Identification of gene targets for morphine & other drugs of abuse

吗啡靶基因的鉴定

• 批准号: 7241523
• 负责人: RICHARD H. GOODMAN
• 金额: $ 29.2万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-28 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7241523
• 关键词: Actins; Acute; Address; Adenylate Cyclase; Affect; Aftercare; Amphetamines; Antisense RNA; Attention; Behavior; Behavioral; Binding; Binding Sites; Bioinformatics; Biological Assay; CREB1 gene; Cells; Chromatin; Chronic; Class; Cocaine; Code; Complement; Custom; Cyclic AMP; Cytoskeleton; DNA; Dependence; Drug Addiction; Drug effect disorder; Elements; Enhancers; Exposure to; Forskolin; Gene Expression; Gene Expression Profile; Gene Targeting; Genes; Genomics; Goals; Growth; Human; Laboratories; Link; Longevity; Measures; Mediating; Mediator of activation protein; Methodology; Methods; MicroRNAs; Modeling; Modification; Morphine; Morphology; N-Methylaspartate; Nature; Nerve Growth Factor Receptors; Neurites; Neuroblastoma; Neurons; Opiates; PC12 Cells; Pathway interactions; Personal Satisfaction; Phenocopy; Phosphorylation; Population; Process; Proteins; RNA; Regulation; Research Personnel; Ribonucleotides; Second Messenger Systems; Sequence Analysis; Signal Transduction; Standards of Weights and Measures; Testing; Transcript; Translations; addiction; chromatin immunoprecipitation; concept; design; drug of abuse; in vivo; inhibitor/antagonist; novel strategies; programs; promoter; receptor coupling; research study; response; rho; second messenger; serial analysis of gene expression; transcription factor

DESCRIPTION (provided by applicant): Long term changes in gene expression are believed to contribute importantly to the mechanisms underlying drug addiction. Considerable attention has been devoted to the cAMP second messenger pathway because it is upregulated by opiates and other drugs of abuse. Both tolerance and dependence have been attributed to changes in function of the transcription factor CREB, which mediates cAMP-dependent gene expression. It is currently believed that CREB binds constitutively to a promoter element termed the CRE. This proposal challenges this model and tests a new hypothesis-that chronic exposure to morphine induces CREB binding to some genes but not others. To address this hypothesis the lab has developed a novel approach termed SACO for examining CREB binding to target genes in vivo. This method combines chromatin immunoprecipitation with a modification of Long SAGE (an approach designed for analysis of mixtures of RNA). SACO will be used to identify the entire complement of CREB targets and measure how the selection of these targets is affected by agents, such as morphine, that upregulate the cAMP pathway. Additional studies will address the mechanisms underlying the morphological changes in dendritic processes induced by CREB that occur after treatment with other drugs of abuse, namely cocaine and amphetamine. Specific goals of this project are to identify the entire set of CREB targets in human neuroblastoma cells and determine whether this set is altered by acute exposure to cAMP or chronic exposure to morphine. Previous studies have indicated that CREB regulates the expression of both protein-coding and noncoding transcripts. Many protein-coding transcripts and most noncoding transcripts are missing from conventional microarrays, however. Studies in this proposal will examine both classes of RNAs by developing a custom microarray representing the CREB transcriptome. One specific microRNA, designated miR132, was found to be induced by CREB in PC12 cells and to stimulate changes in dendritic morphology in neurons that are highly reminiscent of those caused by CREB activators. A candidate target for this miRNA has been identified and experiments are designed to elucidate the mechanism of miR132 action. The concept that CREB signaling induces mediators of translational arrest has profound implications for the understanding of drug action.

描述（由申请人提供）：据信基因表达的长期变化对药物成瘾的基本机制有重要作用。由于鸦片和其他虐待药物的上调，人们对第二通信途径进行了大量关注。耐受性和依赖性都归因于转录因子CREB功能的变化，后者介导了cAMP依赖性基因表达。目前认为CREB与称为CRE的启动子元素结合。该提议挑战了该模型并检验了一种新的假设 - 长期暴露于吗啡会诱导CREB与某些基因的结合，而不是其他基因的结合。为了解决这一假设，实验室开发了一种新的方法，该方法称为Saco，用于检查体内与靶基因的CREB结合。该方法将染色质免疫沉淀与长鼠的修饰（一种用于分析RNA混合物的方法）结合在一起。 SACO将用于识别CREB靶标的整个补体，并测量这些靶标的选择如何受到上调cAMP途径的代理（例如吗啡）的影响。其他研究将探讨CREB诱导的形态学变化的基础机制，Creb诱导的其他滥用药物（即可卡因和苯丙胺）治疗后发生的机制。 该项目的具体目标是确定人类神经母细胞瘤细胞中的整个CREB靶标，并确定该组是否会因急性暴露于cAMP还是长期暴露于吗啡来改变。先前的研究表明，CREB调节蛋白质编码和非编码转录本的表达。但是，常规微阵列中缺少许多蛋白质编码的转录本和大多数非编码转录本。该提案中的研究将通过开发代表CREB转录组的自定义微阵列来检查两类RNA。发现一种特定的microRNA，指定为miR132，是由PC12细胞中的CREB诱导的，并刺激神经元中的树突形态变化，这些神经元高度让人联想到CREB激活剂引起的神经元。已经确定了该miRNA的候选目标，并设计了实验以阐明miR132作用的机理。 Creb信号传导引起转化停滞的介体的概念对理解药物作用具有深远的影响。