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症过程/疾病中具有重要作用，包括帕金森病、阿尔茨海默病和神经性疼痛。在联合抗逆转录病毒治疗 (cART) 时代，1 型人类免疫缺陷病毒 (HIV-1) 现在被认为是一种慢性疾病，专门针对大脑并导致轻度神经认知障碍的高患病率，也称为 HIV 相关神经认知障碍 (HAND) [1, 2]。 HAND 的最佳相关性已被证明是突触树突损伤与突触和树突损伤，这是 HIV-1 阳性个体中神经认知损伤的基础 [3]。有人提出，增加内源性大麻素信号传导将在此类神经元损伤模型中起到保护作用。我们实验室的初步研究表明，用作直接配体的花生四烯酸乙醇胺 (AEA) 和 2-花生四烯酰甘油 (2-AG) 对 HIV-1 Tat 引起的功能障碍和损伤具有神经保护作用。然而，体内使用内源性大麻素并不是最佳/可行的，因为它们会被分解代谢酶快速降解：负责降解 AEA 的主要酶，脂肪酸酰胺水解酶 (FAAH)，以及负责降解 2-AG 的主要酶，单酰基甘油脂肪酶 (MAGL)。最近，开发出一类新的这些酶的选择性抑制剂，具有神经保护、抗炎、镇痛和抗焦虑作用[4-6]。因此，本提案的重点是确定 1) 使用内源性大麻素分解代谢酶抑制剂作为工具，内源性大麻素在 HIV-1 Tat 诱导的损伤中的神经保护作用，并确定 2) 受体介导的机制，通过该机制内源性大麻素对 HIV-1 Tat 神经毒性具有天然保护作用。我们假设内源性大麻素通过突触前 CB1 受体依赖性机制对神经获得性免疫缺陷综合征 (neuroAIDS) 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-1 Tat 毒性具有神经保护作用的机制。对培养的前额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 诱导的离子稳态紊乱（即 [Ca2]I 和 [Na]i 增加）、突触神经传递、突触树突损伤和细胞死亡的影响。在具体目标 2 中，我们将确定内源性大麻素是否通过突触前 CB1 受体介导的途径对 HIV-1 Tat 诱导的毒性具有天然的神经保护作用。全细胞膜片钳记录将在 CB1 和 CB2 敲除小鼠的前额皮质切片上进行离体记录，包括配对脉冲刺激、双全膜片记录和细胞外细胞附着记录。通过关注兴奋和抑制的突触可塑性的变化，我们将获得关于内源性大麻素系统如何保护神经元免受 HIV-1 Tat 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手部疾病和其他发生认知缺陷疾病的新治疗靶点。

 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.