组中观察到的基因-环境相互作用的基础。 严重的情绪障碍在系主任Eric Nestler博士的主要指导下， 他是神经科学教授，也是纽约西奈山医学院弗里德曼脑研究所的主任， 独立之路奖将为候选人提供扩展和发展其 下一代测序技术和细胞类型特异性信号分析方面的培训和专业知识。的 奖励将推进候选人的长期职业目标：（1）确定环境如何 与基因相互作用，并确定经验赋予基因持久变化的机制 表达，（2）发现新的易感性（或弹性）基因，以了解它们如何影响 行为尽管抑郁症的流行及其相当大的影响， 病理生理学是基本的。因此，迫切需要发现新的信号通路 有助于抑郁症的发展，以便更好的诊断测试，治疗和预防 可以采取措施。该研究计划的目标是评估SIRT 1的作用及其对 下游靶点作为潜在的新候选治疗神经精神障碍， 染色质免疫沉淀，然后在核中进行全基因组分析（ChIP-seq） 来自对照组和社交失败的应激小鼠的组织。我们实验室的初步数据显示， 失败压力，一种行为学上证实的抑郁症和其他压力相关疾病的模型， SIRT 1水平在NAc和是一个pro-tumor足以增加应激敏感性。SIRT 1蛋白是 NAD+依赖性脱乙酰酶和ADP-核糖基转移酶家族的创始成员，称为 sirtuins。在这项研究中，我们提出了一个假设，即SIRT 1的调制构成了一个新的候选人， 抗抑郁药的治疗靶点在本补助金的辅导K阶段（具体目标1和具体目标 2)我们将直接确定sirtuins在调节抑郁和焦虑样行为中的作用， 药理学和遗传学工具。首先，我们将通过直接内NAc抑制或增加sirtuins的活性， 输注药理学抑制剂（sirtinol）或激活剂（白藜芦醇）以评估sirtuins对 压力诱发的抑郁和焦虑样行为的发展。接下来，我们将专门针对SIRT 1 使用遗传方法在floxed SIRT 1（SIRT 1flx）小鼠的NAc中过表达HSV-Cre病毒载体， 敲低SIRT 1水平，或通过使用HSV-SIRT 1载体在细胞中过表达SIRT 1来增加SIRT 1水平。 的NAC。在具体目标2中，我们还将确定SIRT 1在NAc中的转录靶点， 通过对SIRT 1进行ChIP-Seq， 基因激活和抑制的标志物。在独立阶段（R 00），具体目标3将表征 使用drd 1-GFP在NAc中以细胞类型特异性方式研究社交失败对沉默调节蛋白信号传导的影响， drd 2-GFP转基因小鼠，允许鉴定纹状体黑质和纹状体苍白球中棘 神经元（MSN）。总之，在这个独立之路奖提出的研究将准备 候选人开发一个完全独立的研究计划，能够整合广泛的分子 和行为的方法，在技术上先进和高影响力的方式，包括：（一）线的遗传 （ii）在暴露于慢性应激后靶向沉默调节蛋白信号传导的细胞类型特异性调节的工程化小鼠， 从脑组织中分离染色质的能力，（iii）ChIP，然后进行全基因组分析，（iv）啮齿动物 抑郁症模型具有高有效性，和（v）自动行为测定测量许多抑郁症-和 类似焦虑的行为反应 .

Project Summary Epigenetic factors may play a key role in unraveling the molecular mechanisms underlying the pathogenesis of several neuropsychiatric disorders, including depression and drug addiction. Genes appear to explain only part of the risk factor for developing these disorders. The NIH Pathway to Independence Award (K99/R00) will significantly facilitate the candidate's ability to begin his career as an independent scientist, allowing him to study the epigenetic mechanisms underlying the gene-environment interactions observed in the onset of major mood disorders. Under the primary mentorship of Dr. Eric Nestler, Chairman of the Department of Neuroscience, and Director of the Friedman Brain Institute, at Mount Sinai School of Medicine in New York, the Pathway to Independence Awards would provide the candidate the opportunity to extend and develop his training and expertise in next generation sequencing technologies and cell type specific signaling analysis. The Award would advance the candidate's long term career objectives to: (1) determine how the environment interacts with genes and identify mechanisms by which experience confers enduring changes in gene expression, and (2) discover novel susceptibility (or resilience) genes to understand how they influence behavior. Despite the prevalence of depression and its considerable impact, knowledge about its pathophysiology is rudimentary. Thus, there is an urgent need to discover novel signaling pathways contributing to the development of depression so that better diagnostic tests, treatments, and preventive measures can be attained. The objectives of this program of research are to evaluate the role of SIRT1 and its downstream targets as potential new candidates for the treatment of neuropsychiatric disorders by performing chromatin immunoprecipitation followed by genome-wide profiling (ChIP-seq) in nucleus accumbens (NAc) tissue from control and socially defeated stressed mice. Preliminary data from our lab show that chronic social defeat stress, an ethologically validated model of depression and other stress-related disorders, modulates SIRT1 levels in the NAc and is a pro-depressant sufficient to increase stress sensitivity. The SIRT1 protein is the founding member of a family of NAD+-dependent deacetylases and ADP-ribosyltransferases, termed sirtuins. In this grant we propose to test the hypothesis that modulation of SIRT1 constitutes a novel candidate therapeutic target for antidepressants. In the mentored K-phase of this grant (Specific Aim 1 and Specific Aim 2) we will directly determine the role of sirtuins in regulating depressive- and anxiety-like behaviors using pharmacological and genetic tools. First, we will inhibit or increase the activity of sirtuins by direct intra-NAc infusion of a pharmacological inhibitior (sirtinol) or activator (resveratrol) to assess the effects of sirtuins on the development of stress-induced depressive and anxiety-like behaviors. Next, we will specifically target SIRT1 using a genetic approach to overexpress HSV-Cre viral vectors in the NAc of floxed SIRT1 (SIRT1flx) mice to knock-down SIRT1 levels, or to increase SIRT1 levels by overexpressing SIRT1 using HSV-SIRT1 vectors in the NAc. In Specific Aim 2 we also will identify transcriptional targets of SIRT1 in the NAc regulated by social defeat and antidepressants in susceptible and resilient mice by performing ChIP-Seq for SIRT1 in addition to markers of gene activation and repression. In the independent phase (R00), Specific Aim 3, will characterize the effects of social defeat on sirtuin signaling in a cell type specific manner in the NAc using drd1-GFP and drd2-GFP transgenic mice, allowing for the identification of striatonigral and striatopallidal medium spiny neurons (MSNs). In summary, the research proposed in this Pathway to Independence Award will prepare the candidate to develop a fully independent research program capable of integrating a wide range of molecular and behavioral approaches in a technically advanced and high impact manner, including: (i) lines of genetically engineered mice to target cell type specific regulation of sirtuin signaling after exposure to chronic stress, (ii) the ability to isolate chromatin from brain tissue, (iii) ChIP followed by genome-wide profiling, (iv) a rodent model of depression with high validity, and (v) automated behavioral assays measuring many depression- and anxiety-like behavioral responses. .