glutaminase and HIV-Infections

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.. 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.. 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.. 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.

Topics: Aniline Compounds; Benzeneacetamides; Benzylidene Compounds; Brain; Calcium-Binding Proteins; Cell Cycle Proteins; Cells, Cultured; Central Nervous System; Ceramides; Dose-Response Relationship, Drug; Endosomal Sorting Complexes Required for Transport; Enzyme Inhibitors; Extracellular Vesicles; Glutamates; Glutaminase; Glutamine; HIV Infections; Humans; Lipopolysaccharides; Macrophages; Membrane Proteins; Microglia; Sulfides; Thiadiazoles

Aberrant excitatory neurotransmission associated with overproduction of glutamate has been implicated in the development of HIV-associated neurocognitive disorders (HAND). The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 14) attenuates glutamate synthesis in HIV-infected microglia/macrophages, offering therapeutic potential for HAND. We show that 14 prevents manifestation of spatial memory deficits in chimeric EcoHIV-infected mice, a model of HAND. 14 is not clinically available, however, because its development was hampered by peripheral toxicities. We describe the synthesis of several substituted N-(pivaloyloxy)alkoxy-carbonyl prodrugs of 14 designed to circulate inert in plasma and be taken up and biotransformed to 14 in the brain. The lead prodrug, isopropyl 6-diazo-5-oxo-2-(((phenyl(pivaloyloxy)methoxy)carbonyl)amino)hexanoate (13d), was stable in swine and human plasma but liberated 14 in swine brain homogenate. When dosed systemically in swine, 13d provided a 15-fold enhanced CSF-to-plasma ratio and a 9-fold enhanced brain-to-plasma ratio relative to 14, opening a possible clinical path for the treatment of HAND.

Topics: Aminocaproates; Animals; Azo Compounds; Blood; Brain; Diazooxonorleucine; Drug Stability; Female; Glutamic Acid; Glutaminase; HIV Infections; Humans; Male; Mice, Inbred C57BL; Neurocognitive Disorders; Nootropic Agents; Prodrugs; Swine; Viral Load

HIV-1 associated neurocognitive disorders (HAND) develop during progressive HIV-1 infection and affect up to 50% of infected individuals. Activated microglia and macrophages are critical cell populations that are involved in the pathogenesis of HAND, which is specifically related to the production and release of various soluble neurotoxic factors including glutamate. In the central nervous system (CNS), glutamate is typically derived from glutamine by mitochondrial enzyme glutaminase. Our previous study has shown that glutaminase is upregulated in HIV-1 infected monocyte-derived-macrophages (MDM) and microglia. However, how HIV-1 leads to glutaminase upregulation, or how glutaminase expression is regulated in general, remains unclear. In this study, using a dual-luciferase reporter assay system, we demonstrated that interferon (IFN) α specifically activated the glutaminase 1 (GLS1) promoter. Furthermore, IFN-α treatment increased signal transducer and activator of transcription 1 (STAT1) phosphorylation and glutaminase mRNA and protein levels. IFN-α stimulation of GLS1 promoter activity correlated to STAT1 phosphorylation and was reduced by fludarabine, a chemical that inhibits STAT1 phosphorylation. Interestingly, STAT1 was found to directly bind to the GLS1 promoter in MDM, an effect that was dependent on STAT1 phosphorylation and significantly enhanced by IFN-α treatment. More importantly, HIV-1 infection increased STAT1 phosphorylation and STAT1 binding to the GLS1 promoter, which was associated with increased glutamate levels. The clinical relevance of these findings was further corroborated with investigation of post-mortem brain tissues. The glutaminase C (GAC, one isoform of GLS1) mRNA levels in HIV associated-dementia (HAD) individuals correlate with STAT1 (p<0.01), IFN-α (p<0.05) and IFN-β (p<0.01). Together, these data indicate that both HIV-1 infection and IFN-α treatment increase glutaminase expression through STAT1 phosphorylation and by binding to the GLS1 promoter. Since glutaminase is a potential component of elevated glutamate production during the pathogenesis of HAND, our data will help to identify additional therapeutic targets for the treatment of HAND.

Topics: AIDS Dementia Complex; Animals; Base Sequence; Brain; Cells, Cultured; Dose-Response Relationship, Drug; Gene Expression Regulation; Glutamic Acid; Glutaminase; HEK293 Cells; HIV Infections; HIV-1; Host-Pathogen Interactions; Humans; Interferon-alpha; Isoenzymes; Macrophages; Molecular Sequence Data; Monocytes; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Rats; Reverse Transcriptase Polymerase Chain Reaction; STAT1 Transcription Factor

Human immunodeficiency virus (HIV) induces a neurological disease culminating in frank dementia referred to as HIV-associated dementia (HAD). Neurotoxins from HIV-1-infected and activated mononuclear phagocytes contribute to the neuropathogenesis of HAD. Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS) and functions through activation of multiple receptors. Excessive glutamate production by HIV-infected macrophages in HAD may contribute to neuronal injury. Our previous studies have suggested that mitochondrial glutaminase is responsible for the excessive production of glutamate. However, how HIV-1 infection regulates glutamate over-production remains unclear. In this study, we propose that HIV infection-induced oxidative stress contributes to mitochondrial glutaminase release, which results in the excessive production of glutamate and subsequent neuronal injury. We collected conditioned media from HIV-1 infected macrophages and analyzed glutamate concentration in the media by RP-HPLC, and found that the cyclosporine A (CsA), an inhibitor of HIV-1 replication and mitochondrial permeability transition pore, and N-acetylcysteine (NAC), a remover of reactive oxygen species (ROS), not only blocked the excessive glutamate production, but also decreased the glutamate-mediated neurotoxicity. In addition, HIV-infection-induced ROS generation was accompanied with the excessive glutamate production, suggesting that oxidative stress was involved in glutamate regulation. Using the isolated rat brain mitochondria as an ex vivo model and over-expressing GFP-glutaminase fusion protein in mammalian cells as a cell model, we confirm oxidative stress-mediated mitochondrial glutaminase release during HIV-1 infection contributes to glutamate over-production and the subsequent neurotoxicity. These results may provide insight into HAD pathogenesis and a therapeutic strategy for HAD treatment.

Topics: Animals; Cells, Cultured; Cerebral Cortex; Fetus; Glutamic Acid; Glutaminase; HIV Infections; HIV-1; Humans; Mitochondria; Oxidative Stress; Rats; Rats, Sprague-Dawley

Microglia represent the main cellular targets of HIV-1 in the brain. Infected and/or activated microglia play a pathogenic role in HIV-associated neurocognitive disorders (HAND) by instigating primary dysfunction and subsequent death of neurons. Although microglia are known to secrete neurotoxins when infected with HIV-1, the detailed mechanism of neurotoxicity remains unclear. Using a human microglia primary culture system and macrophage-tropic HIV-1 strains, we have now demonstrated that HIV-1 infection of microglia resulted in a significant increase in extracellular glutamate concentrations and elevated levels of neurotoxicity. RNA and protein analysis revealed upregulation of the glutamate-generating enzyme glutaminase isoform glutaminase C in HIV-1-infected microglia. The clinical relevance of these findings was further corroborated with investigation of postmortem brain tissues. The glutaminase C levels in the brain tissues of HIV dementia individuals were significantly higher than HIV serum-negative control and correlated with elevated concentrations of glutamate. When glutaminase was subsequently inhibited by siRNA or by a small molecular inhibitor, the HIV-induced glutamate production and the neuronal loss was diminished. In conclusion, these findings support glutaminase as a potential component of the HAND pathogenic process as well as a novel therapeutic target in their treatment.

Topics: Analysis of Variance; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Brain; Caspase 3; Cells, Cultured; Chromatography, High Pressure Liquid; Disintegrins; Dizocilpine Maleate; Enzyme-Linked Immunosorbent Assay; Excitatory Amino Acid Antagonists; Fetus; Gene Expression Regulation, Viral; Glutamic Acid; Glutaminase; Glutamine; HIV Infections; HIV-1; Humans; Microglia; Microtubule-Associated Proteins; Receptors, Cell Surface; RNA, Small Interfering; Tetrazolium Salts; Thiazoles; Time Factors

Mononuclear phagocyte (MP, macrophages and microglia) dysfunction plays a significant role in the pathogenesis of HIV-1-associated dementia (HAD) through the production and release of soluble neurotoxic factors including glutamate. Glutamate production is greatly increased following HIV-1 infection of cultured MP, a process dependent upon the glutamate-generating enzyme glutaminase. Glutaminase inhibition was previously found to significantly decrease macrophage-mediated neurotoxicity. Potential mechanisms of glutaminase-mediated excitotoxicity including enzyme up-regulation, increased enzyme activity and glutaminase localization were investigated in this report. RNA and protein analysis of HIV-infected human primary macrophage revealed up-regulation of the glutaminase isoform GAC, yet identified no changes in the kidney-type glutaminase isoform over the course of infection. Glutaminase is a mitochondrial protein, but was found to be released into the cytosol and extracellular space following infection. This released enzyme is capable of rapidly converting the abundant extracellular amino acid glutamine into excitotoxic levels of glutamate in an energetically favorable process. These findings support glutaminase as a potential component of the HAD pathogenic process and identify a possible therapeutic avenue for the treatment of neuroinflammatory states such as HAD.

Topics: AIDS Dementia Complex; Cells, Cultured; Glutamic Acid; Glutaminase; HIV Infections; HIV-1; Humans; Macrophages

Dysfunction in mononuclear phagocyte (MP, macrophages and microglia) immunity is thought to play a significant role in the pathogenesis of HIV-1 associated dementia (HAD). In particular, elevated extracellular concentrations of the excitatory neurotransmitter glutamate, produced by MP as a consequence of viral infection and immune activation, can induce neuronal injury. To determine the mechanism by which MP-mediated neuronal injury occurs, the concentration and rates of production of extracellular glutamate were measured in human monocyte-derived macrophage (MDM) supernatants by reverse phase high-performance liquid chromatography (RP-HPLC). Measurements were taken of supernatants from MDM infected with multiple HIV-1 strains including ADA and DJV (macrophage tropic, M-tropic), and 89.6 (dual tropic). High levels of glutamate were produced by MDM infected with M-tropic viruses. AZT, an inhibitor of HIV-1 replication, inhibited glutamate generation, demonstrating a linkage between HIV-1 infection and enhanced glutamate production. In our culture system, glutamate production was dependent upon the presence of glutamine and was inhibited by 6-diazo-5-oxo-L-norleucine, a glutaminase inhibitor. Supernatants collected from HIV-1-infected MP generated more glutamate following glutamine addition than supernatants isolated from uninfected MP. These findings implicate the involvement of a glutamate-generating enzyme, such as phosphate-activated mitochondrial glutaminase (PMG) in MP-mediated glutamate production.

Topics: AIDS Dementia Complex; Anti-HIV Agents; Cell Survival; Cells, Cultured; Chromatography, High Pressure Liquid; Culture Media, Conditioned; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Space; Glutamic Acid; Glutaminase; Glutamine; HIV Infections; HIV-1; Humans; Macrophages; Mitochondria; Monocytes; RNA, Messenger; Zidovudine