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

描述(由申请人提供)：遗传学、药理学和成像研究表明，多巴胺(DA)信号变化是注意力缺陷多动障碍(ADHD)和ADHD共病的风险决定因素。突触前DA转运体(DAT，SLC6A3)由于与最常用的ADHD药物、哌甲酸甲酯(MPH，利他林)和苯丙胺(AMPH)制剂(如Adderall)相互作用而引起人们的特别兴趣。MPH是一种DAT拮抗剂，而AMPH与DA竞争DAT转运，并诱导DAT介导的非囊泡性DA释放。这些药物在ADHD治疗中的有效性的分子/细胞基础，以及它们与以DA为基础的ADHD病因学的关系尚不清楚，这在很大程度上是由于对该疾病的病因学缺乏了解。这一事实也导致了缺乏具有构造有效性的动物模型， 有必要使用具有特征相似性(例如，多动)的模型，但这些模型的起源可能与疾病机制几乎没有关系。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基因变异引起的。虽然常见的遗传变异通常很难建模，但由于缺乏物种间的外显性和保守性，罕见的变异，特别是影响蛋白质编码序列的多态，提供了靶向蛋白质保守元件以实现分子、细胞和电路功能变化的前景。最近，我们在两个患有ADHD的男性兄弟姐妹的SLC6A4基因中发现了一个罕见的编码变异(Val559)，该变异也在双相情感障碍和自闭症患者中被发现。我们对DAT Val559在转基因细胞和神经元中表达的研究表明，尽管DAT蛋白表达和DA摄取正常，但该变异体表现出显著的、电压依赖的、DAT介导的异常DA外流(ADE)表型。AMPH显著抑制DAT Val559的ADE，而诱导WT DAT(Ala559)的DA外流。DAT Val559在正常情况下只有在AMPH作用后才被磷酸化的N末端位点也被磷酸化，而DAT Val559中这些位点的突变阻止了磷酸化消除了ADE。这些研究使我们提出了一种新的假说，即强直性DA“泄漏”是由于DAT调节网络的扰动引起的，该网络通常维持集中的DA摄取，导致以多种神经行为障碍为显著特征的DA相关特征。为了验证这一假设，我们培育了DAT Val559敲入小鼠，并启动了一个多学科计划来描述它们的生化、生理和行为特征。我们的初步研究提供了体内ADE导致局部和全身给药AMPH异常行为的证据。在目前的提案中，我们试图扩展这些研究，以建立在模型中出现的分子、生理和行为扰动，这些研究可以将该领域重新引导到一个新的框架，用于RDoC反应性诊断和治疗潜在的多种精神障碍。

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.