Background
HIV-1-associated neurocognitive disorders (HAND) are currently prevalent in spite of major advances in combination anti-retroviral therapy. Therefore, novel therapeutic targets are required to be developed to treat the disease [
1‐
4]. The HIV-1-infected and immune-activated mononuclear phagocytes (MPs, including macrophages and microglia) are critical to HAND pathogenesis, producing a variety of inflammatory and neurotoxic factors, including excess levels of the excitatory neurotransmitter glutamate [
5,
6]. Glutamate is a major mediator of excitatory synaptic transmission and has a vital role in mediating learning and memory [
7‐
9]. Early studies reported that the concentrations of glutamate in the plasma and cerebrospinal fluid were significantly higher in HIV-1-infected patients than in uninfected controls [
10‐
13]. Studies also showed that excessive levels of extracellular glutamate induce excitotoxicity and augment neuroinflammation and neuronal injury, which may play a role in the pathogenesis of HAND [
14‐
19].
Glutaminase (GLS), a resident mitochondria enzyme, is specialized in the
de novo synthesis of the neurotransmitter glutamate [
20‐
24]. Two major types of GLS exist in mammals, which include “kidney-type” (GLS1) and “liver-type” (GLS2) transcribed from different genes. GLS has also been found to be abundant in the brain tissue [
15]. In human brain, GLS1 has two allozymes: kidney-type glutaminase (KGA) [
25] and glutaminase C (GAC) [
26]. The allozymes are generated through tissue-specific alternative splicing from the same gene and have the identical core GLS1 enzyme domain but different 3′ tails [
27]. Our previous studies suggested that GAC and KGA are differentially upregulated in HAND brain samples, HIV-1-infected MPs and inflammatory neurons [
6,
28‐
31]. Increased extracellular levels of glutamate from activated MPs could cause excitotoxicity to neurons through NMDA receptor activation [
28,
29]. Therefore, regulation of GLS1 isoforms is of importance to HAND research. A key molecular event associated with the elevation of glutamate is the release of GLS1 [
28,
30,
31]. Although several early observations of GLS1 release were linked with cell death, more recent data from our lab suggested that mitochondrial stress could lead to membrane destabilization and relocation of GLS1 from the mitochondrial matrix to the cytosol through the permeability transition pore [
32]. Because further release of GLS1 into extracellular supernatants contributes to excess glutamate production, it is imperative to understand the molecular mechanism of cellular GLS1 release.
Recent evidence indicates that microvesicles (MVs), unconventional cellular secretory vesicles, are shed from the plasma membrane and range from 100 nm to 1 μm in diameter [
33]. Interestingly, MVs are abundant in the central nervous system (CNS) and are derived from multiple brain cell types, including neurons, microglia, oliogodendrocytes, and astrocytes [
34]. Therefore, there is a growing appreciation of the important role of MVs in regulating the brain microenvironment [
35,
36]. CNS-derived MVs may contribute to neuroinflammation through secretion of signaling molecules, nucleic acids, lipids, and proteins, and may participate in inter- and intra-cellular communication [
33,
37‐
42]. Release of MVs is increased upon neural cancer progression, neuroinflammation, and acute neurological disorders. MVs could serve as a useful biomarker for CNS diseases including ischemic stroke, multiple sclerosis, glioblastoma, and other neurological and neurodegenerative disorders [
43‐
45]. However, the role of MVs in the pathogenesis of neurodegenerative disorders, especially HAND, remains to be elucidated. In our current study, we identified MVs as a primary mechanism of GLS1 release, which subsequently mediates excess glutamate generation and neurotoxicity from HIV-1-infected macrophages and immune-activated microglia. The investigation of the function of GLS1-containing MVs is important for understanding a potentially pathological event in HAND, and it may provide possible therapeutic targets and a unique biomarker.
Discussion
Our previous reports have described the release of GLS1 into extracellular space during neuroinflammation or HIV-1 infection. However, key molecular mechanisms that regulate GLS1 release remain unknown. Our current study presents two important new findings regarding GLS1 release. First, MVs contain GLS1, which is a key enzyme for generating glutamate in the brain, and GLS1 is released into the extracellular fluid primarily via MVs in HIV-1-infected cells and immune-activated microglia. Second, HIV-1 infection and LPS activation increase the magnitude of MV release from macrophages and microglia. Interestingly, increased release of GLS1-containing MVs also induces excitotoxicity in RCN. The toxic effect of MVs was reversed by glutaminase inhibitors and MV inhibitors. These observations suggest that MVs may contribute to excess glutamate production in macrophages in the context of HIV-1 neurotoxicity.
The physiological relevance of this observation is significant, where elevated endogenous levels of GLS1 have been reported in the post mortem brain tissues of HIV-1-associated dementia patients [
29,
49]. Furthermore, it has been demonstrated that both of the upregulated GLS1 isoforms, KGA and GAC, are released from the inner membrane of mitochondria into the cytosol through the permeability transition pore [
31,
32]. However, it is the extracellular glutamate that causes neurotoxicity, and the mechanisms by which cytosolic GLS1 is released into the cell supernatant have not been previously established. The current study provides strong evidence that GLS1-containing MVs are the main mechanism for the release of GLS1 from the macrophage and microglia cytosol into the extracellular compartment, where the extracellular glutamate subsequently induces neurotoxicity. It is unclear how GLS1 hydrolyzes extracellular glutamine inside MVs. We have detected vesicular glutamate transporter in the MV lysates from HIV-1 infected MDM (Additional file
1: Figure S1), suggesting that MVs may transport glutamate across its lipid bilayer through glutamate transporters.
Our studies were designed to rigorously establish the purity and characterization of the isolated microvesicles. To confirm complete separation of MVs from mitochondria, the absence of the mitochondrial marker protein voltage-dependent anion-selective channel and cytochrome C was confirmed through Western blots (data not shown). Secondly, to characterize the MVs, both scanning and transmission electron microscopies were used, which showed vesicles ranging in size from 100 nm to 500 nm. Western blot could not detect the presence of GLS1 in exosomes collected from commercially available exosome kits, suggesting that GLS1 is selectively packed into MVs rather than into smaller exosomes. Furthermore, because the MV-free supernatants and the pellets that contained cellular debris had minimum GLS activities and GLS1 was found predominantly in the isolated MVs fraction, we concluded that MVs are the primary instigator in facilitating GLS1 release in HIV-1-infected macrophages and LPS-activated microglia. Chronic activation of the immune system is a hallmark of progressive HIV infection yet its etiology remains obscure. Circulating microbial products such as LPS, possibly derived from the gastrointestinal tract, was significantly increased in chronically HIV-infected individuals and may be a cause of HIV-related systemic immune activation [
50]. In our report, when LPS was used to treat BV2 microglia cell line, high levels of GLS1 were found in the supernatants leading to neurotoxicity. These results support the pathogenic effect of the immune activation in the CNS during HIV-1 infection. The discovery that immune activation induces GLS1 release via MVs may have a broader implication to other neurological diseases, where excitotoxicity and GLS1 are involved. Because MVs are abundantly expressed in the CNS, it is tempting to speculate whether qualitative or quantitative changes of MVs contribute more broadly to neurological diseases. In addition, the regulation of MVs could be exploited as a novel therapeutic target.
In the current study, we also utilized a MV inhibitor, GW4869, to block the release of the MVs. Both GLS1 and MV markers were decreased in a dose-dependent manner by GW4869, suggesting that GLS1 release is through MVs. GW4869 is an inhibitor for nSMase2, which is responsible for the production of ceramide. Ceramide has been found enriched in MVs and involved in the formation of vesicles. Our results point to a possible mechanism of MVs release in the microglia and macrophages through the endosomal sorting complex required for transport (ESCRT) machinery or a ceramide-dependent pathway [
47,
48,
51]. These interesting possibilities remain the subject of future investigation. Furthermore, we have demonstrated that the neurotoxicity by HIV-1-infected MDM was abolished by pre-treatment with GW4869, which indicates that GLS1-containing MVs are the neurotoxic factors in HIV-1-infected macrophages and immune-activated microglia. Therefore, inhibiting the release of MVs might become a potential therapeutic approach for the treatment of HAND patients.
Three aspects of the studies merit further investigation. First, how is cytosolic GLS1 loaded into MVs for extracellular secretion from macrophages? Second, how are GLS1-containing MVs formed? Third, what mediates the release of GLS1-containing MVs from the plasma membrane? Autophagosomes, inflammasomes or mitochondria-derived vesicles may provide possible mechanisms for these events to occur. It is possible that HIV-1 infection and immune activation of macrophages and microglia directly lead to release of GLS1-containing MVs, however, other possibilities also need to be explored.
Methods
Culture, HIV-1 infection and LPS activation of macrophages and microglia
Human peripheral blood-derived mononuclear cells were isolated through leukopheresis from healthy donors. Human macrophages were differentiated in Dulbecco’s Modified Eagle’s Media (DMEM) (Sigma Chemical Co., St. Louis, MO) with 10 % human serum, 50 μg/ml gentamycin, 10 μg/ml ciprofloxacin (Sigma), and 1000 U/ml recombinant human macrophage colony-stimulating factor (MCSF) for 7 days. The HIV-1
ADA strain was used to infect the macrophages at a multiplicity of infection (MOI) of 0.1, respectively. The HIV-1
ADA strain was originally isolated from the PBMCs of an HIV-infected patient with Kaposi’s sarcoma [
52,
53]. MDM were infected with HIV-1
ADA at a multiplicity of infection (MOI) of 0.05 virus/target cell. For mock-infection, MDM were incubated with same volume of medium without virus. After 24 h, the culture medium was changed to remove any remnant virus. Seven days after HIV-1-infection, culture medium was changed to glutamine-free neurobasal medium for 24 h and supernatants were collected for subsequent RP-HPLC or Western blot analysis. BV2 cell lines were obtained from ATCC, and both cell lines were grown in DMEM with 10 % fetal bovine serum and antibiotics. Lipopolysaccharide (LPS) (50 ng/ml) (Sigma) was used to immune activate BV2 cells for 24 h and supernatants were collected for HPLC and Western blot analysis.
Rat cortical neuron cultures
Cerebral cortices were dissected from Sprague–Dawley rat (Charles River Laboratories International Inc., Wilmington, MA) between embryonic days 15 and 17 and triturated with a pipet to generate cell suspension. The cell suspension was passed through a 70-μm nylon membrane (Becton Dickinson Labware, Franklin Lakes, NJ) and then plated at a density of 40,000 cells/well in 96-well plates pre-coated with 5 μg/ml poly-D-lysine. The cells were then cultured at 37 °C in a 5 % CO2 atmosphere for 7 days in neurobasal medium containing B27 supplement (Life Technologies), 0.5 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin.
Ethics statement
Primary RCN were prepared in accordance with ethical guidelines for care and use of laboratory animals set forth by the National Institutes of Health (NIH), with Institutional Animal Care and Use Committee (IACUC) #: 04-097-01; Monocytes were used in full compliance with the University of Nebraska Medical Center and NIH ethical guidelines, with the Institutional Review Board (IRB) #: 162-93-FB. We have the informed written consent from all participants involved in this study.
Isolation of MVs
MVs were isolated from the supernatants of HIV-1-infected macrophages and LPS-activated microglia through differential centrifugations with or without neutral sphingomyelinases inhibitor GW4869 (Sigma) at different dosages, 2, 5 and 10 μM. Briefly, the supernatants were first centrifuged at 300 × g for 10 min to remove free cells, at 3000 × g for 20 min to remove cellular debris and then 10,000 × g for 30 min to remove free organelles. Lastly, MVs were collected by ultracentrifugation at 100,000 × g for 2 h at 4 °C. To prepare MVs for Western blot, the MVs pellets were lysed in M-PER mammalian protein extraction reagent (Thermo Scientific, Pittsburgh, PA). For negative staining, MVs were fixed in 2 % glutaraldehyde and 2 % paraformaldehyde. For glutaminase activity assay and neurotoxicity, the MVs were resuspended in 1 ml of glutamine-free neurobasal medium.
Negative staining and electron microscopy
MVs were negatively stained with onscreen measurements. Briefly, MVs were fixed and then spread on the silicon monoxide and nitro-cellular film coated copper grid. The droplets of MVs were removed with filter paper, air-dried at room temperature and then subjected to transmission electron microscopy (TEM) (FEI Tecnai G2 Spirit TWIN). For the scanning electron microscope (SEM) (FEI Quanta 200), cells were fixed in 2 % glutaraldehyde and 2 % paraformaldehyde and point dried, mounted and coated with gold/palladium. The investigator in the EM core facility was blinded for image acquisition and quantification.
Neurotoxicity assays
Resuspended MVs were added to neuronal cultures for 48 h with or without 10 μM of bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) (a generous gift presented by Dr. Tsukamoto from Colorado State University) and cell viability was assessed by MTT assays in 96-well plates. MTT (Sigma) was added to the cultures to a final concentration of 125 μg/ml. The plates were incubated for 30 min at 37 °C with 5 % CO2 and the medium was aspirated. The insoluble formazan was solubilized in DMSO, and the concentrations were determined by optical density at 490 nm with an ELX808 densitometer (Bio-Tek Instruments, Winooski, and VT). MAP2 ELISA was performed on primary RCN cultures as previously described. Briefly, fixed neurons were blocked with 3 % normal goat serum in phosphate buffered saline and incubated for 2 h with antibodies against MAP-2 (Millipore-Chemicon International, Atlanta, GA), followed by anti-mouse biotinylated antibody (Vector Laboratories, Burlingame, CA) for 1 h. Avidin/biotin complex solution was added for 30 min, and then color was developed using TMB substrate (Sigma Chemical Co., St. Louis, MO) and terminated with 1 M sulfuric acid (Sigma Chemical Co., St, Louis, MO). The absorbance was read at 450 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA). For morphological data that demonstrated neuronal damage after exposed to supernatant of HIV-1-infected macrophages or immune-activated microglia, MAP2 immunostaining was examined by a Nikon Eclipse TE2000E fluorescent microscope and photographed by a digital camera (CoolSNAP EZ, Photometrics). All obtained images were imported into Image-ProPlus, version 7.0 (Media Cybernetics, Sliver Spring, MD) for quantifying levels of MAP2 staining. The assessors were blinded during image acquisition or quantification.
Western blot
Protein concentrations were determined by Bradford protein assay. SDS PAGE separated proteins from whole cell and MVs lysates. After electrophoretically transferred to polyvinyldifluoridene membranes (Millipore, Billerica, MA and Bio-Rad, Hercules, CA). Membranes were incubated overnight at 4 °C with polyclonal antibodies for GAC (Dr. N. Curthoys, Colorado State University, Fort Collins, CO), Alix (Santa Cruz Biotechnology, CA) and flotillin-2 (Cell Signaling Technology, Danvers, MA), followed by horseradish peroxidase-linked secondary anti-rabbit or anti-mouse secondary antibodies (Cell signaling Technology). Antigen-antibody complexes were visualized by Pierce ECL Western Blotting Substrate. For quantification of the data, films were scanned with a CanonScan 9950 F scanner and images were analyzed using the public domain NIH image program (developed at the U.S. National Institutes of Health and available on the internet at
http://rsb.info.nih.gov/nih-image/).
Analysis of glutamate and glutamine by RP-HPLC
Glutamate levels were analyzed by RP-HPLC using an Agilent 1200 liquid chromatograph and fluorescence detector as previously described [
29] with a few modifications. The experiments utilized 4.6 × 75 mm, 3.5 μm ZORBAX Eclipse AAA analytical columns (Agilent). A gradient elution program was optimized for glutamate measurement with a flow rate 0.75 ml/min.
Statistical analysis
Data are expressed as means ± SD unless otherwise specified. Statistical analysis was performed using ANOVA, followed by the Tukey-post-test for paired observations. Significance was determined by a p value < 0.05. All experiments were performed with cells from at least three donors to account for any donor-specific differences. Assays were performed at least three times in triplicate or quadruplicate within each assay.
Acknowledgments
We kindly thank Dr. Norman Curthoys for providing the KGA and GAC antibodies. We thank Dr. Hui Peng, Dr. Changhai Tian, Dr. Santhi Gorantla and Ms. Li Wu, Yi Wang and Tom Bargar for the technical support of this work; Dr. Dan Monaghan, Shelly Smith, Andrew Dudley for sincere sciencific suggestions and mentoring; Dr. Myron Toews for critical scientific writing; Julie Ditter, Lenal Bottoms, Jaclyn Ostronic, Myhanh Che, Johna Belling, and Robin Taylor provided outstanding administrative and secretarial support. This work was supported by grants from National Key Basic Research Program of China (973 Program Grant No. 2014CB965000, project 1 No. 2014CB965001 and project 3 No. 2014CB965003) and Innovative Research Groups of the National Natural Science Foundation of China (#81221001 to JZ), and Joint Research Fund for Overseas Chinese, Hong Kong and Macao Young Scientists of the National Natural Science Foundation of China (#81329002 to JZ); National Institutes of Health: R01 NS41858-01, 2R56NS041858-15A1 (JZ), and R03 NS094071-01 (YH).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
BW, YH and JZ: conception and design, data analysis and interpretation, writing and final approval of the manuscript; AB, ZT, RZ, YL and FL: participation in the design, data collection, provision of study material, data analysis and interpretation of the studies. All authors read and approved the final manuscript.