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

调节药物滥用倾向的机制尚未完全了解， 对精神兴奋剂成瘾的干预措施基本上仍然无效。我们进行了一个全基因组关联 在果蝇中进行的研究，并确定了基因Ctr 9与运动反应显着相关， 安非他明已知Ctr 9作为Paf 1C转录的一部分在调节基因表达中起作用 复杂.在一篇文章中，它还显示出与多巴胺转运蛋白（多巴胺转运蛋白的主要分子）相互作用。 精神兴奋剂的目标我们已经验证了Ctr 9在安非他明诱导的果蝇多动症中的作用; 多巴胺神经元特异性敲除Ctr 9导致对安非他明的反应急剧增加， 甲基苯丙胺，而它的过度表达减弱了反应。我们还表明，Ctr 9共定位 与成年果蝇脑中多巴胺神经元的核外多巴胺转运蛋白（DAT）。我们现在的目标是 以表征其对多巴胺神经传递和甲基苯丙胺影响的机制- 诱导的行为，并开发一个遗传模型，以功能验证Ctr 9的作用， 甲基苯丙胺和可卡因诱导的大鼠过度运动，这是唯一适合模型的特点， 模仿人类的强迫性药物寻求行为，从而将有助于我们的长期目标， Ctr 9在成瘾行为中的作用为实现这些目标，我们提出以下具体目标： (1)阐明Ctr 9调节甲基苯丙胺诱导的运动活动的机制 苍蝇。(a)我们将使用多巴胺生物传感器的双光子成像来确定Ctr 9敲低的影响。 以及过表达对光遗传学诱发的和甲基苯丙胺诱导的多巴胺释放的影响。(b)到 确定Ctr 9的核定位是否对其在多巴胺介导的运动活性中的作用至关重要， 在果蝇中，我们将表达一种截短的Ctr 9蛋白，该蛋白被阻止易位到细胞核（Ctr 9 Δ SH 2）。 并测试其对基础运动和甲基苯丙胺诱导的运动的影响。(c)我们会推翻 Ctr 9基因敲除在成年期和发育过程中的不同时间，以确定Ctr 9基因敲除是否 干扰早期神经发育，影响后期对甲基苯丙胺的易感性， 在成年期急性调节甲基苯丙胺诱导的行为。 (2)为了确定Ctr 9在大鼠对甲基苯丙胺的行为反应中的作用，并确定其在大鼠中的作用。 影响多巴胺神经传递和DAT功能。我们将（a）生成条件性Ctr 9敲除 使用CRISPR/Cas9介导的“生殖系基因靶向”方法对大鼠进行检测，以及（B）测试缺失Ctr 9的效果 选择性地在多巴胺神经元基础和甲基苯丙胺和可卡因诱导的多动症。(c)我们 将测试选择性敲除多巴胺神经元中的Ctr 9对DAT细胞表面定位、纹状体脑切片中诱发的多巴胺释放、摄取和甲基苯丙胺诱导的多巴胺流出的影响。

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.