Glutaminase regulates extracellular vesicles
Beiqing Wu1, 2, 4, Runze Zhao1, 2, 4, Alexander Braun4, Yuju Li1, 2, 4,
1The Laboratory of Neuroimmu
2Dept. Pharmacology & Experiment
and Microbiology, 4University of Nebras
release in HIV-1-infected macrophages
Yi Wang1, 2, 4, , Yunlong Huang1, 2, 4,Jialin Zheng1, 2, 3, 4
nology & Regenerative Therapy,
al Neuroscience, 3Dept. of Pathology
ka Medical Center, Omaha, NE 68198-5930
Abstract
Background: Extracellular vesicles (EVs) are important in the
intercellular communication in the central nervous system and their
release is increased upon neuroinflammation and neurological
disorders. Our previous data demonstrated an increased release of
EVs from HIV-1-infected macrophages that have neurotoxic effects.
However, the mechanism which elevates EV release in those HIV-1infected cells remains unknown. In the current studies, we
investigated glutaminase 1 (GLS1), which is a mitochondria enzyme
critical for glutamine metabolism. GLS is upregulated in HIV-1infected macrophages and microglia. We propose that HIV-1
infection increases GLS1, leading to a metabolic status that favors
the EVs generation and release. The new understanding of the
metabolic control of EV release in HIV-1-infected cells will shed light
on HIV-1 pathobiology and neurological complications.
Methods: Human primary microglia and monocyte-derived
macrophage culture systems and macrophage-tropic HIV-1ADA were
used to study the regulation of EVs during HIV-1 infection. EVs were
isolated through differential centrifugations. A gene overexpression
system, delivered via adenovirus vector, was utilized to overexpress
GLS1 in the cell culture to mimic the upregulation of GLS1 during
HIV-1 infection. A brain-specific GLS1 transgenic mouse line was
created to model GLS1 elevation in vivo. BPTES was used to
specifically inhibit GLS1 activity. Transmission electron microscopy
and Western blot were used to quantify the EVs released from cells
and brain tissues. Glutamate and glutamine levels were determined
by reverse phase high performance liquid chromatography.
Results: An elevated number of EVs were found in the supernatants
of HIV-1-infected macrophages and microglia when compared with
controls. Overexpression of GLS1 in macrophages and microglia
cultures led to increased release of EVs. Conversely, blocking the
GLS1 activity by BPTES significantly reduced EV release and
glutamate generation in HIV-1-infected macrophages and microglia,
suggesting a critical role of GLS1 in EV release. Interestingly, we
detected an elevated release of EVs in the brain tissues of GLS1
transgenic mice, suggesting that GLS1 is also important for EV
release in vivo.
Conclusion: GLS1 is essential for EVs release in HIV-1-infected
macrophages and microglia. Therefore, blocking EV release through
GLS1 inhibitors may serve as a novel therapeutic strategy against
the HIV-1 pathobiology and neurological complications.
Introduction
• Extracellular vesicles (EVs) are cellular secretory vesicles
shed from the plasma membrane of various cell types in
physiological conditions; they are abundant in the central
nervous system (CNS).
• CNS-derived EVs contribute to neuroinflammation
through secretion of various signaling molecules, nucleic
acid, lipids, and proteins. EVs release increases upon
neural cancer progression, neuroinflammation, and acute
neurological disorders. However, the regulation of EV
release remain clear.
• HIV-associated neurocognitive disorder (HAND) is a
chronic neurological disorder associated with HIV
infections and AIDS. HIV associated dementia (HAD) is the
most severe form of HAND occurring in the late stages of
HIV-1 infection. The characteristic symptoms of HAD
include, cognitive impairment, behavioral disorders, and
potential progressive motor abnormality.
• Mitochondria glutaminase 1 (GLS1) is the primary enzyme
in the brain responsible for converting glutamine into
glutamate. Previously, our studies showed that GLS1 is
upregulated in the HAND brain tissues and in the
supernatant and cells of HIV-1-infected macrophages. The
GLS1 upregulation may directly increase glutamate
production and extracellular release. However, our data
suggest that mitochondrial stress during HIV infection
may lead to membrane destabilization and GLS1
translocation from the mitochondrial matrix to the cytosol,
and even further to the extracellular fluid.
• Two of the GLS1 isoforms, glutaminase C (GAC) and
kidney-type glutaminase (KGA), are present in brain
tissue, and shares much of the functional GLS1 regions
but each possesses a unique 3’ tail.
• Despite the association of EVs with neurodegenerative
disorders and neuroinflammation, the role of EVs and
their regulation in HIV-1-associated neurocognitive
disorders (HAND) remains to be elucidated.
Hypothesis
The regulation of EV release through glutaminase is
dependent on glutamine metabolism as a critical
pathogenic event in HIV-1-mediated neuronal injury
and cell death.
Methods
Results
Fig 1. HIV-1 infection and immune activation increase EV
release from macrophages and microglia.
Macrophage EVs
BV2 EVs
Figure 1. (A) MDM were fixed at 7th day post HIV-1 infection and subsequently
subjected to SEM for EV detection. (B) High-magnification image of the
corresponding small box area in panel A was shown. Magnification, 24000 X.
(C-H) EVs were isolated through differential centrifugation from normalized
volumes of cultural supernatants and observed under TEM using negative
staining. Representative TEM images of EVs from mock-infected MDM (C),
HIV-1 infected MDM (D), untreated microglia (F), and LPS-stimulated microglia
(G) were shown. (E, H) EVs numbers in C, D and E, F were quantified by
manually counting from a total of 10 random vision fields. ** denotes p < 0.01
in comparison to controls. (I, J) EVs were isolated from normalized volumes
of supernatants in mock-infected and HIV-1-infected MDM cultures (I) or
untreated and LPS-stimulated microglia cultures (J). The levels of tissue
transglutaminase (tTG), Alix, and flotillin-2 in EVs were determined by
Western blotting.
Figure 2. The release of EVs is dependent on glutamine
metabolism.
Figure 2. (A) Nanosight tracking analysis (NTA) showed the size and
concentration of EVs extracted from BV2 cells treated with LPS. (B) Different
glutamine concentrations were used to treat cells 12 hours prior to LPS
treatment in in the serum-free media. Concentrations of EVs extracted from
BV2 cells were quantified from NTA analysis. (C-F) EVs were isolated through
differential centrifugation from normalized volumes of cultural supernatants
and observed under TEM using negative staining. Representatives are TEM
images of EVs from HIV-1-infected MDM with GLN levels of 0 mM (C), 1 mM
(D), 2 mM (E) and 5 mM (F).
Figure 3. EVs release is increased in GLS-overexpressing
microglia.
Figure 3. (A) Both GLS1 isoforms, KGA and GAC, were overexpressed
through an adenovirus vector in microglia culture. Two days after infection,
proteins were collected and the levels of KGA and GAC were analyzed by
Western blotting. Actin was used as loading control. (B) GLS1 activities were
determined by enzyme activity assay. (C) Intracellular glutamate levels in
KGA- or GAC-overexpressing microglia were determined by Glutamic
Acid/Glutamate Oxidase Assay Kit. ***, p < 0.001, compared with GFP control
group. (D) Glutamate concentrations in the culture supernatant of KGA- or
GAC-overexpressing microglia were determined by RP-HPLC. ***, p < 0.001
(E) EVs were isolated from KGA- or GAC-overexpressing culture supernatants
through differential centrifugation. The levels of tissue transglutaminase (tTG)
and flotillin-2 were analyzed by Western blotting. (F, G) Levels of tTG and
flotillin-2 were quantified and normalized to control group. **, p < 0.01.
Figure 4. GLS inhibitor reduces EV release in LPS-treated
microglia and HIV-1-infected macrophages.
Figure 4. (A) Human primary microglia were treated with 10 μM BPTES
overnight prior to the treatment of 50 ng/ml of LPS for 24 hours. Cell viability
was determined by MTT assay. (B) BPTES were added to cultures 12 hours
prior to LPS treatment in serum-free media. EVs were isolated from culture
supernatant through differential centrifugation. EV marks, flotillin-2 and CD9,
were analyzed by Western blotting. (C, D) Levels of flotillin-2 and CD9 were
quantified and normalized to control group. *, p < 0.05, # and **, p < 0.01. (E,
F) Human macrophages were differentiated from monocytes and were
infected with HIV-1 for 7 days. 10 μM BPTES was added to serum-free media
24 hours prior to EV isolation. Levels of Alix and tTG were determined by
Western blotting (E) and densimetrically quantified by image J (F).
Fig 5. GLS-overexpression increases EV release in mice
brain.
Figure 5. (A) Generation of Nestin-GAC transgenic mouse to specifically
overexpress GAC in the CNS. Plasmid vector was engineered to overexpress
GAC then microinjected into the fertilized egg. The egg was implanted in the
uterus of a pseudopregnant female to create CAG-loxp-GAC mouse. CAGloxp-GAC mice were mated with Nestin-Cre mice to produce Nestin-GAC
mice. (B, C) Brain tissues from adult Nestin-GAC mice were dissected and
EVs were isolated through differential centrifugation. Alix (A) and flotillin-2
(B) levels in the EV protein lysates were determined through Western blotting
and densimetrically quantified using Image J. Statistical comparisons were
made with two-tailed Student's t test. (D-F) EVs were extracted from Thy-1
GAC mice using the same techniques described above. EVs collected from
control (D) and Thy1-GAC (E) were evaluated with TEM using negative
staining. EVs numbers per vision fields (N = 10) under TEM were quantified.
***, p < 0.001.
Summary
• Overexpression of GLS1 increases EV release in
vitro and in vivo.
• GLS regulates the release of EVs in HIV-1-infected
macrophages and immune-activated microglia.
• EV release in macrophages and microglia is
dependent on the glutamine metabolism.
Acknowledgements
This work was supported in part by research grants by the
National Institutes of Health: R01 NS 41858-01, R01 NS 06164201, the State of Nebraska.
Dr. Jialin Zheng
University of Nebraska Medical Center
985930 Nebraska Medical Center
Omaha, NE 68198-5930
[email protected]
Tel (402) 559-5656
Fax (402) 559-3744