Role of ABHD6 in 2-AG Signaling

描述(申请人提供)：内源性大麻酰胺和2-花生四烯基甘油(2-AG)通过大麻素CB1和CB2受体起作用，并通过两步过程失活：两者都被输送到细胞内，然后Anandide被脂肪酸酰胺水解酶(FAAH)和2-AG被单酰甘油脂肪酶(MGL)水解酶水解。选择性抑制FAAH或MGL会分别导致ANANDAME或2-AG的蓄积，且治疗效果不重叠。在一项由NIDA-CEBRA拨款资助的研究中，我们发现小胶质细胞系BV-2不表达MGL，但却有效地降解2-AG，这表明存在一种新的2-AG水解酶，可以作为治疗与神经炎症相关的病理的靶点。在与Ben Cravatt的合作中，我们使用了功能蛋白质组学的方法，并确定ABHD6是BV-2细胞中2-AG水解酶的候选酶。最近，我们使用shRNA敲除和一种新开发的ABHD6抑制剂，并表明该酶在BV-2细胞和神经元的2-AG水解中发挥主要作用。由于洪廷顿病(HD)与早期CB1受体下调和慢性神经炎性反应有关，抑制ABHD6可能是治疗这种退行性疾病的有效方法。在这份赠款提案中，我们概述了旨在：1)绘制ABHD6在健康和HD小鼠大脑中的表达和活性的图谱，2)确定ABHD6的药理特性并开发新型抑制剂3)测试ABHD6是否控制小胶质细胞和神经元中的2-AG信号，并构成治疗HD小鼠的有效靶点。这三个目标的完成将使我们更全面地了解ABHD6在健康和退化大脑中的小胶质细胞和神经元中的表达、活性和作用。我们的长期目标是开发有选择性地抑制内源性大麻素失活的药理和遗传工具，以开发出不像大麻素激动剂那样容易滥用和产生不良影响的治疗方法。 与公众健康相关：在这项拨款提案中，我们概述了旨在1)绘制新型内源性大麻素水解酶ABHD6在健康小鼠和两种亨廷顿病小鼠模型的大脑中的表达和活性的实验，2)确定这种新型酶的药理特性和开发抑制剂，以及3)测试它在控制内源性大麻素调节小胶质细胞和神经细胞功能的能力方面的作用，以及它的抑制是否影响亨廷顿病小鼠模型的疾病进展。

DESCRIPTION (provided by applicant): The endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) act through cannabinoid CB1 and CB2 receptors, and are inactivated by a two-step process: Both are transported into cells, and then anandamide is hydrolyzed by fatty acid amide hydrolase (FAAH) and 2-AG by monoacylglycerol lipase (MGL). Selective inhibition of either FAAH or MGL results in the accumulation of either anandamide or 2-AG, respectively, and in non-overlapping therapeutic effects. In a study funded by a NIDA-CEBRA grant, we found that the microglial cell line, BV-2, does not express MGL and yet efficiently hydrolyzes 2-AG, suggesting the existence of a new 2-AG-hydrolyzing enzyme that could be targeted for the treatment of pathologies associated with neuroinflammation. In collaboration with Ben Cravatt, we used a functional proteomics approach and identified ABHD6 as a candidate enzyme for 2-AG hydrolysis in BV-2 cells. More recently, we used shRNA knockdown and a newly developed ABHD6 inhibitor and showed that this enzyme plays a major role in 2-AG hydrolysis in both BV-2 cells and neurons. Because Hungtington's disease (HD) is associated with an early down-regulation of CB1 receptors and a chronic neuroinflammatory response, inhibiting ABHD6 might represent a valid therapeutic approach to treat this degenerative disease. In this grant proposal, we outline experiments designed to: 1) Map ABHD6 expression and activity in healthy and HD mice brains, 2) Determine the pharmacological profile of ABHD6 and develop novel inhibitors 3) Test if ABHD6 controls 2-AG signaling in microglia and neurons, and constitutes a valid target for treating HD mice. The completion of these three aims will provide a comprehensive understanding of the expression, activity and role of ABHD6 in microglia and neurons in healthy and degenerating brain. Our long-term goal is to generate pharmacological and genetic tools that selectively inhibit endocannabinoid inactivation as means to develop therapeutics that lack the abuse liability and adverse effects produced by cannabinoid agonists. PUBLIC HEALTH RELEVANCE: In this grant proposal, we outline experiments designed to 1) Map the expression and activity of the novel endocannabinoid-hydrolyzing enzyme, ABHD6, in the brains of healthy mice, and of two mice models of Huntington's disease, 2) Determine the pharmacological profile and develop inhibitors of this novel enzyme and 3) Test its role in controlling endocannabinoid's ability to regulate microglial and neuronal cell functions, and whether its inhibition affects disease progression in a mouse model of Huntington's disease.