ENDOCANNABINOID ANALGESIA: AN IN VIVO GENE TRANSFER - LIPIDOMICS APPROACH

内源性大麻素镇痛：体内基因转移 - 脂组学方法

• 批准号: 7286847
• 负责人: Andrea Grace Hohmann
• 金额: $ 14.57万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-15 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7286847
• 关键词: 1,2-diacylglycerol; 2-arachidonylglycerol; Absence of pain sensation; Address; Anabolism; Analgesics; Appendix; Arachidonic Acids; Behavioral; Biochemical; Brain; Cannabinoids; Cells; Condition; Development; Diglycerides; Distal; Drug Delivery Systems; Endocannabinoids; Enzymes; Fatty Acids; Figs - dietary; Future; Gene Silencing; Gene Transfer; Genes; Glycerol; Goals; Human; Hydrolysis; In Vitro; Ligands; Lipids; Localized; Mass Spectrum Analysis; Measurement; Mediating; Mediator of activation protein; Membrane; Messenger RNA; Methodology; Methods; Microinjections; Midbrain structure; Modeling; Molecular; Monoacylglycerol Lipases; Nature; Neurons; Nociception; Opioid; Opioid Peptide; Pain; Pathway interactions; Pattern; Phosphatidylinositols; Phospholipase C; Physiological; Play; Positioning Attribute; Process; Protein Isoforms; Proteins; RNA Interference; Rattus; Reporting; Research; Risk; Role; Shock; Signal Pathway; Signal Transduction; Small Interfering RNA; Source; Stimulus; Stress; Structure; System; Testing; Therapeutic; Time; Validation; Work; adenoviral-mediated; anandamide; base; cannabinoid receptor; environmental stressor; foot; gene therapy; genetic manipulation; in vivo; inhibitor/antagonist; innovation; lipid mediator; lipoprotein lipase; midbrain central gray substance; non-opioid analgesic; novel; pain inhibition; programs; receptor; relating to nervous system; research study; response; stressor; transmission process; uptake

DESCRIPTION (provided by applicant): Environmental stressors transiently activate neural systems that inhibit pain responsiveness, thereby inducing a phenomenon known as stress-induced analgesia (SIA). We have recently reported that nonopioid SIA may be mediated by the mobilization of endogenous cannabinoid lipids, such as 2-arachidonoylglycerol (2-AG), in the midbrain periaqueductal gray (PAG) [Hohmann et al., Nature 435: 1108-1112, 2005]. However, the molecular mechanisms governing 2-AG signaling in the brain under physiological conditions remain unknown. Our central hypothesis is that stress stimuli that result in nonopioid SIA activate diacylglycerol lipase (DGL) in select neurons of the PAG, causing the rapid mobilization of 2-AG. Newly- released 2-AG engages local cannabinoid receptors to induce nonopioid SIA, and is then subsequently deactivated through monoacylglycerol lipase (MGL)-mediated hydrolysis. We will test this hypothesis by developing a novel, though high-risk, methodology that unites, for the first time, in vivo virally-mediated gene transfer with targeted lipidomic analyses. Our work will identify the functional consequences of virally- mediated RNA silencing of enzymes implicated in 2-AG formation (DGL) and deactivation (MGL) using three independent complementary approaches. We will use behavioral, neuroanatomical and mass spectrometric analyses (of related lipid mediators, their precursors and metabolites) to evaluate direct and indirect consequences of DGL and MGL silencing on lipid signaling pathways and SIA. Specifically, we will determine whether virally mediated silencing of the DGL and MGL genes within the PAG (i) influences 2-AG signaling and SIA; and (ii) causes distal changes in other endocannabinoid and non-cannabinoid lipid pathways. These studies are expected to demonstrate that 1) in vivo virally-mediated RNA silencing of DGL- beta - a DGL isoform recently shown to colocalize with phospholipase C, an enzyme implicated in formation of the 2-AG precursor diacylglycerol (DAG) - suppresses 2-AG formation in the PAG and SIA, and 2) in vivo virally-mediated RNA silencing of MGL stimulates 2-AG accumulation in the PAG and SIA. Validation of our in vivo gene transfer - targeted lipidomic approach should offer a critical advantage over existing methods which rely solely on neuroanatomical measurements of mRNA or protein. The results of these studies should elucidate, for the first time, the molecular, biochemical and physiological mechanisms governing 2-AG signaling in the brain and determine the role of this endocannabinoid mediator in pain modulation. Furthermore, the results will validate targeted lipidomics as a means to quantify changes in lipid signaling that occur downstream of targeted genetic manipulations. Successful validation of our technological approach and hypotheses may facilitate development of endocannabinoid-based pharmacological and gene therapies for pain.

描述（由申请人提供）：环境应激源瞬间激活神经系统，抑制疼痛反应，从而诱发应激性镇痛（SIA）现象。我们最近报道了非阿片类SIA可能是由内源性大麻素脂质的动员介导的，如2-花生四烯醇甘油（2-AG），在中脑导水管周围灰质（PAG） [Hohmann等人，Nature 435: 1108- 1112,2005]。然而，生理条件下大脑中2-AG信号传导的分子机制尚不清楚。我们的中心假设是，应激刺激导致非阿片类SIA激活PAG特定神经元中的二酰基甘油脂肪酶（DGL），导致2-AG的快速动员。新释放的2-AG与局部大麻素受体结合诱导非阿片类SIA，随后通过单酰基甘油脂肪酶（MGL）介导的水解使其失活。我们将通过开发一种新颖但高风险的方法来验证这一假设，该方法首次将体内病毒介导的基因转移与靶向脂质组学分析结合起来。我们的工作将使用三种独立的互补方法确定病毒介导的RNA沉默对2-AG形成（DGL）和失活（MGL）相关酶的功能影响。我们将使用行为学、神经解剖学和质谱分析（相关脂质介质、其前体和代谢物）来评估DGL和MGL沉默对脂质信号通路和SIA的直接和间接影响。具体来说，我们将确定病毒介导的PAG内DGL和MGL基因的沉默是否会影响2-AG信号和SIA；（ii）引起其他内源性大麻素和非大麻素脂质途径的远端改变。这些研究有望证明：1)体内病毒介导的DGL- β RNA沉默（DGL- β是DGL的一种异构体，最近被证明与磷脂酶C共定位，磷脂酶C参与2- ag前体二酰基甘油（DAG）的形成）抑制PAG和SIA中2- ag的形成；2)体内病毒介导的MGL RNA沉默刺激PAG和SIA中2- ag的积累。验证我们的体内基因转移靶向脂质组学方法应该提供一个关键的优势比现有的方法，仅依赖于mRNA或蛋白质的神经解剖学测量。这些研究的结果将首次阐明大脑中2-AG信号传导的分子、生化和生理机制，并确定这种内源性大麻素介质在疼痛调节中的作用。此外，结果将验证靶向脂质组学作为一种量化靶向遗传操作下游发生的脂质信号变化的手段。我们的技术方法和假设的成功验证可能促进以内源性大麻素为基础的疼痛药理和基因治疗的发展。