Dopamine Transporter: Tools for in vivo molecular replacement

多巴胺转运蛋白：体内分子替代的工具

• 批准号: 9975977
• 负责人: Haley E Melikian
• 金额: $ 25.13万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9975977
• 关键词: Acute; Address; Adult; Amphetamines; Antidepressive Agents; Anxiety; Attention deficit hyperactivity disorder; Behavior; Behavioral; Brain; Cell membrane; Cocaine; Code; Consumption; Corpus striatum structure; Data; Development; Disease model; Dopamine; Dorsal; Elements; Endocytosis; Enterobacteria phage P1 Cre recombinase; Epidemic; Event; Excision; Exhibits; Female; Financial compensation; Functional disorder; Future; Gene Mutation; Genes; Goals; Half-Life; Health; Homeostasis; Human; In Situ; Influentials; Investigation; Knock-in; Knock-in Mouse; Knowledge; Laboratories; Lead; Learning; LoxP-flanked allele; Measures; Mediating; Modeling; Molecular; Mood Disorders; Motor Activity; Movement; Mus; Mutant Strains Mice; Mutation; Neurotransmitters; Nucleus Accumbens; Parkinson Disease; Periodicity; Physiological; Play; Psychostimulant dependence; Recycling; Research Personnel; Research Support; Resources; Rewards; Ritalin; Role; Scanning; Schizophrenia; Short-Term Memory; Signal Transduction; Sleep; Slice; Surface; Synaptic Transmission; System; Testing; Tetanus Helper Peptide; Therapeutic; Time; Transgenes; Transgenic Mice; United States; Variant; Viral; Work; addiction; autism spectrum disorder; cost; dopamine transporter; dopaminergic neuron; expectation; extracellular; gain of function; high risk; in vivo; in vivo evaluation; loss of function; male; molecular transporter; mouse model; mutant; nervous system disorder; neuropsychiatric disorder; neurotransmission; novel strategies; novel therapeutic intervention; presynaptic; protein function; psychostimulant; reuptake; success; tool; trafficking; transmission process; uptake

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.

多巴胺（DA）是一种具有多种生理影响的主要神经递质，是运动所必需的， 睡眠、工作记忆和奖励。在诱发释放后，DA细胞外半衰期通过以下测定： 突触前重摄取，由SLC 6质膜DA转运蛋白（DAT）介导。上瘾和 治疗性精神兴奋剂，如安非他明、可卡因和哌醋甲酯（利他林）， DA摄取，维持DA信号传导并影响DA依赖性行为。DAT编码变异体与 各种神经精神疾病和转基因小鼠研究清楚地表明，DA能信号传导 和行为，以及精神兴奋剂功效，对DAT表达水平高度敏感。DAT是 在质膜上不是静止的，而是经历稳健的组成性和受调节的内吞再循环。 然而，目前尚不清楚是否调节DAT内化影响DA能功能和/或DA依赖性行为。我们的中心假设是，受管制的DAT贩运可能是一个关键的和有影响力的 DA信号传导和DA依赖性行为的决定因素。为了直接验证这一假设，我们的目标是取代 内源性小鼠DAT与成年小鼠中运输失调的DAT突变体。然而，种系DAT 扰动有明显的发展补偿问题，强调需要一个系统， DAT突变体在体内，但规避了生殖系突变体表达的陷阱。为此我们 开发了一种小鼠模型，其将具有floxed DAT基因（DATfl/fl）的小鼠与DA能TET-OFF整合， 小鼠（Pitx 3 IRES 2-tTA）。所得DATfl/fl; Pitx 3 IRES 2-tTA小鼠将促进AAV介导的小鼠DAT 用Cre重组酶切除，和转基因置换，由Tet-responsive元件驱动，用于体内 分子置换研究我们认为，这种系统提供了相当大的优势，超过种系 敲入方法来测试突变蛋白质功能，因为它规避了发育补偿， 此外，还允许使用AAV进行电路特定替换。这种方法的可行性很强。 得到了有力的初步数据支持。这个为期两年的项目的主要目标是：1）优化条件， 体内DAT分子替代，和2）测试DAT“功能获得”和“功能丧失” 内吞突变体影响男性和女性的DAT运输、DA能信号传导和DA依赖行为 小鼠我们预计，在完成这些研究，我们将开发出一个强大的鼠标系统 以迄今为止不可行的方式询问DAT突变体在体内的影响。而且我们 我希望我们的新小鼠模型将有广泛的实用性，为一些研究人员旨在评估突变 在成年小鼠DA能回路中的功能。