Protective Effects of Cannabinoids in HIV Neuropathogenesis

描述（由申请人提供）：HIV-1的神经发病机制涉及病毒蛋白、胶质和神经元介质以及环境因素（如滥用药物）之间复杂的相互作用。然而，令人惊讶的是，尽管大麻素受体在纹状体中大量表达，黑质纹状体多巴胺能区对HIV-1相关神经病理高度敏感，但人们对大麻素对HIV-1神经发病机制的影响知之甚少。此外，多巴胺能神经元的明显耗竭、激活的小胶质细胞、反应性星形胶质细胞和该脑区域的氧化损伤是HAD的显著特征。小胶质细胞是炎症介质的重要来源，包括细胞因子、趋化因子和活性氧，所有这些都有助于脑损伤。当受到白细胞介素(IL)-12的刺激时，星形胶质细胞产生神经毒性活性氮和趋化因子，如CCL2和CX3CL1，它们可能会招募巨噬细胞/小胶质细胞到纹状体。在病毒相关因子中，病毒毒素HIV-1 gp120蛋白在小胶质细胞和星形胶质细胞中引发几种神经致病反应。基于我们实验室之前的研究表明，合成大麻素抑制人类小胶质细胞中HIV-1的表达，抑制人类星形胶质细胞炎症介质的产生，以及来自非人类系统的大量文献证明大麻素具有神经保护特性，本研究项目的核心假设是，合成大麻素通过抑制激活的小胶质细胞和星形胶质细胞的机制，保护多巴胺能神经元免受gp120的毒性作用。在承认gp120对神经元具有直接毒性作用的同时，我们打算填补关于大麻素对gp120与参与HIV-1神经发病的胶质细胞相互作用的影响的重要知识空白。在这个项目中，我们将使用两种新开发的人类细胞培养模型：中脑细胞培养模型和神经前体细胞（NPC）衍生的多巴胺能神经元模型。我们将使用这些模型，以及高度纯化的人类小胶质细胞和星形胶质细胞，在实验中研究合成大麻素对gp120诱导的多巴胺能神经元损伤的保护作用，通过表征多巴胺能神经元的功能损伤、凋亡、氧化损伤和胶质细胞的趋化因子产生。在两种细胞培养模型中，评估大麻素改善gp120介导的多巴胺能神经元损伤和抑制趋化因子产生的能力，并研究大麻素抑制小胶质细胞介导的多巴胺能神经元损伤和抑制gp120和il -12介导的星形胶质细胞功能影响的具体机制。通过对原代人脑细胞培养模型的创新使用，我们希望进一步了解合成大麻素如何改变gp120对参与HIV-1神经发病机制的脑细胞的毒性作用，并将这些研究的发现转化为HAD的新治疗方法。尽管抗逆转录病毒疗法（ART）具有治疗效果，但HIV-1相关痴呆（HAD）仍然是艾滋病患者的严重威胁，目前还没有针对HIV-1神经系统表现的特异性治疗方法。根据最近的研究，黑质纹状体多巴胺能区是HAD中神经元功能障碍和死亡的关键脑区，合成大麻素抑制人类小胶质细胞中HIV-1的表达，抑制人类星形胶质细胞中炎症介质的产生，以及大量文献证明大麻素在其他系统中具有神经保护特性。该实验旨在验证合成大麻素通过抑制激活的小胶质细胞和星形胶质细胞的机制来保护多巴胺能神经元免受HIV蛋白gp120的毒性作用的中心假设。通过对原代人脑细胞培养模型的创新使用，我们希望进一步了解合成大麻素如何改变gp120对参与HIV-1神经发病机制的脑细胞的毒性作用，并将这些研究的发现转化为HAD的新治疗方法。

DESCRIPTION (provided by applicant): The neuropathogenesis of HIV-1 involves a complex interaction between viral proteins, glial and neuronal mediators, and environmental factors, such as drugs of abuse. However, surprisingly little is known about the effects of cannabinoids on HIV-1 neuropathogenesis, despite the facts that cannabinoid receptors are abundantly expressed in the striatum and that the nigrostriatal dopaminergic area is highly susceptible to HIV-1-related neuropathology. In addition, marked depletion of dopaminergic neurons, activated microglia, reactive astrocytes, and oxidative damage within this brain region are salient features of HAD. Microglia are an important source of inflammatory mediators, including cytokines, chemokines, and reactive oxygen species, all of which contribute to brain damage. When stimulated by interleukin (IL)-12, astrocytes produce neurotoxic reactive nitrogen species and chemokines, such as CCL2 and CX3CL1, which may recruit macrophages/microglia to the striatum. Among the viral-related factors, the virotoxin HIV-1 gp120 protein triggers several neuropathogenic responses in microglia and astrocytes. Based upon previous work in our laboratory demonstrating that synthetic cannabinoids inhibit expression of HIV-1 in human microglia and suppress production of inflammatory mediators by human astrocytes, and a substantial body of literature from non-human systems demonstrating neuroprotective properties of cannabinoids, the central hypothesis underlying this research project is that synthetic cannabinoids will protect dopaminergic neurons against the toxic effects of gp120 through a mechanism involving inhibition of activated microglia and astrocytes. While acknowledging the direct toxic effect gp120 can have on neurons, we intend to fill important gaps in knowledge regarding the effects of cannabinoids on the interaction of gp120 with the glial cells involved in HIV-1 neuropathogenesis. For this project, we will use two newly developed human cell culture models: a midbrain cell culture model and a neural precursor cell (NPC)-derived dopaminergic neuron model. We will use these models, as well as highly purified human microglial cells and astrocytes in experiments that investigate the protective effects of synthetic cannabinoids against gp120-induced damage to dopaminergic neurons through characterization of functional impairment, apoptosis, and oxidative damage of dopaminergic neurons and chemokine production by glial cells, assess the ability of cannabinoids to ameliorate gp120-mediated damage to dopaminergic neurons and to inhibit chemokine production in both cell culture models, and investigate the specific mechanisms by which cannabinoids inhibit microglia-mediated damage to dopaminergic neurons and inhibit gp120- and IL-12-mediated functional effects on astrocytes. Through the innovative use of primary human brain cell culture models, we hope to advance the understanding of how synthetic cannabinoids alter the toxic effects of gp120 on the brain cells involved in the neuropathogenesis of HIV-1, with the long term objective of translating discoveries arising from these studies into a new treatment method for HAD. Project Narrative Despite the therapeutic impact of anti-retroviral therapy (ART), HIV-1-associated dementia (HAD) remains a serious threat to AIDS patients, and there currently remains no specific therapy for the neurological manifestations of HIV-1. Based upon recent work that the nigrostriatal dopaminergic area is a critical brain region for the neuronal dysfunction and death seen in HAD, that synthetic cannabinoids inhibit HIV-1 expression in human microglia and suppress production of inflammatory mediators in human astrocytes, as well as a substantial literature demonstrating neuroprotective properties of cannabinoids in other systems, experiments have been designed to test the central hypothesis that synthetic cannabinoids will protect dopaminergic neurons against the toxic effects of the HIV protein gp120 through a mechanism involving inhibition of activated microglia and astrocytes. Through the innovative use of primary human brain cell culture models, we hope to advance the understanding of how synthetic cannabinoids alter the toxic effects of gp120 on the brain cells involved in the neuropathogenesis of HIV-1, with the long term objective of translating discoveries arising from these studies into a new treatment method for HAD.