DK, D1K and D2K: CREB and drug addiction

DK、D1K 和 D2K：CREB ​​和毒瘾

• 批准号: 7149340
• 负责人: Cristina Backman
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON DRUG ABUSE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7149340
• 关键词: behavioral /social science research tag; behavioral genetics; cAMP response element binding protein; dopamine receptor; dopamine transporter; drug addiction; gene expression; gene mutation; genetically modified animals; laboratory mouse; neural plasticity; psychopharmacology; reinforcer; tetracyclines

CREB (cAMP response element binding protein) is a protein member of the bZIP subfamily of transcription factors. Activation of CREB occurs via phosphorylation of a single serine residue. Once phosphorylated, CREB dimmers, bind to specific CRE (cAMP response element) sites on target genes and regulates gene expression. CREB is of particular interest in drug addiction because its activation is downstream of the cAMP-signaling pathway, whose upregulation has been extensively characterized as an adaptation to chronic exposure to drugs of abuse. Upregulation of the cAMP pathway and activation of CREB seems to be crucial for the effects of drugs on the brain reward and motivational systems, such as the nucleus accumbens, ventral tegmental area, amygdala and frontal cortex, to name a few. In several of these regions, activation of CREB occurs in response to acute and chronic administration of different drugs of abuse, such as opiates, stimulants and alcohol. The regulation of CREB phosphorylation and the function of CREB in addiction vary with respect to multiple parameters, including the identity of the substance (opiates versus cocaine), the nature of the exposure (acute versus chronic), and the CNS region or neuronal pathways involved. A leading hypothesis is that drug-induced activation of CREB in these motivation centers of the brain underlies some of the common core features of drug addiction seen clinically, by inducing gene expression that leads to permanent molecular changes or drug induced plasticity. Kandel and collaborators were able to demonstrate that inactivation of CREB in hippocampal CA1 neurons impairs learning in the Morris water maze by interfering with some forms of long-term memory formation. To perturb CREB function, a transgenic mouse that expresses KCREB (a mutant of human CREB that is a potent dominant-negative inhibitor) through a forebrain specific promoter was generated. In accordance, over expression of CREB in CA1 neurons facilitated the establishment of long-lasting LTP in hippocampal slices. Interestingly, recent studies by Bonci and collaborators have shown that a single exposure to cocaine induces long-term potentiation in dopamine neurons, a mechanism that, as in memory formation, may contribute to synaptic plasticity and permanent neuronal changes, which may progressively lead to drug addiction. Whether CREB affects the formation of LTP in dopaminergic cells remains to be investigated. Also, Nestler and collaborators have shown that over expression of a dominant negative mutant CREB in the area of the nucleus accumbens (through the neuron specific-enolase promoter) increases the rewarding effects of cocaine, possibly by regulating dynorphin expression. As CREB is a molecular component of ubiquitous nature in the mesolimbic dopamine system, it would be advantageous if we could dissociate its effects by conditionally inactivating its function in separate sets of mesolimbic neurons, one set at the time. This approach would allow us to better understand the specific roles that CREB plays in addiction and other conditions affecting the mesolimbic system. In our laboratory, we are developing genetically modified mice containing a tetracycline inducible dominant inhibitor CREB gene (KCREB) under the control of specific endogenous neuronal promoters. By using a system for temporal, spatial and cell-type specific control of gene expression, KCREB will be induced only in neurons expressing either the dopamine transporter (DAT), the dopamine receptor 1 (Drd1), or 2 (Drd2) and only after treatment with doxycycline (a tetracycline analog). These different knock-in mice will help us determine the functions that CREB mediates during addiction in specific neuronal types of the mesolimbic dopamine system. These conditional animals are being developed with the use of a single DNA construct that has been tested in vitro. We will first determine if the genetic modifications introduced in this new animal strain produce the expected phenotype. Upon verification of a biologically functional mutation, these animals will be used for further in depth studies of CREB function during drug addiction. Exhaustive behavioral, morphological and functional studies of these animals will generate data that correlates CREB inactivation in D1-, D2- or DAT-positive neurons to addiction.

CREB（cAMP 反应元件结合蛋白）是转录因子 bZIP 亚家族的蛋白质成员。 CREB ​​的激活是通过单个丝氨酸残基的磷酸化来实现的。一旦磷酸化，CREB ​​二聚体就会与靶基因上的特定 CRE（cAMP 反应元件）位点结合并调节基因表达。 CREB ​​在药物成瘾方面特别令人感兴趣，因为它的激活位于 cAMP 信号通路的下游，而 cAMP 信号通路的上调已被广泛描述为对长期接触滥用药物的适应。 cAMP 通路的上调和 CREB ​​的激活似乎对于药物对大脑奖赏和激励系统（例如伏核、腹侧被盖区、杏仁核和额叶皮质等）的影响至关重要。在其中几个区域，CREB ​​的激活是由于急性和长期服用不同的滥用药物（例如阿片类药物、兴奋剂和酒精）而发生的。 CREB ​​磷酸化的调节和 CREB ​​在成瘾中的功能因多个参数而异，包括物质的特性（阿片类药物与可卡因）、暴露的性质（急性与慢性）以及所涉及的中枢神经系统区域或神经元通路。一个主要的假设是，药物诱导的大脑这些动机中心的 CREB ​​激活是临床上看到的药物成瘾的一些常见核心特征的基础，通过诱导基因表达，导致永久性分子变化或药物诱导的可塑性。 Kandel 和合作者能够证明，海马 CA1 神经元中 CREB ​​的失活会通过干扰某些形式的长期记忆形成来损害莫里斯水迷宫中的学习。为了扰乱 CREB ​​功能，我们制备了一种转基因小鼠，它通过前脑特异性启动子表达 KCREB（人类 CREB ​​的突变体，是一种有效的显性失活抑制剂）。相应地，CA1 神经元中 CREB ​​的过度表达促进了海马切片中持久 LTP 的建立。有趣的是，Bonci 和合作者最近的研究表明，单次接触可卡因会诱导多巴胺神经元的长期增强，这种机制与记忆形成一样，可能有助于突触可塑性和永久性神经元变化，从而可能逐渐导致药物成瘾。 CREB是否影响多巴胺能细胞LTP的形成仍有待研究。此外，Nestler 和合作者还表明，伏隔核区域显性失活突变体 CREB ​​的过度表达（通过神经元特异性烯醇化酶启动子）可能通过调节强啡肽表达来增加可卡因的奖赏效果。由于 CREB ​​是中脑边缘多巴胺系统中普遍存在的分子成分，如果我们能够通过有条件地使其在不同组中脑边缘神经元（一次一组）中的功能失活来分离其作用，那将是有利的。这种方法将使我们能够更好地了解 CREB ​​在成瘾和影响中脑边缘系统的其他疾病中发挥的具体作用。 在我们的实验室中，我们正在开发含有四环素诱导显性抑制剂 CREB ​​基因（KCREB）的转基因小鼠，该基因受特定内源性神经元启动子的控制。通过使用基因表达的时间、空间和细胞类型特异性控制系统，KCREB ​​仅在表达多巴胺转运蛋白 (DAT)、多巴胺受体 1 (Drd1) 或 2 (Drd2) 的神经元中被诱导，并且仅在用强力霉素（四环素类似物）治疗后才会被诱导。这些不同的敲入小鼠将帮助我们确定 CREB ​​在成瘾过程中中脑边缘多巴胺系统特定神经元类型中介导的功能。这些条件动物是使用经过体外测试的单一 DNA 构建体开发的。我们将首先确定这种新动物品系中引入的基因修饰是否产生预期的表型。在验证生物学功能突变后，这些动物将用于进一步深入研究药物成瘾期间的 CREB ​​功能。对这些动物进行详尽的行为、形态和功能研究将产生将 D1、D2 或 DAT 阳性神经元中 CREB ​​失活与成瘾相关的数据。