Investigation of endocannabinoid-mediated neuroprotection in models of neuroAIDS

 描述(由申请人提供)：内源性大麻素系统在许多神经退行性和神经炎性过程/疾病中发挥重要作用，包括帕金森氏病、阿尔茨海默病和神经病理性疼痛。在联合抗逆转录病毒疗法(CART)时代，人类免疫缺陷病毒1型(HIV-1)现在被认为是一种专门针对大脑的慢性疾病，并导致轻度神经认知障碍的高发，也称为艾滋病毒相关神经认知障碍(HAND)[1，2]。在HIV-1阳性个体的神经认知损害中，突触和树突的损伤与突触和树突的损伤之间的相关性最好。有人认为，在这种神经元损伤的模型中，增加内源性大麻素信号将具有保护作用。我们实验室的初步研究表明，作为直接配体的双胺(AEA)和2-花生四烯酸甘油(2-AG)对HIV-1Tat诱导的功能障碍和损伤具有神经保护作用。然而，在体内使用内源性大麻素并不是最理想/可行的，因为它们被分解代谢酶迅速降解：负责降解AEA的主要酶--脂肪酸酰胺水解酶(FAAH)和负责降解2-氨基酚的主要酶--单酰甘油脂肪酶(MAGL)。最近，已经开发出一类新的选择性抑制这些酶的药物，它们具有神经保护、抗炎、抗伤害和缓解焦虑的作用[4-6]。因此，本研究的重点是以内源性大麻素分解酶抑制剂为工具，确定内源性大麻素在HIV-1TAT诱导的损伤中的神经保护作用，以及2)内源性大麻素在HIV-1TAT神经毒性中发挥天然保护作用的受体介导机制。我们假设内源性大麻素通过突触前CB1受体依赖机制在神经获得性免疫缺陷综合征(NeuroAIDS)中具有保护作用。在具体目标1中，我们将确定FAAH抑制剂PF3845和MAGL抑制剂MJN110对HIV-1Tat毒性的神经保护作用的机制。对培养的前额叶皮质神经元的细胞成像和电生理学研究将评估酶抑制剂对HIV-1 Tat诱导的离子稳态(即[Ca+]i和[Na+]i增加)、突触神经传递、突触树突状细胞损伤和细胞死亡的影响。在特定的目标2中，我们将通过突触前CB1受体介导的途径来确定内源性大麻素在HIV-1TAT诱导的毒性中是否具有天然的神经保护作用。全细胞膜片钳记录将在CB1和CB2基因敲除小鼠的前额叶皮层切片上进行体外实验，包括成对脉冲刺激、双完整膜片记录和细胞外细胞连接记录。通过关注突触兴奋和抑制可塑性的变化，将获得关于内源性大麻素系统如何保护神经元免受HIV-1Tat诱导的毒性的新知识。了解酶抑制剂在神经艾滋病背景下的作用，可能会发现出现认知缺陷的手部和其他疾病的新治疗靶点。

 DESCRIPTION (provided by applicant): The endocannabinoid system has an important role in many neurodegenerative and neuroinflammatory processes/diseases including Parkinson's disease, Alzheimer's disease, and neuropathic pain. In the era of combined antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) is now considered a chronic disease that specifically targets the brain and causes a high prevalence of mild forms of neurocognitive impairments, also referred to as HIV-associated neurocognitive disorders (HAND) [1, 2]. The best correlate of HAND has been shown to be synaptodendritic damage with injury to synapses and dendrites underlying the neurocognitive impairments seen in HIV-1 positive individuals [3]. It is proposed that increasing endocannabinoid signaling will be protective in models of such neuronal damage. Preliminary studies from our laboratory have shown that anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) used as direct ligands are neuroprotective against HIV-1 Tat-induced dysfunction and injury. However, use of endocannabinoids in vivo is not optimal/feasible because of their rapid degradation by catabolic enzymes: the main enzyme responsible for degradation of AEA, fatty acid amide hydrolase (FAAH), and the main enzyme responsible for degradation of 2-AG, monoacylglycerol lipase (MAGL). Recently, a new class of selective inhibitors of those enzymes have been developed that show neuroprotective, anti-inflammatory, antinociceptive and anxiolytic effects [4-6]. Thus, the focus of this proposal is to determine 1) the neuroprotective effects of endocannabinoids in HIV-1 Tat-induced injury using endocannabinoid catabolic enzyme inhibitors as a tool, and to define 2) the receptor- mediated mechanisms by which endocannabinoids are naturally protective in HIV-1 Tat neurotoxicity. We hypothesize that endocannabinoids will be protective in neuro-acquired immunodeficiency syndrome (neuroAIDS) via a presynaptic CB1 receptor-dependent mechanism. In Specific Aim 1, we will determine the mechanisms by which FAAH inhibitor PF3845 and MAGL inhibitor MJN110 are neuroprotective against HIV-1 Tat toxicity. Cell imaging and electrophysiology studies on cultured prefrontal cortex neurons will assess the effects of the enzyme inhibitors on HIV-1 Tat-induced disturbances of ion homeostasis (i.e. increases in [Ca2+]I and [Na+]i), synaptic neurotransmission, synaptodendritic injury and cell death. In Specific Aim 2, we will determine if endocannabinoids are naturally neuroprotective in HIV-1 Tat-induced toxicity via a presynaptic CB1 receptor-mediated pathway. Whole cell-patch clamp recordings will be conducted ex vivo on prefrontal cortex slices of CB1 and CB2 knockout mice, including paired-pulse stimulation, dual whole patch recordings, and extracellular cell-attached recordings. New knowledge will be gained about how the endocannabinoid system protects neurons from HIV-1 Tat-induced toxicity by focusing on changes in synaptic plasticity of excitation and inhibition. Understanding the effects of enzyme inhibitors in the context of neuroAIDS may uncover novel therapeutic targets for HAND and other diseases in which cognitive deficits occur.