DK, D1K and D2K: CREB and drug addiction

CREB（cAMP应答元件结合蛋白）是转录因子bZIP亚家族的蛋白质成员。CREB的激活通过单个丝氨酸残基的磷酸化发生。一旦磷酸化，CREB二聚体结合靶基因上的特异性CRE（cAMP反应元件）位点并调节基因表达。CREB在药物成瘾中特别令人感兴趣，因为它的激活是cAMP信号通路的下游，cAMP信号通路的上调已被广泛表征为对慢性暴露于滥用药物的适应。cAMP途径的上调和CREB的激活似乎对于药物对大脑奖励和动机系统的影响至关重要，例如，延髓核、腹侧被盖区、杏仁核和额叶皮质等。在这些区域中的几个中，CREB的激活响应于不同滥用药物（例如阿片类药物、兴奋剂和酒精）的急性和慢性施用而发生。CREB磷酸化的调节和CREB在成瘾中的功能随着多个参数而变化，包括物质的身份（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CNS区域或神经元通路。一个主要的假设是，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的基础，通过诱导基因表达，导致永久的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区神经元中CREB的失活通过干扰某些形式的长期记忆形成而损害Morris水迷宫中的学习。为了扰乱CREB功能，产生了通过前脑特异性启动子表达KCREB（人CREB的突变体，是有效的显性负抑制剂）的转基因小鼠。CA1区神经元CREB的过度表达促进了海马脑片长时程增强的建立。有趣的是，Bonci及其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时程增强，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的形成仍有待研究。此外，Nestler和合作者已经表明，在延髓核区域中显性负突变CREB的过度表达（通过神经元特异性烯醇化酶启动子）增加了可卡因的奖励作用，可能是通过调节强啡肽表达。由于CREB是中脑边缘多巴胺系统中普遍存在的分子成分，如果我们能够通过在单独的中脑边缘神经元组中有条件地失活其功能来分离其作用，这将是有利的，一次一组。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发挥的具体作用。 在我们的实验室中，我们正在开发的转基因小鼠含有四环素诱导的显性抑制剂CREB基因（KCREB）的控制下，特定的内源性神经元启动子。通过使用时间，空间和细胞类型特异性控制基因表达的系统，KCREB将仅在表达多巴胺转运蛋白（DAT），多巴胺受体1（Drd 1）或2（Drd 2）的神经元中诱导，并且仅在用强力霉素（四环素类似物）处理后诱导。这些不同的基因敲入小鼠将帮助我们确定CREB在成瘾过程中介导的中脑边缘多巴胺系统特定神经元类型的功能。这些条件动物正在使用已经在体外测试的单一DNA构建体进行开发。我们将首先确定在这种新动物品系中引入的遗传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与D1、D2或DAT阳性神经元中CREB失活与成瘾相关的数据。

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.