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

描述(申请人提供)：皮质纹状体通路中的谷氨酸信号异常与人类的渴求和大鼠的可卡因寻找有关。不幸的是，我们对谷氨酸的了解有限，导致许多中枢神经系统疾病缺乏有效的、耐受性良好的治疗方法，包括药物成瘾。虽然谷氨酸被描述为大脑中主要的兴奋性神经递质，但目前尚不清楚这个复杂的转运体和释放机制网络的许多组成部分如何以一种集成的方式调节兴奋信号。由于缺乏有选择性地针对这些新机制的可用工具，很难令人信服地证明这些新机制的重要性。其中一个这样的成分是系统XC-，它是一种主要在星形胶质细胞上表达的非囊泡性谷氨酸释放来源。它的功能是将细胞外的半胱氨酸交换为细胞内的谷氨酸。XC系统通过以下途径影响突触活性和可塑性 谷氨酸和多巴胺在大脑多个区域的释放。反复吸食可卡因会导致系统XC活性持续下降，这似乎是谷氨酸诱导的强迫性药物寻求所必需的。相反，阻止或逆转可卡因诱导的系统XC活性变化的操作可以使谷氨酸水平正常化，并钝化可卡因诱导的恢复。在人类身上，N-乙酰半胱氨酸在治疗药物成瘾和相关的强迫症方面表现出了希望。这类研究表明，系统XC的功能可能对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的话。在大鼠体内操纵XC-系统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。通过将半胱氨酸直接注入大脑或全身注射半胱氨酸前体药物(如N-乙酰半胱氨酸)来提高XC系统的活性都是有效的，因为半胱氨酸-谷氨酸交换的速度是其底物的相对细胞外/细胞内浓度梯度的函数。基因突变给人 Rise to XCT是XC系统的活性亚基，存在于多种品系的小鼠体内。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老鼠或灵长类动物身上进行的。这项建议的目的是使用新的锌指核酸酶(ZFN)方法在大鼠中突变编码XCT的SLC7a11基因。在建立XCT缺陷大鼠模型(目标1)后，我们将验证和表征一般表型(目标2)以及成瘾特异性表型(目标3)。这些产生转基因大鼠品系的技术的开发和应用，可能会通过在一个物种中实现更复杂和高度特定的操作，更好地模拟人类成瘾的关键方面，从而导致研究成瘾的神经基础的重大范式转变。

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.