Glutaminase 1 regulates the release of extracellular vesicles during neuroinflammation through key metabolic intermediate alpha-ketoglutarate

Abstract

BackgroundExtracellular 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.MethodsHuman 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.ResultsOur 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.ConclusionsThese 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.Electronic supplementary materialThe online version of this article (10.1186/s12974-018-1120-x) contains supplementary material, which is available to authorized users.

Propofol reduces microglia activation and neurotoxicity through inhibition of extracellular vesicle release

“…EV release can be modulated by reactive oxygen species (Blanc et al, 2007), inflammation (Bianco et al, 2005), and by ATP (Savina et al, 2003). Our previous work found that immune activation induced by LPS or TNF-α can increase EV release from microglia and astrocytes, Wu et al, 2015;Wu et al, 2018). In the current study, we used LPS as the inflammationinducing agent for the microglia.…”

“…EVs can be found in interstitial fluid, cerebrospinal fluid (Vella et al, 2008), circulating blood (Suetsugu et al, 2013), and primarily assert biological function through delivering cytokines, nucleic acids, lipids, and proteins to nearby or distant cells, suggesting that they play an important role in cell-to-cell communication (Thery et al, 2006). EV secretion is increased during neuroinflammation; our previous studies, along with others', have suggested that EVs fuel pathogenic processes in the brain during neuroinflammation (Gupta and Pulliam, 2014;Huang et al, 2018;Wang et al, 2017;Wu et al, 2015;Wu et al, 2018).…”

“…EVs were isolated from the serum-free culture medium of BV2 cells by a differential centrifugation protocol described previously using the same BV2 cells (Suetsugu et al, 2013;Wu et al, 2015;Wu et al, 2018). Briefly, the supernatants were first centrifuged at 500 ×g for 5 min to remove the free cells, followed by 2000 ×g for 20 min to remove cellular debris, and then at 10,000 ×g for 30 min to remove intracellular organelles.…”

Glutaminase 1 Regulates Neuroinflammation After Cerebral Ischemia Through Enhancing Microglial Activation and Pro-Inflammatory Exosome Release

“…Because our previous findings indicate that GLS1 is a key enzyme in exosomes release of glial cells including microglia and astrocytes in vitro (16,17), we examined whether or not the alteration of exosome release in focal cerebral ischemic brains is mediated by GLS1. NTA demonstrated that GLS1 inhibition by CB839 administration significantly reduced the concentration of exosome in ischemic rat brains vs. the DMSO-treated ischemia group (Figure 5A).…”

Neurodegenerative Susceptibility During Maternal Nutritional Programing: Are Central and Peripheral Innate Immune Training Relevant?

“…For instance, in murine models, intracerebral injection of IL-1β promotes the release of extracellular vesicles from astrocytes, leading to leukocyte migration into the brain and regulation of the liver cytokines profile by inhibition of the peroxisome proliferator-activated receptor α (Dickens et al, 2017). It is important to mention that extracellular vesicles released by central immune activation depend on glutamine modulation by glutaminase enzyme activation (Dickens et al, 2017;Wang et al, 2017;Wu et al, 2018). In fact, substantial programing of metabolic related pathways coordinates innate immunity.…”