Role of System xc- in Addiction: Developing & Phenotyping a Slc7a11 knockout rat

描述（由申请人提供）：皮质纹状体通路内的异常谷氨酸信号传导与人类的渴望和大鼠的可卡因寻求有关。不幸的是，我们对谷氨酸的有限了解导致许多CNS疾病（包括药物成瘾）缺乏有效，耐受性良好的治疗方法。虽然谷氨酸被描述为大脑中的主要兴奋性神经递质，但尚不清楚这种复杂的转运蛋白和释放机制网络的许多组分如何以整合的方式调节兴奋性信号传导。由于缺乏选择性靶向这些新机制的可用工具，因此难以令人信服地证明这些新机制的重要性。一个这样的组成部分是系统xc-，一个来源的非囊泡谷氨酸释放，主要表达在星形胶质细胞。它通过将细胞外半胱氨酸交换为细胞内谷氨酸来发挥作用。系统xc通过以下方式影响突触活动和可塑性 谷氨酸和多巴胺在多个大脑区域的释放。重复使用可卡因会使xc系统活性持续降低，这似乎是谷氨酸诱导的强迫性药物寻求所必需的。相反，防止或逆转可卡因诱导的系统xc活性变化的操作使谷氨酸水平正常化，并使可卡因诱导的恢复迟钝。在人类中，N-乙酰半胱氨酸已显示出治疗药物成瘾和相关强迫症的前景。诸如此类的研究表明，系统xc-功能可能在揭示成瘾的细胞基础以及星形胶质细胞在中枢神经系统活动中的作用方面具有深远的意义-特别是如果确定系统xc-是N-乙酰半胱氨酸的主要作用机制。 在大鼠中操纵系统xc-的努力通常涉及使用与可预测的药理学问题相关的药理学工具。通过将胱氨酸直接输注到脑中或全身给予半胱氨酸前药（例如，N-乙酰半胱氨酸）都是有效的，因为半胱氨酸-谷氨酸交换的速率是其底物的相对细胞外/细胞内浓度梯度的函数。基因突变会导致 系统xc的活性亚基xCT存在于多种小鼠品系中。然而，基本上每一项将xc系统与谷氨酸稳态或成瘾联系起来的研究都是在大鼠或灵长类动物中进行的。本提案的目标是使用新的锌指核酸酶（ZFN）方法突变大鼠中编码xCT的Slc 7a 11基因。在创建xCT缺陷大鼠模型（目的1）后，我们将验证和表征一般表型（目的2）以及成瘾特异性表型（目的3）。这些技术的发展和应用，以产生转基因大鼠品系可能会导致一个重大的范式转变，在研究成瘾的神经基础，使更复杂和高度具体的操作，在一个物种，更好地模拟人类成瘾的关键方面。

DESCRIPTION (provided by applicant): Abnormal glutamate signaling within corticostriatal pathways has been linked to craving in humans and cocaine seeking in rats. Unfortunately, our limited understanding of glutamate has contributed to the lack of effective, well-tolerated treatments for many CNS diseases, including drug addiction. While glutamate is described as the primary excitatory neurotransmitter in the brain, it is unclear how the many components of this complex network of transporters and release mechanisms function in an integrated manner to regulate excitatory signaling. Due to a lack of available tools that selectively target these novel mechanisms, it has been difficult to convincingly demonstrate the importance of these novel mechanisms. One such component is system xc-, a source of nonvesicular glutamate release that is primarily expressed on astrocytes. It functions by exchanging extracellular cysteine for intracellular glutamate. System xc influences synaptic activity and plasticity through the release of glutamate and dopamine in multiple brain regions. Repeated cocaine produces a persistent reduction in system xc- activity, which appears to be necessary for glutamate-induced compulsive drug seeking. In contrast, manipulations that prevent or reverse cocaine-induced changes in system xc- activity normalize glutamate levels and blunt cocaine-induced reinstatement. In humans, N-acetylcysteine has shown promise in the treatment of drug addiction and related compulsive disorders. Studies such as these indicate that system xc- function may have profound implications in revealing the cellular basis of addiction, as well as the role of astrocytes in central nervous system activity - especially if it is determined that system xc- is the primary mechanism of action for N-acetylcysteine. Efforts to manipulate system xc- in rats typically involve the use of pharmacological tools that are associated with predictable pharmacological concerns. Increasing system xc activity by direct infusion of cystine into the brain or systemic administration of a cysteine prodrug (e.g., N acetylcysteine) are both effective since the rate of cysteine-glutamate exchange is a function of the relative extracellular/intracellular concentration gradients of its substrates. Mutations in the gene giving rise to xCT, the active subunit for system xc, is present in multiple mouse strains. However, essentially every study linking system xc to glutamate homeostasis or addiction has been conducted in rats or primates. The goal of this proposal is to use the novel Zinc Finger Nucleases (ZFN) approach to mutate the Slc7a11 gene encoding xCT in rat. After creating an xCT deficient rat model (aim 1), we will verify and characterize the general phenotype (aim 2) as well as addiction-specific phenotypes (aim 3). The development and application of these technologies to generate transgenic rat strains may result in a major paradigm shift in studying the neural basis of addiction by enabling more sophisticated and highly specific manipulations in a species that better models critical aspects of human addiction.