Latent‐HIV‐1 Exosome Blockade Mitophagy Flux in Human Brain Microvascular Endothelial Cells

Abstract

BackgroundInsufficient removal of dysfunctional mitochondria, due to a defective mitophagy pathway, leads to increased ROS, oxidative stress, and neuroinflammation which triggers the pathogenesis of various neurodegenerative diseases. HIV‐1‐associated neurocognitive disorders (HAND) is a common disease with variable reported prevalence in different populations (20– 69%). We investigated how exosomes from latent‐HIV‐1 infected cells impaired mitophagy flux in the human brain microvascular endothelial cells (HBMVEC).MethodsExosomes were isolated by ultracentrifugation from latent‐HIV‐1 infected T‐cells [exo‐J Lat(9.2)] and monocytes (exo‐U1) and characterized by the qNano‐IZON and NanoSight 300 systems by measuring the concentrations (3.2×1012 particles/mL), particle diameter (Mean: 100.5±28.8 nm), and size‐distribution (Mode: 75.5±3.5 nm) of exosomes from J Lat(9.2). To determine whether exposure to exo‐J Lat(9.2) could induce mitophagy, HBMVECs were exposed to varying doses of HIV‐exosomes (10, 25, 50 and 100 μg/mL) for 24 and 48 h, after which the expression level of mitophagy markers PINK1, Parkin, and Drp1 were determined using western blotting. Carbonyl cyanide 4‐(trifluoromethoxy) phenylhydrazone (FCCP; 5 μM for 3 h), a known mitophagy inducer, was used as a positive control.ResultsMitophagy is a two‐step process by which damaged mitochondria are first primed by PINK1/Parkin proteins and then eliminated via the autophagic machinery. We then examined the expression levels of autophagy (macroautophagy) proteins: the autophagosome initiation marker (Beclin 1), autophagosome formation marker (LC3B‐II), and autophagy degradation marker (SQSTM1 or p62) in HBMVECs exposed to latent‐HIV‐1‐exosomes. Interestingly, following exposure of HBMVECs to exo‐J Lat(9.2), the expression level of PINK1/Parkin proteins were upregulated in a dose dependent manner, with a concomitant increase in the formation of autophagosomes, shown by increased expression of Beclin 1 and LC3B‐II. FCCP treated cells showed decreased expression of p62 indicated by the active presence of mitophagy flux. Remarkably, cell exposure to HIV‐exosome resulted in increased expression of p62, indicating a possible blockage of the mitophagy flux, leading to the accumulation of mitophagosomes.ConclusionsOur preliminary study indicated that latent‐HIV‐1 exosome increased mitochondrial damage via defective mitophagy.Support or Funding InformationGrant Numbers: U54 GM104940, AHA 17SDG33410366, HL‐093554.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.