Dopamine Transporter: Tools for in vivo molecular replacement

多巴胺转运蛋白：体内分子替代的工具

基本信息

批准号：
10133035
负责人：
Haley E Melikian
金额：
$ 20.94万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
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/10133035
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

项目摘要

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）是具有不同生理影响的主要神经递质，并且是运动所必需的睡眠，工作记忆和奖励。突触前再摄取，由SLC6质膜DA转运蛋白（DAT）介导治疗性心理刺激剂，例如苯丙胺，可卡因和哌醋甲酯（Ritalin），可有效抑制 DA吸收，维持DA信号传导和影响DA依赖性行为。多种神经精神疾病和转基因小鼠清楚地证明了Daergic信号传导行为以及心理刺激效率对DAT表达水平高度敏感。在质膜上不是静态的，而是要受到稳健的征宪和常规内吞回收的约束。霍弗（Howver）尚不清楚调节的dat内在化是否会影响daergic功能和/或DA依赖性行为。 DA信号传导和DA依赖性行为的决定因素。内源性小鼠DAT患有成年小鼠失调的DATS侵蚀剂失调。扰动有明确的发展补偿问题，强调了对评估系统的需求 DAT突变体在体内，但绕过Germaline突变体的陷阱。开发了一种将鼠标与Floxed Dat基因（DATFL/FL）相结合的鼠标模型鼠标（pitx3ires2-tta）通过TET响应元件驱动的CRE重组酶切除和转基因替换，用于体内分子替代研究。由于它规避发育压缩和另外，允许使用AAV进行特定的电路替换。在强大的初步数据的支持下。体内DAT分子置换和2）测试DAT是否“功能获得”和“功能丧失”是否内吞突变体会影响男性和女性的DAT运输，DAERGIC信号传导和DA的行为小鼠。我们以一种不可行的方式询问Dats在体内的影响预计我们的新鼠标模型将为旨在评估突变体的研究人员提供广泛的实用程序成年小鼠Daergic电路的功能。