KNOCK-IN MOUSE MODEL OF DOPAMINE DYSFUNCTION UNDERLYING TRAITS OF ADHD

描述（由申请人提供）：遗传学、药理学和成像研究指出，多巴胺（DA）信号传导改变是注意力缺陷多动障碍（ADHD）和ADHD共病障碍的风险决定因素。突触前DA转运蛋白（DAT，SLC6A3）由于其与最常见的ADHD处方药物哌甲酯（MPH，利他林）和安非他明（AMPH）制剂（例如Adderall）的相互作用而引起了特别的兴趣。MPH是DAT拮抗剂，而AMPH与DA竞争DAT转运并诱导DAT介导的非囊泡DA释放。这些药物在ADHD治疗中有效性的分子/细胞基础及其与基于DA的ADHD病因学的关系尚不清楚，这在很大程度上是由于缺乏对该疾病病因学的了解。这一事实也导致缺乏具有结构效度的动物模型， 需要使用具有性状相似性的模型（例如，多动），但其起源可能与疾病机制关系不大或没有关系。包括ADHD在内的神经精神疾病的遗传风险被认为是由常见和罕见的遗传变异引起的。由于缺乏跨物种的遗传多样性和保守性，常见的遗传变异通常难以建模，而罕见的变异，特别是影响蛋白质编码序列的多态性，提供了靶向蛋白质保守元件以实现分子、细胞和电路的功能变化的前景。最近，我们在两名患有ADHD的男性同胞的SLC6A4基因中发现了一种罕见的编码变体（Val559），这种变体也在双相情感障碍和自闭症患者中发现。我们对转染细胞和神经元中表达的DAT Val559的研究表明，尽管DAT蛋白表达和DA摄取是正常的，但该变体显示出显著的电压依赖性、DAT介导的异常DA外排（ADE）表型。值得注意的是，AMPH抑制DAT Val559的ADE，而它诱导WT DAT（Ala 559）的DA流出。DAT Val 559也在N-末端位点磷酸化，这些位点通常仅在AMPH应用后磷酸化，并且DAT Val 559中这些位点的突变以阻止磷酸化消除了ADE。这些研究使我们提出了一个新的假设，即紧张性DA "泄漏"，由DAT调节网络的扰动引起，该网络通常维持集中的DA摄取，导致DA相关的特征在多种神经行为障碍中突出。为了验证这一假设，我们已经产生了DAT Val559基因敲入小鼠，并启动了一个多学科的计划，以分析其生化，生理和行为特征。我们的初步研究提供了体内ADE导致局部和全身给予AMPH的异常作用的证据。在目前的提案中，我们试图扩展这些研究，以建立模型中出现的分子，生理和行为扰动，这些研究可以将该领域重新导向一个新的框架，用于RDoc响应性诊断和治疗多种精神疾病。

DESCRIPTION (provided by applicant): Genetic, pharmacological and imaging studies point to altered dopamine (DA) signaling as a risk determinant of Attention-deficit Hyperactivity Disorder (ADHD) and ADHD comorbid disorders. The presynaptic DA transporter (DAT, SLC6A3) has drawn particular interest due to its interactions with the most commonly prescribed ADHD medications, methylphenidate (MPH, Ritalin) and amphetamine (AMPH) formulations (e.g. Adderall). MPH is a DAT antagonist, whereas AMPH competes with DA for transport by DAT and induces DAT-mediated, non-vesicular DA release. The molecular/cellular basis for the effectiveness of these agents in ADHD treatment, and their relationship to a DA-based etiology of ADHD is unclear, owing in large part to a lack of understanding of the etiology of the disorder. This fact has also contributed to a lack of animal models with construct validity, necessitating the use of models with trait similarities (e.g., hyperactivity) but where origins likly have little or no relationship to disease mechanisms. The genetic risk for neuropsychiatric disorders, including ADHD, is believed to arise from contributions of both common and rare genetic variation. Whereas common genetic variation is often difficult to model, owing to a lack of penetrance and conservation across species, rare variation, particularly polymorphisms that impact protein coding sequence, offers the prospect of targeting conserved elements of proteins to achieve functional changes in molecules, cells and circuits. Recently, we identified a rare, coding variant (Val559) in the SLC6A4 gene of two male siblings with ADHD, a variant also identified in subjects with bipolar disorder and autism. Our studies of DAT Val559 expressed in transfected cells and neurons revealed that although DAT protein expression and DA uptake are normal, the variant displays a striking, voltage-dependent, DAT-mediated, anomalous DA efflux (ADE) phenotype. Remarkably, AMPH suppresses the ADE of DAT Val559, whereas it induces DA efflux from WT DAT (Ala559). DAT Val559 is also phosphorylated at N-terminal sites that are normally phosphorylated only after AMPH application, and the mutation of these sites in DAT Val559 to preclude phosphorylation eliminates ADE. These studies led us to the novel hypothesis that tonic DA "leak", arising from perturbations of a DAT regulatory network that normally sustains concentrative DA uptake, leads to DA-linked traits prominently featured in multiple neurobehavioral disorders. To test this hypothesis, we have generated DAT Val559 knock-in mice and initiated a multi-disciplinary program to profile their biochemical, physiologica and behavioral features. Our Preliminary Studies provide evidence of ADE in vivo that leads to anomalous actions of local and systemically administered AMPH. In the current proposal, we seek to extend these studies to establish molecular, physiological and behavioral perturbations that arise in the model, studies that can redirect the field toward a novel framework for RDoc-responsive diagnoses and treatments underlying multiple psychiatric disorders.