BACKGROUND Extracellular vesicles (EVs) are nano-sized particles present in most body fluids including cerebrospinal fluid (CSF). Little is known about CSF EV proteins in HIV+ individuals. Here, we characterize the CSF EV proteome in HIV+ subjects and its relationship to neuroinflammation, stress responses, and HIV-associated neurocognitive disorders (HAND). METHODS CSF EVs isolated from 20 HIV+ subjects with (n = 10) or without (n = 10) cognitive impairment were characterized by electron microscopy, nanoparticle tracking analysis, immunoblotting, and untargeted LC/MS/MS mass spectrometry. Functional annotation was performed by gene ontology (GO) mapping and expression annotation using Biobase Transfac and PANTHER software. Cultured astrocytic U87 cells were treated with hydrogen peroxide for 4 h to induce oxidative stress and EVs isolated by ultracentrifugation. Selected markers of astrocytes (GFAP, GLUL), inflammation (CRP), and stress responses (PRDX2, PARK7, HSP70) were evaluated in EVs released by U87 cells following induction of oxidative stress and in CSF EVs from HIV+ patients by immunoblotting. RESULTS Mass spectrometry identified 2727 and 1626 proteins in EV fractions and EV-depleted CSF samples, respectively. CSF EV fractions were enriched with exosomal markers including Alix, syntenin, tetraspanins, and heat-shock proteins and a subset of neuronal, astrocyte, oligodendrocyte, and choroid plexus markers, in comparison to EV-depleted CSF. Proteins related to synapses, immune/inflammatory responses, stress responses, metabolic processes, mitochondrial functions, and blood-brain barrier were also identified in CSF EV fractions by GO mapping. HAND subjects had higher abundance of CSF EVs and proteins mapping to GO terms for synapses, glial cells, inflammation, and stress responses compared to those without HAND. GFAP, GLUL, CRP, PRDX2, PARK7, and HSP70 were confirmed by immunoblotting of CSF EVs from subjects with HAND and were also detected in EVs released by U87 cells under oxidative stress. CONCLUSIONS These findings suggest that CSF EVs derived from neurons, glial cells, and choroid plexus carry synaptic, immune/inflammation-related, and stress response proteins in HIV+ individuals with cognitive impairment, representing a valuable source for biomarker discovery.

中文翻译：

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对脑脊液细胞外囊泡的蛋白质组学分析显示，HIV认知障碍患者的突触损伤，炎症和应激反应标志物。

背景技术细胞外囊泡（EVs）是存在于包括脑脊髓液（CSF）在内的大多数体液中的纳米级颗粒。对HIV +个体中的CSF EV蛋白知之甚少。在这里，我们表征了HIV +受试者中的CSF EV蛋白质组及其与神经炎症，压力反应以及与HIV相关的神经认知障碍（HAND）的关系。方法通过电子显微镜，纳米粒子跟踪分析，免疫印迹和非靶向LC / MS / MS质谱对从20名具有（n = 10）或无（n = 10）认知障碍的HIV +受试者中分离出的CSF电动汽车进行表征。使用Biobase Transfac和PANTHER软件通过基因本体（GO）定位和表达注释进行功能注释。培养的星形胶质细胞U87细胞用过氧化氢处理4 h，以诱导氧化应激和通过超速离心分离的EV。在诱导氧化应激后U87细胞释放的电动汽车中以及通过免疫印迹从HIV +患者中获得的CSF电动汽车中评估星形胶质细胞（GFAP，GLUL），炎症（CRP）和应激反应（PRDX2，PARK7，HSP70）的选定标志物。结果质谱分别鉴定了EV馏分和EV耗尽的CSF样品中的2727和1626蛋白。与EV耗竭的CSF相比，CSF EV馏分富含外泌体标志物，包括Alix，Syntenin，四跨膜蛋白和热休克蛋白，以及一部分神经元，星形胶质细胞，少突胶质细胞和脉络丛标志物。与突触，免疫/炎症反应，应激反应，代谢过程有关的蛋白质，通过GO作图还可以确定脑脊液EV级分中的线粒体功能和血脑屏障。与没有HAND的受试者相比，HAND受试者的CSF EV和映射到GO术语的蛋白具有更高的突触，神经胶质细胞，炎症和应激反应。GFAP，GLUL，CRP，PRDX2，PARK7和HSP70已通过对具有HAND的受试者的CSF EV进行免疫印迹得到了证实，并且在氧化应激下由U87细胞释放的EV中也得到了检测。结论这些发现表明，源自神经元，神经胶质细胞和脉络丛的CSF EV在具有认知障碍的HIV +个体中携带突触，免疫/炎症相关和应激反应蛋白，代表了生物标志物发现的宝贵来源。与没有HAND的受试者相比，HAND受试者具有更高的CSF EV和映射到GO项的突触，神经胶质细胞，炎症和应激反应蛋白。GFAP，GLUL，CRP，PRDX2，PARK7和HSP70已通过对具有HAND的受试者的CSF EV进行免疫印迹得到了证实，并且在氧化应激下由U87细胞释放的EV中也得到了检测。结论这些发现表明，源自神经元，神经胶质细胞和脉络丛的CSF EV在具有认知障碍的HIV +个体中携带突触，免疫/炎症相关和应激反应蛋白，代表了生物标志物发现的宝贵来源。与没有HAND的受试者相比，HAND受试者具有更高的CSF EV和映射到GO项的突触，神经胶质细胞，炎症和应激反应蛋白。GFAP，GLUL，CRP，PRDX2，PARK7和HSP70已通过对具有HAND的受试者的CSF EV进行免疫印迹得到了证实，并且在氧化应激下由U87细胞释放的EV中也得到了检测。结论这些发现表明，源自神经元，神经胶质细胞和脉络丛的CSF EV在具有认知障碍的HIV +个体中携带突触，免疫/炎症相关和应激反应蛋白，代表了生物标志物发现的宝贵来源。HSP70和HSP70可以通过免疫印迹法从患有HAND的受试者身上获得证实，并且还可以在氧化应激下由U87细胞释放的EVs中检测到。结论这些发现表明，源自神经元，神经胶质细胞和脉络丛的CSF EV在具有认知障碍的HIV +个体中携带突触，免疫/炎症相关和应激反应蛋白，代表了生物标志物发现的宝贵来源。HSP70和HSP70可以通过免疫印迹法从患有HAND的受试者身上获得证实，并且还可以在氧化应激下由U87细胞释放的EVs中检测到。结论这些发现表明，源自神经元，神经胶质细胞和脉络丛的CSF EV在具有认知障碍的HIV +个体中携带突触，免疫/炎症相关和应激反应蛋白，代表了生物标志物发现的宝贵来源。