Latent HIV‐1 Exosomes Induce Mitochondrial Hyperfusion due to Loss of Phosphorylated Dynamin‐related Protein 1 in Brain Endothelium

Abstract

Background Damage to the cerebral vascular endothelium is a critical initiating event in the development of HIV-1-associated neurocognitive disorders (HAND). To study the role of mitochondria in cerebral endothelial dysfunction, we investigated how exosomes, isolated from both cell lines with integrated provirus and HIV-1 infected primary cells (HIV-exosomes), accelerate the dysfunction of either primary human brain microvascular endothelial cells (HBMVECs) in vitro or mice brain microvessels (MVs), ex vivo. Methods Exosomes were isolated by either ultracentrifugation from uninfected and latent HIV-1 infected T-cells (Jurkat Exo and J-Lat(9.2) Exo, respectively) and monocytes (U937 Exo and U1 Exo, respectively) or by using QIAGEN exoEasy Maxi kit from HIV-1 infected human peripheral blood mononuclear cells (HIV+PBMCs Exo). Exosomes were characterized by the qNano-IZON system. Initially, the assessment of mitochondrial characteristics was performed in primary HBMVECs, and this study was further extended in extracted cerebral MVs from mice brain expressing mito-Dendra2+ fluorescence protein (Green) in endothelial mitochondria. Results Quantitative analysis of the extracellular vesicles (EVs) indicates that the isolated EVs were predominantly exosomes. This was further supported by the detection of exosomal markers, e.g. CD63, CD81, Alix, TSG101 and the absence of large EV-related protein, GRP94. These exosomes were readily taken up by both primary HBMVECs and mice cerebral MVs. HIV-exosomes induced cellular and mitochondrial superoxide production but reduced mitochondrial membrane potential in HBMVECs. HIV-exosomes increased mitochondrial hyperfusion, possibly due to the loss of phosphorylated dynamin-related protein 1 (p-DRP1). We further confirmed that HIV-exosomes, containing the HIV-Tat protein, and viral Tat protein accelerate brain endothelial dysfunction due to reduction in the expressions of (a) p-DRP1, (b) phosphorylated endothelial nitric oxide synthase (p-eNOS), and (c) blood brain barrier related tight junction proteins. Finally, HIV-1 infected primary human PBMCs produced more exosomes than uninfected controls, and exposure to HIV+PBMCs Exo also reduced both p-DRP1 and p-eNOS expressions in primary HBMVECs. Conclusions Our novel findings reveal the significant role of HIV-exosomes on dysregulation of mitochondrial function, which induces adverse changes in the function of the brain microvascular endothelium. These mechanistic studies using HIV-exosomes may elucidate the etiology of HAND and provide novel therapeutic strategies.