Functional validation of a role for the candidate gene Ctr9 in psychostimulant action

调节物质滥用责任的机制还不完全被理解和治疗。 对精神刺激性成瘾的干预在很大程度上仍然无效。我们进行了全基因组关联 在果蝇身上进行的研究，并发现Ctr9基因与运动反应显著相关 安非他命。众所周知，Ctr9作为Paf1C转录的一部分，在调节基因表达方面发挥了作用 很复杂。一份出版物还显示，它与主要分子多巴胺转运体相互作用 精神刺激剂的目标。我们已经证实了Ctr9在安非他明诱导的果蝇多动中的作用； 多巴胺神经元特异性Ctr9基因敲除导致苯丙胺和 甲基苯丙胺，而它的过度表达会钝化反应。我们还展示了Ctr9共本地化 与核外的多巴胺转运蛋白(DAT)在成虫果蝇大脑中的多巴胺神经元。我们现在的目标是 为了表征其对多巴胺神经传递和甲基苯丙胺影响的潜在机制- 诱导果蝇行为并建立遗传模型以从功能上验证Ctr9在果蝇中的作用 甲基苯丙胺和可卡因诱导的大鼠多动症，这是唯一适合于 在人类身上模拟强迫寻求药物的行为，从而有助于我们研究 Ctr9在成瘾性行为中的作用。为了实现这些目标，我们提出了以下具体目标： (1)阐明Ctr9调节甲基苯丙胺诱导的运动活动的机制 在苍蝇中。(A)我们将使用多巴胺生物传感器的双光子成像来确定Ctr9基因敲除的影响 以及光基因诱发和甲基苯丙胺诱导的多巴胺释放的过度表达。(B)至 确定Ctr9的核定位是否对其在多巴胺介导的运动活动中的作用是必要的 ，我们将表达一个截短的ctr9蛋白，该蛋白被阻止转移到细胞核(ctr9Δsh2)。 并测试其对基础运动和甲基苯丙胺诱导的运动的影响。(C)我们将删除该短语 在成年期和发育的不同时期严格检测Ctr9基因，以确定Ctr9基因是否被敲除 干扰早期神经发育，以影响生命后期对甲基苯丙胺的敏感性，或者如果它起作用 在成年期间强烈地调节甲基苯丙胺诱导的行为。 (2)建立Ctr9在大鼠对甲基苯丙胺的行为反应中的作用，并测定其 对多巴胺神经传递和DAT功能的影响。我们将(A)生成有条件的Ctr9淘汰赛 使用CRISPR/Cas9介导的“生殖系基因打靶”方法和(B)检测缺失Ctr9的效果 选择性地在多巴胺神经元的基础和甲基苯丙胺和可卡因诱导的多动。(C)我们 将测试选择性敲除多巴胺神经元Ctr9对纹状体脑片DAT细胞表面定位、诱发的多巴胺释放、摄取和甲基苯丙胺诱导的多巴胺外流的影响。

The mechanisms that mediate substance abuse liability are incompletely understood and therapeutic interventions for psychostimulant addiction remain largely ineffective. We conducted a genome-wide association study in Drosophila and identified the gene Ctr9 to be significantly associated with the locomotor response to amphetamine. Ctr9 is known to play a role in regulating gene expression as part of the Paf1C transcription complex. It was also shown in one publication to interact with the dopamine transporter, the major molecular target of psychostimulants. We have validated the role of Ctr9 in amphetamine-induced hyperactivity in flies; dopamine neuron-specific knockdown of Ctr9 leads to a dramatic increase in the response to amphetamine and methamphetamine, whereas its overexpression blunts the response. We have also shown that Ctr9 co-localizes with dopamine transporter (DAT) outside of the nucleus in dopamine neurons in the adult fly brain. We now aim to characterize the mechanisms underlying its effects on dopamine neurotransmission and methamphetamine- induced behavior in Drosophila and to develop a genetic model to functionally validate the role of Ctr9 in methamphetamine- and cocaine-induced hyperlocomotion in rats, which are uniquely suited to model traits that mimic compulsive drug-seeking behavior in humans and will thus facilitate our long-term goal to investigate the role of Ctr9 in addiction-like behavior. In pursuit of these goals, we propose the following specific aims: (1) To elucidate the mechanisms by which Ctr9 modulates methamphetamine-induced locomotor activity in flies. (a) We will use 2-photon imaging of a dopamine biosensor to determine the impact of Ctr9 knockdown and overexpression on optogenetically-evoked and methamphetamine-induced dopamine release. (b) To determine if the nuclear localization of Ctr9 is essential for its role in dopamine-mediated locomotor activity in Drosophila, we will express a truncated Ctr9 protein that is blocked from translocating to the nucleus (Ctr9ΔSH2) and test its impact on basal and methamphetamine-induced locomotion. (c) We will knock down the expression of Ctr9 strictly during adulthood, and at different times during development, to determine if Ctr9 knockdown interferes with early neurodevelopment to influence susceptibility to methamphetamine later in life or if it acts acutely during adulthood to modulate methamphetamine-induced behavior. (2) To establish a role for Ctr9 in the behavioral response to methamphetamine in rats and determine its impact on dopamine neurotransmission and DAT function. We will (a) generate a conditional Ctr9 knockout rat using a CRISPR/Cas9-mediated “Germline Gene Targeting” approach and (b) test the effects of deleting Ctr9 selectively in dopamine neurons on basal and methamphetamine- and cocaine-induced hyperactivity. (c) We will test the impact of selective knockout of Ctr9 in dopamine neurons on DAT cell-surface localization, evoked dopamine release, uptake and methamphetamine-induced dopamine efflux in striatal brain slices.