Sami Saribas A, Cicalese Stephanie, Ahooyi Taha Mohseni, Khalili Kamel, Amini Shohreh, Sariyer Ilker Kudret
Department of Neuroscience, Center for Neurovirology, Lewis Katz School of Medicine, Temple University, Philadelphia, 19140 PA, USA.
Cell Death Dis. 2017 Jan 12;8(1):e2542. doi: 10.1038/cddis.2016.467.
Human immunodeficiency virus-associated neurological disorders (HANDs) affect the majority of AIDS patients and are a significant problem among HIV-1-infected individuals who live longer because of combined anti-retroviral therapies. HIV-1 utilizes a number of viral proteins and subsequent cytokine inductions to unleash its toxicity on neurons. Among HIV-1 viral proteins, Nef is a small protein expressed abundantly in astrocytes of HIV-1-infected brains and has been suggested to have a role in the pathogenesis of HAND. In order to explore its effect in the central nervous system, HIV-1 Nef was expressed in primary human fetal astrocytes (PHFAs) using an adenovirus. Our results revealed that HIV-1 Nef is released in extracellular vesicles (EVs) derived from PHFA cells expressing the protein. Interestingly, HIV-1 Nef release in EVs was enriched significantly when the cells were treated with autophagy activators perifosine, tomaxifen, MG-132, and autophagy inhibitors LY294002 and wortmannin suggesting a novel role of autophagy signaling in HIV-1 Nef release from astrocytes. Next, Nef-carrying EVs were purified from astrocyte cultures and neurotoxic effects on neurons were analyzed. We observed that HIV-1 Nef-containing EVs were readily taken up by neurons as demonstrated by immunocytochemistry and immunoblotting. Furthermore, treatment of neurons with Nef-carrying EVs induced oxidative stress as evidenced by a decrease in glutathione levels. To further investigate its neurotoxic effects, we expressed HIV-1 Nef in primary neurons by adenoviral transduction. Intracellular expression of HIV-1 Nef caused axonal and neurite degeneration of neurons. Furthermore, expression of HIV-1 Nef decreased the levels of phospho-tau while enhancing total tau in primary neurons. In addition, treatment of primary neurons with Nef-carrying EVs suppressed functional neuronal action potential assessed by multielectrode array studies. Collectively, these data suggested that HIV-1 Nef can be a formidable contributor to neurotoxicity along with other factors, which leads to HAND in HIV-1-infected AIDS patients.
人类免疫缺陷病毒相关神经障碍（HANDs）影响大多数艾滋病患者，并且在因联合抗逆转录病毒疗法而寿命延长的HIV-1感染者中是一个重大问题。HIV-1利用多种病毒蛋白以及随后的细胞因子诱导来对神经元释放其毒性。在HIV-1病毒蛋白中，Nef是一种在HIV-1感染大脑的星形胶质细胞中大量表达的小蛋白，并且已被认为在HAND的发病机制中起作用。为了探究其在中枢神经系统中的作用，使用腺病毒在原代人胎儿星形胶质细胞（PHFAs）中表达HIV-1 Nef。我们的结果显示，HIV-1 Nef从表达该蛋白的PHFA细胞衍生的细胞外囊泡（EVs）中释放出来。有趣的是，当用自噬激活剂哌立福辛、托瑞米芬、MG-132以及自噬抑制剂LY294002和渥曼青霉素处理细胞时，EVs中HIV-1 Nef的释放显著增加，这表明自噬信号在HIV-1 Nef从星形胶质细胞释放中具有新作用。接下来，从星形胶质细胞培养物中纯化携带Nef的EVs，并分析其对神经元的神经毒性作用。我们观察到，通过免疫细胞化学和免疫印迹证明，含HIV-1 Nef的EVs很容易被神经元摄取。此外，用携带Nef的EVs处理神经元会诱导氧化应激，谷胱甘肽水平降低就是证据。为了进一步研究其神经毒性作用，我们通过腺病毒转导在原代神经元中表达HIV-1 Nef。HIV-1 Nef的细胞内表达导致神经元的轴突和神经突退化。此外，HIV-1 Nef的表达降低了原代神经元中磷酸化tau的水平，同时增加了总tau的水平。另外，用携带Nef的EVs处理原代神经元会抑制通过多电极阵列研究评估的功能性神经元动作电位。总体而言，这些数据表明，HIV-1 Nef与其他因素一起可能是导致HIV-1感染的艾滋病患者发生HAND的神经毒性的一个重要因素。
