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

候选基因 Ctr9 在精神兴奋作用中的作用的功能验证

• 批准号: 10392183
• 负责人: Jonathan A Javitch
• 金额: $ 19.99万
• 依托单位: NEW YORK STATE PSYCHIATRIC INSTITUTE DBA RESEARCH FOUNDATION FOR MENTAL HYGIENE, INC
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10392183
• 关键词: Acute; Adult; Amphetamines; Animal Model; Behavior; Biosensor; Brain; CRISPR/Cas technology; Candidate Disease Gene; Cell Nucleus; Cell membrane; Cell surface; Cells; Chromosomes; Cocaine; Complex; Corpus striatum structure; Development; Dopamine; Drosophila genus; Drug Addiction; Drug usage; Gene Expression; Gene Expression Profiling; Gene Targeting; Genes; Genetic; Genetic Models; Genetic Transcription; Goals; Human; Hyperactivity; Image; In Vitro; Inbreeding; Knock-out; Life; Link; Locomotion; Mediating; Medical; Membrane; Methamphetamine; Modeling; Molecular Target; Motor Activity; Mus; Nervous system structure; Nuclear; Pharmaceutical Preparations; Physiological; Play; Predisposition; Property; Proteins; Public Health; Publications; Rattus; Reporting; Rewards; Role; Single Nucleotide Polymorphism; Slice; Substance abuse problem; Testing; Therapeutic Intervention; Time; Validation; Ventral Tegmental Area; abuse liability; addiction; behavioral response; conditional knockout; dopamine transporter; dopaminergic neuron; drug seeking behavior; fly; genome wide association study; knock-down; neurodevelopment; neurotransmission; novel; optogenetics; overexpression; pancreatic differentiation 2 protein; psychosocial; psychostimulant; response; stimulant abuse; stimulant dependence; stimulant use disorder; tool; trafficking; trait; two-photon; uptake

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.

介导药物滥用倾向的机制尚未完全了解和治疗 我们对精神兴奋剂成瘾的干预措施仍然基本上无效。 在果蝇中进行的研究发现基因 Ctr9 与运动反应显着相关 已知 Ctr9 作为 Paf1C 转录的一部分在调节基因表达中发挥作用。 一份出版物还表明它与主要分子多巴胺转运蛋白相互作用。 我们已经验证了 Ctr9 在苯丙胺诱导的果蝇多动中的作用； 多巴胺神经元特异性敲低 Ctr9 导致对安非他明和 甲基苯丙胺，而其过度表达会减弱反应。我们还表明 Ctr9 共定位。 我们现在的目标是在成年果蝇大脑的多巴胺神经元的细胞核外使用多巴胺转运蛋白（DAT）。 表征其对多巴胺神经传递和甲基苯丙胺影响的机制 诱导果蝇行为并开发遗传模型以功能验证 Ctr9 在果蝇中的作用 甲基苯丙胺和可卡因诱导的大鼠过度运动，特别适合模型特征 模仿人类强迫性的药物寻求行为，从而有助于我们研究药物的长期目标 Ctr9 在成瘾样行为中的作用 为了实现这些目标，我们提出以下具体目标： (1) 阐明Ctr9调节甲基苯丙胺诱导的运动活性的机制 (a) 我们将使用多巴胺生物传感器的 2 光子成像来确定 Ctr9 敲低的影响。 以及光遗传学诱发和甲基苯丙胺诱导的多巴胺释放的过度表达（b）To。 确定 Ctr9 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 果蝇，我们将表达截短的 Ctr9 蛋白，该蛋白被阻止转位至细胞核 (Ctr9ΔSH2) 并测试其对基础和甲基苯丙胺诱导的运动的影响 (c) 我们将敲低表达。 Ctr9 严格在成年期和发育过程中的不同时间进行，以确定 Ctr9 敲低是否 干扰早期神经发育，影响以后生活中对甲基苯丙胺的敏感性，或者影响其是否发挥作用 在成年期急性调节甲基苯丙胺诱导的行为。 (2) 建立Ctr9在大鼠冰毒行为反应中的作用并确定其作用 对多巴胺神经传递和 DAT 功能的影响 我们将 (a) 产生条件性 Ctr9 敲除。 使用 CRISPR/Cas9 介导的“种系基因靶向”方法的大鼠并 (b) 测试删除 Ctr9 的效果 选择性地在多巴胺神经元中对基础和甲基苯丙胺和可卡因诱导的多动症（c）我们。 将测试多巴胺神经元中选择性敲除 Ctr9 对纹状体脑切片中 DAT 细胞表面定位、诱发多巴胺释放、摄取和甲基苯丙胺诱导的多巴胺流出的影响。