Investigation of Novel Drug Targets for Stimulant Use Disorder

项目摘要 在美国，兴奋剂使用障碍（SUD）的发病率正在上升。值得注意的是，没有FDA批准的 有治疗SUD的药物。可卡因和甲基苯丙胺（METH）是吸毒者使用的主要兴奋剂。 个人SUD因为这些兴奋剂通过单胺转运蛋白（MAT）发挥作用， 调节MAT功能的干预可能是有效的SUD治疗。我们的团队鉴定了大脑- 表达蛋白RGS 12作为单胺转运蛋白的调节剂，并且RGS蛋白被认为 成为可下药的目标因此，我的第一个目标（我的论文研究项目）是评估RGS 12抑制作为一种 使用疾病的小鼠模型的SUD的潜在治疗策略。我们的研究小组发现， RGS 12功能对各种刺激剂的运动激活作用具有抗性，包括3，4- 亚甲二氧基甲基苯丙胺（MDMA）。我早期的论文工作集中在理解为什么RGS 12- 无效小鼠对MDMA诱导的过度运动有抗性。MDMA的主要目标是血清素转运体 （SERT），我假设SERT在RGS 12-null小鼠中失调。为了验证我的假设，我测量了 使用来自野生型和RGS 12缺失小鼠的脑组织的SERT表达和功能。我发现， 与野生型小鼠相比，RGS 12缺失小鼠表现出SERT表达和功能的增加。给定 小鼠RGS 12的缺失伴随着SERT表达和功能的增加（我的发现）， 多巴胺转运蛋白（DAT;以前的研究生的发现），因为可卡因的目标都SERT 和DAT，我假设RGS 12缺失小鼠对可卡因戒断相关行为具有抵抗力。 为了验证我的假设，我评估了先前报道的野生型和非野生型可卡因戒断的躯体体征。 RGS 12缺失小鼠。我的研究结果表明，虽然野生型小鼠在服用可卡因期间表现出更强的梳理能力， 戒断，RGS 12-null小鼠则没有。在F99培训阶段，我将继续努力验证RGS 12作为 通过测量可卡因或METH静脉自我给药（IVSA）（金标准）， 在实验室动物中建立SUD模型的行为范例。我会确定是否有Rgs 12基因型- 对可卡因或METH自我给药的获得和维持的相关影响， 恢复已熄灭的可卡因或METH自我给药。我的第二个目标（我的博士后研究 方向）是表征SUD相关电路中的GPCR转录组，因为GPCR是极好的靶点 用于调节细胞/电路的活动。在K 00训练阶段，我将使用逆行示踪剂标记单个 这些细胞投射到与SUD相关的大脑区域。然后，我将使用单细胞RNA-seq， 标记细胞的转录组。理想情况下，这些数据将揭示GPCR，在激活或失活时， 抑制多巴胺在丘脑核的释放所提议的研究阶段将为我提供充足的 学习新技术的机会。此外，拟议的培训计划将帮助我提高我的技能， 演示，写作，指导，教学和领导实验室小组。

PROJECT SUMMARY The incidence of stimulant use disorder (SUD) in the United States is on the rise. Notably, no FDA-approved medications exist to treat SUD. Cocaine and methamphetamine (METH) are the primary stimulants used by individuals with SUD. Because those stimulants exert their effects via monoamine transporters (MATs), interventions that modulate MAT function could be effective SUD treatments. Our group identified the brain- expressed protein RGS12 as being a modulator of monoamine transporters, and RGS proteins are thought to be druggable targets. Thus, my first aim (my dissertation research project) is to evaluate RGS12 inhibition as a potential treatment strategy for SUD using a mouse model of the disease. Our group discovered that mice lacking RGS12 function are resistant to the locomotor-activating effects of various stimulants, including 3,4- methylendedioxymethamphetamine (MDMA). My early dissertation work focused on understanding why RGS12- null mice are resistant to MDMA-induced hyperlocomotion. As MDMA’s primary target is the serotonin transporter (SERT), I hypothesized that SERT was dysregulated in RGS12-null mice. To test my hypothesis, I measured SERT expression and function using brain tissue from wild-type and RGS12-null mice. I discovered that, compared with wild-type mice, RGS12-null mice exhibited increases in SERT expression and function. Given that loss of RGS12 in mice is accompanied by increased expression and function of SERT (my finding) and the dopamine transporter (DAT; a previous graduate student’s finding), and because cocaine targets both SERT and DAT, I hypothesized that RGS12-null mice would be resistant to cocaine withdrawal-associated behaviors. To test my hypothesis, I assessed previously reported somatic signs of cocaine withdrawal in wild-type and RGS12-null mice. My results showed that while wild-type mice display increased grooming during cocaine withdrawal, RGS12-null mice do not. In the F99 training phase, I will continue my efforts to validate RGS12 as a target for SUD by measuring cocaine or METH intravenous self-administration (IVSA), the gold standard behavioral paradigm for modeling SUD in laboratory animals. I will determine if there are Rgs12 genotype- associated effects on the acquisition and maintenance of cocaine or METH self-administration, as well as on reinstatement of extinguished cocaine or METH self-administration. My second aim (my postdoctoral research direction) is to characterize the GPCR transcriptome in SUD-relevant circuitry, as GPCRs are excellent targets for modulating the activities of cells/circuits. In the K00 training phase, I will use retrograde tracers to label single cells that project to SUD-relevant brain regions. I will then identify, using single-cell RNA-seq, the GPCR transcriptome of labeled cells. Ideally, those data will reveal GPCRs that, upon activation or inactivation, will dampen dopamine release in the nucleus accumbens. The proposed research phases will provide me ample opportunities to learn new techniques. Additionally, the proposed training plan will help me improve my skills in presenting, writing, mentoring, teaching, and leading a laboratory group.