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

 描述（由申请人提供）：神经递质多巴胺（DA）在运动、奖励、注意力和执行功能中起着关键作用。几种非常重要的神经精神障碍与DA信号传导功能障碍相关，包括精神分裂症、强迫症、双相情感障碍、自闭症和注意缺陷多动障碍（ADHD）。注意力缺陷多动障碍（ADHD）是一种流行的精神疾病，仅根据行为特征进行诊断。虽然ADHD的生物学基础仍不明确，但基础、药理学和遗传学研究的证据已引起人们对DA转运蛋白（DAT）表达/功能改变的潜在贡献的关注。因此，DA信号调节ADHD中改变的行为的关键要素，靶向DAT的精神兴奋剂，包括哌醋甲酯（MPH，利他林）和各种安非他明（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（SLC 6A 3）多态性相关的ADHD风险增加。我们的实验室在ADHD先证者中发现了多种罕见的DAT编码变体，其中之一DAT Val 559是当前提案的主题。有趣的是，DAT Val 559不仅在ADHD受试者中被鉴定，而且在双相情感障碍和自闭症中也被鉴定，这表明对DA依赖性特征的贡献可以表现为不同疾病中的风险或导致其中的ADHD合并症。通过体外研究，我们证明了Val 559变体产生异常DA流出（ADE），这是一种非囊泡DA释放形式，通常在DAT表达细胞的AMPH处理中观察到。在DAT Val 559的情况下，AMPH不能触发DA流出，尽管AMPH和MPH都阻断ADE。这些发现导致了这样一种假设，即强直性DA泄漏可能是一种或多种DA相关行为障碍风险的未知贡献者。为了在体内测试这一概念，我们开发了DAT Val 559敲入小鼠，其表达来自天然Slc 6a 3基因座的变体，提供了与神经精神疾病相关的DAT功能障碍的第一个构建有效模型。在这里，我提出测试以下假设：1）DAT Val 559的体内表达产生改变的神经递质释放以及对ADHD相关药理学操作敏感的突触前和突触后信号传导事件的变化，以及2）DAT Val 559的终身表达导致与ADHD的一个或多个属性一致的DA相关行为的改变。总而言之，我的努力是有组织的，以提供丰富的培训机会，在调查的机制，神经精神疾病的风险，同时推进分析的第一个结构有效的模型，在多动症的DA功能障碍。

 DESCRIPTION (provided by applicant): 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 (SLC6A3) 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.