自噬在HIV-1gp120致海马神经元损伤过程中的作用及信号机制研究

HIV-associated neurocognitive disorders (HAND) are still an important problem in HIV-infected individuals despite the use of combination antiretroviral therapy (ART). HAND is present in 40 - 60% of HIV-infected individuals on ART. Although the exact mechanism by which HIV causes HAND remains unknown, HIV envelope glycoprotein gp120 likely plays an important role in the pathogenesis of HAND, as HIV gp120 is a potent neurotoxin. We and others have reported that HIV gp120 can induce apoptosis of neural cells in hippocampus and cause impairment of spatial learning and memory in Spraque Dawley (SD) rats. Significantly, our recent data demonstrated that autophagy markedly increased during the early stage of interaction between HIV gp120 and SD rat hippocampal neurons, while excessive autophagy accompaning with neuronal apoptosis was observed in the late stage of the interaction. Therefore, we hypothesize that neuronal autophagy increased in the early stage of interaction exerts a neuroprotective effect, and excessive autophagy in the late stage leads to neuronal apoptosis. We also hypothesize that autophagy triggers different signal pathways in the early stage versus the late stage. To test these hypotheses, we will use our established research approaches and techniques in morphological analysis, behavioral science, and molecular biology to disect gp120-triggered autophagy pathways. We will conduct in vitro and in vivo experiments to study key signal molecules that potentially link mTOR, non-mTOR and gp120-triggered autophagy pathways. Specific inhibitors of the key signal molecules will be used to probe their functions. Finally, we will study the molecular mechanisms undrlying neuronal autophagy and apoptosis in the early stage versus late stage of gp120 challenge. Our results will provide critical insights to the understanding of HAND pathogenesis and to the development of preventive and therapeutic interventions of HAND.

研究显示：因HAART治疗延长了艾滋病患者的生存期，40-60%的HIV感染者可出现HIV相关神经认知障碍(HAND)，其中起重要作用的包膜蛋白gp120在神经元损伤中的机制一直是研究的热点。本室及他人研究已证实gp120可致大鼠空间记忆障碍及海马神经元凋亡，近期我们观察自噬在gp120致大鼠海马神经元损伤过程中的变化，发现损伤早期自噬增强；晚期自噬过度增强伴神经元大量凋亡，但机制不清。此预实验结果显示自噬在gp120致神经元作用早期起保护作用，晚期自噬过度促凋亡。这种早、晚期截然不同的损伤差异可能通过各自不同的自噬信号转导通路实现。为验证此假说，本研究拟通过体内、外实验，采用形态学、行为学、分子生物学等技术，寻找自噬mTOR、非mTOR通路中的关键信号分子靶点并加以阻断，探究神经元早、晚期损伤中自噬水平的变化引起神经细胞凋亡变化的信号分子机制，为HAND的防治提供全新的视角和新策略。

研究显示：因HAART治疗延长了艾滋病患者的生存期，40-60%的HIV感染者可出现HIV相关神经认知障碍(HAND)，其中起重要作用的包膜蛋白gp120在神经元损伤中的机制一直是研究的热点。本室及他人研究已证实gp120可致大鼠空间记忆障碍及海马神经元凋亡，预实验观察自噬在gp120致大鼠海马神经元损伤过程中的变化，发现损伤早期自噬增强；晚期自噬过度增强伴神经元大量凋亡，但机制不清。本研究采用形态学、行为学、分子生物学等技术，通过体内、外实验寻找自噬mTOR、非mTOR通路中的关键信号分子靶点并加以阻断，探究神经元早、晚期损伤中自噬水平的变化引起神经细胞凋亡变化的信号分子机制。. 研究首次发现：（1） HIV-1gp120V3环对原代培养的大鼠海马神经元损伤具有显著的时间依赖性，其在损伤过程中可出现自噬与凋亡；（2）HIV-1gp120V3环在损伤神经元过程中可出现早期6小时自噬增强及晚期72小时自噬过度增强，即早期自噬增强保护神经元，晚期自噬过度增强促神经元死亡；（3）在正常情况下，海马神经元即存在基本的自噬现象。无论6小时还是72小时时间点，均有mTOR通路中的ERK、AMPK及非mTOR通路中的calpain信号转导通路参与。信号分子ERK抑制自噬，促凋亡；AMPK促进自噬，抑凋亡；信号分子calpain抑制自噬，促凋亡。实验中以HIV-1gp120V3环作用于海马神经元，在早期自噬增强的过程中有ERK、AMPK及calpain三条信号转导通路参与，它们相互协调导致海马神经元自噬增强而凋亡率无明显变化。在晚期自噬增强的过程中仅有AMPK及calpain两条信号转导通路参与而未见ERK信号通路参与，可能由于ERK信号分子的失活导致海马神经元自噬过度增强通同时凋亡率明显升高。（4）侧脑室灌注HIV-1gp120V3环一天未造成大鼠认知功能障碍，只引起轻度海马自噬增强；连续灌注gp120V3环三天可造成大鼠认知功能障碍，海马自噬过度增强且伴凋亡。. 上述结果证实了我们提出的“自噬在gp120致海马神经元作用中早期起保护作用，晚期自噬过度促凋亡。这种早、晚期截然不同的损伤差异可能通过各自不同的自噬信号转导通路实现”的假说，为HAND的防治提供全新的视角和新策略。