ENDOCANNABINOID ANALGESIA: AN IN VIVO GENE TRANSFER - LIPIDOMICS APPROACH

描述（由申请人提供）：环境应激源短暂激活抑制疼痛反应的神经系统，从而诱导称为应激诱导镇痛（SIA）的现象。我们最近报道了非阿片类物质SIA可能是由中脑导水管周围灰质（PAG）中内源性大麻素脂质（如2-花生四烯酰甘油（2-AG））的动员介导的[Hohmann et al.，Nature 435：1108-1112，2005]。然而，在生理条件下大脑中2-AG信号传导的分子机制仍然未知。我们的中心假设是，应激刺激，导致非阿片类SIA激活二酰基甘油脂肪酶（DGL）在选择的PAG神经元，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以诱导非阿片类SIA，然后通过单酰基甘油脂肪酶（MGL）介导的水解失活。我们将通过开发一种新的，但高风险的方法来测试这一假设，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相结合。我们的工作将使用三种独立的互补方法来鉴定病毒介导的RNA沉默的功能后果，所述RNA沉默的酶涉及2-AG形成（DGL）和失活（MGL）。我们将使用行为，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的直接和间接后果。具体来说，我们将确定病毒介导的PAG内DGL和MGL基因沉默是否（i）影响2-AG信号传导和SIA;以及（ii）导致其他内源性大麻素和非大麻素脂质途径的远端变化。这些研究预期证明1）DGL-β-α DGL同种型的体内病毒介导的RNA沉默最近显示与磷脂酶C共定位，磷脂酶C是一种参与2-AG前体二酰基甘油（DAG）形成的酶，其抑制PAG和SIA中的2-AG形成，和2）MGL的体内病毒介导的RNA沉默刺激PAG和SIA中的2-AG积累。我们的体内基因转移靶向脂质组学方法的验证应提供优于仅依赖于mRNA或蛋白质的神经解剖学测量的现有方法的关键优势。这些研究的结果应该阐明，第一次，在大脑中的2-AG信号的分子，生化和生理机制，并确定这种内源性大麻素介质在疼痛调制的作用。此外，这些结果将验证靶向脂质组学作为量化靶向遗传操作下游发生的脂质信号传导变化的手段。我们的技术方法和假设的成功验证可能有助于开发基于内源性大麻素的疼痛药理学和基因疗法。

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.