Attention deficit/Hyperactivity disorder (ADHD) is one of the most diagnosed psychiatric disorders nowadays. The core symptoms of the condition include hyperactivity, impulsiveness and inattention. The main pharmacological treatment consists of psychostimulant drugs affecting Dopamine Transporter (DAT) function. We have previously shown that genetically modified mice lacking p35 protein (p35KO), which have reduced Cdk5 activity, present key hallmarks resembling those described in animal models useful for studying ADHD. The p35KO mouse displays spontaneous hyperactivity and shows a calming effect of methylphenidate or amphetamine treatment. Interestingly, dopaminergic neurotransmission is altered in these mice as they have an increased Dopamine (DA) content together with a low DA turnover. This led us to hypothesize that the lack of Cdk5 activity affects DAT expression and/or function in this animal model. In this study, we performed biochemical assays, cell-based approaches, quantitative fluorescence analysis and functional studies that allowed us to demonstrate that p35KO mice exhibit decreased DA uptake and reduced cell surface DAT expression levels in the striatum (STR). These findings are supported by in vitro observations in which the inhibition of Cdk5 activity in N2a cells induced a significant increase in constitutive DAT endocytosis with a concomitant increase in DAT localization to recycling endosomes. Taken together, these data provide evidences regarding the role of Cdk5/p35 in DAT expression and function, thus contributing to the knowledge of DA neurotransmission physiology and also providing therapeutic options for the treatment of DA pathologies such as ADHD.

注意缺陷/多动障碍 (ADHD) 是当今诊断最多的精神疾病之一。该病的核心症状包括多动、冲动和注意力不集中。主要的药物治疗包括影响多巴胺转运蛋白 (DAT) 功能的精神兴奋剂。我们之前已经表明，缺乏 p35 蛋白 (p35KO) 的转基因小鼠具有降低 Cdk5 活性的关键特征，这些特征与动物模型中描述的特征相似，可用于研究多动症。p35KO 小鼠表现出自发性活动过度，并表现出哌甲酯或苯丙胺治疗的镇静作用。有趣的是，这些小鼠的多巴胺能神经传递发生了改变，因为它们的多巴胺 (DA) 含量增加且 DA 转换率低。这使我们假设缺乏 Cdk5 活性会影响该动物模型中的 DAT 表达和/或功能。在这项研究中，我们进行了生化分析、基于细胞的方法、定量荧光分析和功能研究，这些研究使我们能够证明 p35KO 小鼠在纹状体 (STR) 中表现出 DA 摄取减少和细胞表面 DAT 表达水平降低。这些发现得到了支持在体外观察中，N2a 细胞中 Cdk5 活性的抑制导致组成型 DAT 内吞作用显着增加，同时 DAT 定位到再循环内体的增加。总之，这些数据提供了关于 Cdk5/p35 在 DAT 表达和功能中的作用的证据，从而有助于了解 DA 神经传递生理学，并为治疗多动症等多动症病理提供治疗选择。