Background: Extracellular vesicles (EVs) are important in the intercellular communication of the central nervous system, and their release is increased during neuroinflammation. Our previous data demonstrated an increased release of EVs during HIV-1 infection and immune activation in glial cells. However, the molecular mechanism by which infection and inflammation increase EV release remains unknown. In the current study, we investigated the role of glutaminase 1 (GLS1)-mediated glutaminolysis and the production of a key metabolic intermediate α-ketoglutarate on EV release.
Methods: Human monocyte-derived macrophage primary cultures and a BV2 microglia cell line were used to represent the innate immune cells in the CNS. Transmission electron microscopy, nanoparticle tracking analysis, and Western blots were used to determine the EV regulation. GLS1 overexpression was performed using an adenovirus vector in vitro and transgenic mouse models in vivo. Data were evaluated statistically by ANOVA, followed by the Bonferroni post-test for paired observations.
Results: Our data revealed an increased release of EVs in GLS1-overexpressing HeLa cells. In HIV-1-infected macrophages and immune-activated microglia BV2 cells, treatment with bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) or CB839, two specific GLS inhibitors, significantly decreased EV release, suggesting a critical role of GLS1 in EV release. Furthermore, addition of α-ketoglutarate or ceramide rescued EV release during BPTES treatment, implicating α-ketoglutarate and ceramide as critical downstream effectors for GLS inhibitors. These findings were further corroborated with the investigation of brain tissues in GLS1-transgenic mice. The EV levels were significantly higher in GLS1 transgenic mice than those in control mice, suggesting that GLS1 increases EV release in vivo.
Conclusions: These findings suggest that GLS1-mediated glutaminolysis and its downstream production of α-ketoglutarate are essential in regulating EV release during HIV-1 infection and immune activation. These new mechanistic regulations may help understand how glutamine metabolism shapes EV biogenesis and release during neuroinflammation.
Keywords: Extracellular vesicles; Glutamine metabolism; HIV-1; Inflammation; α-Ketoglutarate.