ENDOCANNABINOID ANALGESIA: AN IN VIVO GENE TRANSFER - LIPIDOMICS APPROACH

描述(由申请人提供)：环境应激源瞬间激活抑制疼痛反应的神经系统，从而导致一种称为应激诱导止痛(SIA)的现象。我们最近报道，非阿片类SIA可能是通过动员中脑导水管周围灰质(PAG)内的内源性大麻素脂，如2-花生四烯基甘油(2-AG)来介导的[Hohmann等，自然，435：1108-1112,2005]。然而，生理条件下大脑中调控2-AG信号的分子机制仍不清楚。我们的中心假设是，导致非阿片类SIA的应激刺激激活了PAG部分神经元中的二酰甘油脂肪酶(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，然后通过单酰甘油脂肪酶(MGL)介导的水解失活。我们将通过开发一种新的、尽管风险很高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有针对性的脂组分析相结合。我们的工作将使用三种独立的互补方法来确定病毒介导的RNA沉默与2-AG形成(DGL)和失活(MGL)有关的酶的功能后果。我们将使用行为学、神经解剖学和质谱学分析(相关的脂质介质、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。具体地说，我们将确定病毒介导的PAG内DGL和MGL基因的沉默是否(I)影响2-AG信号和SIA；以及(Ii)导致其他内源性大麻素和非大麻素脂途径的远端变化。这些研究有望证明：1)体内病毒介导的DGL-β-DGL亚型与磷脂酶C共定位的RNA沉默可抑制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。我们的体内基因转移靶向脂肪组学方法的验证应该比现有的方法提供一个关键的优势，这些方法仅依赖于对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信号的分子、生化和生理机制，并确定这种内源性大麻素在痛觉调制中的作用。此外，这些结果将验证靶向脂质组学作为量化发生在靶向基因操作下游的脂质信号变化的一种手段。我们的技术方法和假说的成功验证可能会促进基于内源性大麻素的疼痛药理和基因疗法的发展。

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.