Dopamine Transporter: Tools for in vivo molecular replacement

多巴胺(DA)是一种主要的神经递质，具有多种生理影响，是运动所必需的， 睡眠、工作记忆和奖励。在诱发释放后，DA细胞外半衰期由以下因素决定 突触前重摄取，由SLC6质膜DA转运体(DAT)介导。令人上瘾和 治疗性精神刺激剂，如苯丙胺、可卡因和哌醋甲酯(利他林)，有效地抑制 DA摄取，维持DA信号，影响DA依赖行为。DAT编码变体涉及到 各种神经精神障碍和转基因小鼠的研究清楚地表明，DAR能信号 行为和心理刺激效果对DAT的表达水平高度敏感。DAT是 在质膜上不是静止的，但受到强有力的结构性和受调控的内吞细胞循环的影响。 然而，目前尚不清楚受调控的DAT内化是否会影响DAR能功能和/或DA依赖行为。我们的中心假设是，受监管的DAT贩运可能是一个关键和有影响力的 DA信号和DA依赖行为的决定因素。为了直接检验这一假设，我们的目标是替换 内源性DAT在成年小鼠体内运输异常的DAT突变体。然而，生殖系DAT 扰动有明显的发展补偿问题，突显出需要一个评估系统 DAT在体内发生突变，但绕过了生殖系突变表达的陷阱。为此，我们有 建立了一种整合了DAT基因(DATfl/fl)和DAR能Tet-off的小鼠模型 小鼠(Pitx3IRES2-TTA)。得到的DATfl/fl；Pitx3IRES2-TTA小鼠将促进AAV介导的小鼠DAT 体内Cre重组酶切除和Tet反应元件驱动的转基因替换 分子置换研究。我们争辩说，这个系统比生殖系有相当大的优势 检测突变蛋白功能的敲入方法，因为它绕过了发育补偿和 此外，还允许使用AAV更换特定于电路的设备。这种方法的可行性很强。 受强劲的初步数据支撑。这个为期两年的项目的主要目标是：1)优化条件，以 体内DAT分子置换，以及2)检测DAT是否有功能获得和功能丧失 胞内突变对雄性和雌性DAT转运、DAR能信号转导和DA依赖行为的影响 老鼠。我们预计，在这些研究完成后，我们将开发出一种强大的鼠标系统 以一种到目前为止还不可行的方式来询问DAT突变体在体内的影响。此外，我们 预计我们的新小鼠模型将对许多旨在评估突变的研究人员具有广泛的实用价值 在成年小鼠的DAR能回路中起作用。

Dopamine (DA) is a major neurotransmitter with diverse physiological impact, and is required for movement, sleep, working memory, and reward. Following evoked release, DA extracellular half-life is determined by presynaptic reuptake, mediated by the SLC6 plasma membrane DA transporter (DAT). Addictive and therapeutic psychostimulants, such as amphetamine, cocaine and methylphenidate (Ritalin), potently inhibit DA uptake, sustain DA signaling and impact DA-dependent behaviors. DAT coding variants are implicated in a variety of neuropsychiatric disorders, and transgenic mouse studies clearly demonstrate that DAergic signaling and behaviors, as well as psychostimulant efficacy, are highly sensitive to the level of DAT expression. DAT is not static at the plasma membrane, but is subject to robust constitutive and regulated endocytic recycling. However, it remains unclear whether regulated DAT internalization impacts DAergic function and/or DA-dependent behaviors. Our central hypothesis is that regulated DAT trafficking is likely a critical and influential determinant of DA signaling and DA-dependent behaviors. To test this hypothesis directly, we aim to replace endogenous mouse DAT with trafficking dysregulated DAT mutants in adult mice. However, germline DAT perturbations have clear developmental compensatory issues, underlining the need for a system that evaluates DAT mutants in vivo, but circumvents the pitfalls of germline mutant expression. To this end, we have developed a mouse model that integrates mouse with a floxed DAT gene (DATfl/fl) with a DAergic TET-OFF mouse (Pitx3IRES2-tTA). The resulting DATfl/fl;Pitx3IRES2-tTA mouse will facilitate AAV-mediated mouse DAT excision with Cre recombinase, and transgene replacement, driven by the Tet-responsive element, for in vivo molecular replacement studies. We contend that this system offers considerable advantages over germline knock-in approaches to test mutant protein function, as it circumvents developmental compensation and additionally allows for circuit-specific replacement using AAVs. Feasibility for this approach is strongly supported by strong preliminary data. The main aims of this two-year project are: 1) to optimize conditions for in vivo DAT molecular replacement, and 2) to test whether DAT “gain-of-function” and “loss of function” endocytic mutants impact DAT trafficking, DAergic signaling, and DA-dependent behaviors in male and female mice. We anticipate that at the completion of these studies we will have developed a powerful mouse system to interrogate the impact of DAT mutants in vivo in a manner that, heretofore, was not feasible. Moreover, we expect that our new mouse model will have broad utility for a number of researchers aiming to evaluate mutant function in adult mouse DAergic circuits.