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亚家族中的一个蛋白质成员。CREB的激活是通过单个丝氨酸残基的磷酸化来实现的。一旦被磷酸化，CREB二聚体就会与靶基因上的特定Cre(cAMP反应元件)结合，并调节基因的表达。CREB对药物成瘾特别感兴趣，因为它的激活位于cAMP信号通路的下游，cAMP信号通路的上调被广泛地描述为对长期药物滥用的适应。CAMP通路的上调和CREB的激活似乎对药物对大脑奖赏和动机系统的影响至关重要，如伏隔核、腹侧被盖区、杏仁核和额叶皮质等。在这些区域中的几个区域，CREB的激活是对不同滥用药物的急性和长期给药的反应，如阿片剂、兴奋剂和酒精。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(阿片类和可卡因)，暴露的性质(急性和慢性)，以及涉及的中枢神经系统区域或神经通路。一个主要的假说是，药物诱导的CREB在大脑的这些动机中心的激活是临床上看到的药物成瘾的一些共同的核心特征，通过诱导导致永久性分子变化或药物诱导的可塑性的基因表达。Kandel和他的合作者能够证明，海马CA1区神经元CREB的失活通过干扰某些形式的长期记忆形成，损害了在Morris水迷宫中的学习。为了干扰CREB的功能，产生了一种转基因小鼠，它通过前脑特异的启动子表达KCREB(人CREB的突变体，是一种有效的显性负抑制因子)。相应地，在CA1区神经元中CREB的过表达促进了海马片LTP的建立。有趣的是，Bonci和他的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，这一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，这可能会逐渐导致药物成瘾。CREB是否影响多巴胺能细胞LTP的形成还有待研究。此外，Nestler和他的合作者已经证明，在伏隔核区域过度表达显性负突变CREB(通过神经元特异性烯醇化酶启动子)增加了可卡因的奖赏效应，可能是通过调节强啡肽的表达。由于CREB是中脑边缘多巴胺系统中普遍存在的分子成分，如果我们能通过有条件地在不同的一组中脑边缘神经元中失活它的功能来分离它的作用，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条件中所起的具体作用。 在我们的实验室中，我们正在开发含有四环素可诱导显性抑制物CREB基因(KCREB)的转基因小鼠，该基因受特定内源性神经启动子的控制。通过使用时间、空间和细胞类型特异的基因表达控制系统，KCREB仅在表达多巴胺转运体(DAT)、多巴胺受体1(DRD1)或2(DRD2)的神经元上被诱导，并且只有在用多西环素(四环素类似物)处理后才能被诱导。这些不同的敲入小鼠将帮助我们确定CREB在成瘾期间在中脑边缘多巴胺系统的特定神经元类型中所介导的功能。这些有条件的动物是通过使用已在体外测试的单一DNA结构来开发的。我们将首先确定在这种新的动物品系中引入的遗传修饰是否产生预期的表型。在验证了生物功能突变后，这些动物将被用于进一步深入研究CREB在药物成瘾过程中的功能。对这些动物的详尽的行为、形态和功能研究将产生数据，将D1、D2或DAT阳性神经元中CREB的失活与成瘾联系起来。