Identification of gene variants mediating the behavioral and physiological response to THC

大麻是美国使用最广泛的精神药物之一，公共政策的变化 预计将进一步增加使用。目前的大麻品种已被选为高Δ9- 四氢大麻酚(THC)水平和THC水平在过去几十年中增加了两倍。有越来越多的 有证据表明，使用含有高水平THC的高效力大麻会增加有害健康的风险 以及行为后果。许多特定的效应器(例如，受体、受体信号通路) 对THC的调解反应尚未完全确定，而且几乎不知道如何 个体遗传差异会影响反应。这种知识的缺乏是一个严重的障碍 了解与高效大麻使用相关的健康风险和行为。要解决这些问题 我们已经在BXD重组近交系小鼠群体中开发了一种强大的药物基因组学筛选 识别调节大麻素1受体(CB1)水平、信号的效应蛋白中的基因变体 急性暴露于高剂量(10 mg/kg，I.P.)后，信号转导、信号终止和/或运输 太好了。在此之前，我们展示了THC反应的主要效应因子CB1， BXD亲本菌株之间以及亲本对THC急性反应的可遗传变异 菌株和BXD。我们已经确定了提示性数量性状基因座(QTL)和候选因果 在我们的药物基因组筛选中，QTL基因(QTGs)仅使用BXD的一个子集(即20个)。在这里，我们扩展 我们的工作是识别CB1蛋白水平的遗传调节因子(目标1)，并识别和验证基因 大剂量THC急性反应的调节剂(目标2)。在目标1A中，我们使用有针对性的蛋白质组学方法 在双亲、正反F1和BXD中定量纹状体CB1蛋白组分中的CB1水平 菌株。亲本的差异表达、F1的等位基因特异性表达及连锁定位 BXD将识别和验证包含纹状体CB1反式作用调节因子的QTL。在AIM 1B中，我们 将分析扩展到另外两个地区，在AIM 1C中，我们包括接触高剂量THC。在Aim 2A中，我们 筛选BXD的大面板(即，80个)用于量化为低速运动的差异CB1激活， 体温过低和VEH或THC治疗后的抗伤害性感觉，并确定至少可解释 在±2Mb的目标精度下，每个性状的30%的变异。在目标2B中，我们生成匹配的蛋白质组 和转录本，并生成表达QTL(EQTL)的转录组数据 对BXD进行标测，目的是促进因果QTGs的识别。在目标2C中，我们验证 QTGs在大麻素受体信号通路和对THC反应中的生物学作用。这是第一次 药物基因组筛选确定CB1信号效应因子的变异并将揭示新的靶点 啮齿动物和人类对高剂量THC的潜在反应和与大麻使用相关的行为。

Cannabis is one of the most widely used psychotropic drugs in the United States and changes in public policy are expected to further increase use. Current cultivars of cannabis have been selected for high Δ9- tetrahydrocannabinol (THC) levels and THC levels have tripled over the past few decades. There is increasing evidence that use of high potency cannabis containing high levels of THC increases the risk of adverse health and behavioral consequences. Many of the specific effectors (e.g., receptors, receptor signaling pathways) mediating response to THC have not yet been fully identified and virtually nothing is known about how individual genetic differences influence response. This lack of knowledge represents a critical barrier in understanding health risks and behaviors associated with high potency cannabis use. To address these issues we have developed a powerful pharmacogenomic screen in the BXD recombinant inbred mouse population to identify gene variants in effector proteins that regulate cannabinoid 1 receptor (CB1) levels, signal transduction, termination of signaling, and/or trafficking following acute exposure to high-dose (10 mg/kg, i.p.) THC. Previously, we demonstrated strain differences in the level of the major effector of THC responses, CB1, between BXD parental strains and demonstrated heritable variation in acute responses to THC in parental strains and BXDs. We have already identified suggestive quantitative trait loci (QTLs) and candidate causal QTL genes (QTGs) in our pharmacogenomic screen using only a subset (i.e., 20) of BXDs. Here, we expand our work to identify genetic modulators of CB1 protein levels (Aim 1) and to identify and validate genetic modulators of acute responses to high-dose THC (Aim 2). In Aim 1A we use a targeted proteomics approach to quantify striatal CB1 levels in membrane-enriched protein fractions in parents, reciprocal F1, and BXD strains. Differential expression in parental strains, allele-specific expression in the F1 and linkage mapping in the BXDs will identify and validate QTLs containing trans-acting regulators of striatal CB1. In Aim 1B we extend the analysis to two other regions and in Aim 1C we include exposure to high-dose THC. In Aim 2A we screen a large panel (i.e., 80) of BXDs for differential CB1 activation quantified as hypolocomotion, hypothermia, and antinociception following VEH or THC treatment and identify QTLs accounting for at least 30% of the variation in each trait at a target precision of ±2 Mb. In Aim 2B we generate matched proteomes and transcriptomes for parental strains and F1 and generate transcriptome data for expression QTL (eQTL) mapping in BXDs with the goal of facilitating the identification of causal QTGs. In Aim 2C we validate the biological role of QTGs in cannabinoid receptor signaling pathways and responses to THC. This is the first pharmacogenomic screen to identify variation in effectors of CB1 signaling and will reveal new targets underlying responses to high-dose THC and behaviors associated with cannabis use in rodents and humans.