In Vivo Modeling of Heritable Dopamine Transporter Dysfunction Associated with Neuropsychiatric Disorders

项目摘要 神经递质多巴胺(DA)在运动、奖励、注意力和 执行职能。几种严重的神经精神障碍与 DA信号功能障碍包括精神分裂症、强迫症、躁郁症 精神障碍、自闭症和注意力缺陷多动障碍(ADHD)。注意力缺陷 多动症(ADHD)是一种普遍存在的精神障碍，仅根据 行为特征。尽管ADHD的生物学基础仍不明确， 来自基础、药理学和遗传学研究的证据已经引起了人们对潜在 DA转运体(DAT)表达/功能改变的贡献。因此，DA信令 调节ADHD患者行为改变的关键因素，靶向DAT的精神刺激剂， 包括哌酸甲酯(MPH、利他林)和各种苯丙胺(AMPH)制剂(例如 Adderall)，迅速缓解许多受试者的ADHD症状，人类遗传学研究显示 与DAT基因(SLC6A4)多态相关的ADHD风险增加。我们的实验室鉴定出 ADHD先证者中存在多种罕见的DAT编码变体，其中一种是DAT Val559 目前的提案。有趣的是，DAT Val559不仅在ADHD患者中发现， 但在双相情感障碍和自闭症中也是如此，这表明对DA依赖特征的贡献可以 在不同的疾病中表现为风险，或导致其中的ADHD共病。通过体外试验 研究表明，Val559变异体会产生异常的DA外流(ADE)，这是一种 非囊泡性DA释放，通常见于DAT表达细胞的AMPH处理。在 在DAT Val559的情况下，Amph不能触发DA外流，尽管Amph和MPh都能阻断ADE。 这些发现导致了一种假设，即强直性DA泄漏可能是一个意想不到的因素 有患上一种或多种多巴胺相关行为障碍的风险。为了在体内测试这一概念，我们 开发出表达天然SLC6a3基因变异的DAT Val559敲入小鼠， 提供第一个与神经精神疾病相关的DAT功能障碍的有效模型 精神错乱。在这里，我建议检验以下假设：1)DAT Val559在体内表达 产生改变的神经递质释放以及突触前和突触后信号的变化 对ADHD相关药物操作敏感的事件和2)终生 DAT Val559表达导致DA相关行为的改变，这些行为与一个或多个 ADHD的更多特征。总之，我的努力是为了提供一个丰富的培训机会 在研究神经精神障碍风险的机制时 ADHD多巴胺功能障碍的第一个有效模型的推进分析。

Project Summary The neurotransmitter dopamine (DA) plays a critical role in movement, reward, attention, and executive function. Several critically important neuropsychiatric disorders are associated with dysfunction in DA signaling including schizophrenia, obsessive compulsive disorder, bipolar disorder, autism, and attention deficit hyperactivity disorder (ADHD). Attention-Deficit Hyperactivity Disorder (ADHD) is a prevalent psychiatric disorder diagnosed solely on the basis of behavioral features. Although the biological underpinnings of ADHD remain ill-defined, evidence from basic, pharmacological and genetic studies have drawn attention to potential contributions of altered expression/function of the DA transporter (DAT). Thus, DA signaling modulates key elements of the behaviors altered in ADHD, psychostimulants that target DAT, including methylphenidate (MPH, Ritalin) and various amphetamine (AMPH) formulations (e.g. Adderall), rapidly relieve ADHD symptoms in many subjects, and human genetic studies reveal increased ADHD risk associated with DAT gene (SLC6A4) polymorphisms. Our lab identified multiple rare DAT coding variants in ADHD probands, one of which, DAT Val559, is the subject of the current proposal. Interestingly, DAT Val559 has not only been identified in ADHD subjects, but also in bipolar disorder and autism, suggesting contributions to DA-dependent traits that can manifest as risk in distinct disorders or contribute to ADHD comorbidities therein. Through in vitro studies, we demonstrated that the Val559 variant produces anomalous DA efflux (ADE), a form of non-vesicular DA release, typically seen with AMPH treatment of DAT-expressing cells. In the case of DAT Val559, AMPH fails to trigger DA efflux, though both AMPH and MPH block ADE. These findings lead to the hypothesis that tonic DA leak may be an unsuspected contributor to risk for one or more DA-linked behavioral disorders. To test this concept in vivo, we developed DAT Val559 knock-in mice that expresses the variant from the native Slc6a3 locus, affording the first construct valid model of DAT dysfunction associated with neuropsychiatric disorders. Here I propose to test the hypotheses that 1) in vivo expression of DAT Val559 generates altered neurotransmitter release as well as changes in pre and post-synaptic signaling events that are sensitive to ADHD-relevant pharmacological manipulations and 2) that lifelong DAT Val559 expression leads to alterations in DA-associated behaviors that align with one or more attributes of ADHD. Altogether, my efforts are organized to afford a rich training opportunity in the investigation of mechanisms that underlie risk for neuropsychiatric disorders while advancing analysis of the first construct-valid model of DA dysfunction in ADHD.