After primary peripheral infection, the human immunodeficiency virus (HIV) crosses the blood–brain barrier (BBB) using monocytes as a vehicle, gaining access to the central nervous system (CNS) [1
]. After that, the HIV infects both microglia and a small population of astrocytes [3
], adversely affecting proper glial cell function and concomitant survival of neighboring neurons [5
]. The latter has been linked to the release of proinflammatory cytokines and free radicals, cytoplasmic Ca2+
imbalance and glutamate excitotoxicity [5
]. In parallel, infected glial cells release soluble viral proteins into the brain parenchyma, triggering direct neuronal toxicity [7
]. Overall these mechanisms are thought to contribute to a wide range of cognitive, behavioral and motor deficits, collectively known as HIV-associated neurocognitive disorders (HAND) [9
]. Despite that antiretroviral therapy has diminished the severity of HIV cognitive disorders, the prevalence of HAND in HIV-infected patients remains high (50%) as they live longer and due to the relatively poor BBB penetrance of most antiretroviral drugs [11
]. The prominent CNS damage observed in HIV-infected individuals with effective antiretroviral therapy had led to the thought that additional and novel mechanisms of bystander cell death might be implicated.
Embedded in the synaptic cleft, astrocytes govern crucial brain processes that include synaptic function and plasticity, energy supply for neurons, cytoplasmic Ca2+
signaling and homeostatic equilibrium of extracellular pH, neurotransmitters and ions, along with controlling the redox and inflammatory response [15
]. Previous studies have shown that HIV itself or the HIV gp120, the surface glycoprotein responsible for viral entry, impairs astroglial function with particularly detrimental consequences for neuronal survival [19
]. Indeed, in astrocytes HIV gp120 alters the exchange of Na+
and glutamate efflux [21
]; the levels of cytoplasmic Ca2+
] and the production of proinflammatory cytokines/chemokines and free radicals [24
]. Despite the continuous ongoing research in the field, currently, there is no effective treatment for HAND and the precise underlying mechanism behind the pathological action of HIV and/or HIV gp120 remains not well understood.
A growing body of evidence has pointed out that cellular signaling mediated by hemichannels and pannexons might contribute to the dysfunction of astrocytes, with potentially significant repercussions for neuronal function and survival [27
]. Hemichannels are plasma membrane channels containing six connexin subunits that oligomerize around a central pore, allowing autocrine/paracrine signaling via the exchange of ions and small molecules between the cytoplasm and the extracellular space [28
]. Pannexins channels—also called pannexons—result from the oligomerization of pannexins, a three-member family of proteins with similar secondary and tertiary structures than connexins that establish plasma membrane channels permeable to ions and small molecules [29
]. In pathological scenarios, instead of being beneficial, the persistent function of hemichannels and pannexons contributes to cell damage and dysfunction through different mechanisms, such as the release of potentially toxic molecules, intracellular Ca2+
imbalance and transmembrane ionic/osmotic disturbances [30
]. In a prior study, we showed that HIV increases the opening of hemichannels but not pannexin channels in cultured human astrocytes, which result in the collapse of neuronal processes [32
]. However, whether HIV derived viral proteins, including gp120, regulate hemichannel and/or pannexon opening in astrocytes is still unknown.
Here, we demonstrated that gp120 increased the function of Cx43 hemichannels and Panx1 channels in astrocytes. This stimulatory effect took place via the signaling of IL-1β/TNF-α and the activation of p38 MAPK/iNOS/[Ca2+]i-dependent cascades and P2Y1/P2X7 purinergic receptors. Remarkably, the function of Cx43 hemichannels and Panx1 channels evoked by gp120 was crucial for inducing severe alterations in ATP release, NO production and [Ca2+]i dynamics in astrocytes.
Time-lapse recordings of Etd uptake demonstrated that gp120 increased the function of Cx43 hemichannels and Panx1 channels in a time and concentration-dependent form in primary cortical astrocytes. Two well-recognized specific mimetic peptides that antagonize Cx43 hemichannel opening (Tat-L2 and gap19), drastically neutralized the gp120-induced Etd uptake. In addition, the inhibition of Panx1 channels with 10
panx1 or probenecid resulted in similar antagonistic effects, revealing that both Cx43 hemichannels and Panx1 channels were significant protagonists in the gp120-induced Etd uptake. These data are consistent with prior in vitro and ex vivo studies describing the parallel opening of both channels in astrocytes subjected to neuroinflammatory conditions such as FGF-1 [60
], alcohol [61
], ultrafine carbon black [62
], gp120 [44
], familial Alzheimer’s disease [63
], spinal cord injury [64
] and acute infection [65
The functional state of gap junction channels and hemichannels is inversely modulated in inflamed astrocytes [41
]. In discrepancy with this evidence, we observed that gp120 did not reduce astroglial coupling, as recorded by intercellular Etd transfer. As inferred from confocal immunofluorescence studies, gp120 had no effects on the localization of Cx43 in astrocytes, suggesting that the organization of gap junctions remain unaltered. One hypothesis to explain this apparent contradiction is that gap junctions could keep their functional state to spread and amplify gp120-mediated toxic substances. The latter it has been demonstrated to occur between HIV-1-infected and uninfected astrocytes [20
]. The variety of potentially toxic molecules diffusing through gap junction is wide, ranging from free radicals to high concentrations of cytoplasmic Ca2+
, as well as IP3
Mounting evidence has shown that gp120 triggers the persistent activation of astrocytes associated with a broad-spectrum generation of inflammatory signals, including IL-1β and TNF-α [71
]. Certainly, the expression of both cytokines augments in postmortem brains of HIV-1 patients [73
] and their signaling linked to the p38 MAPK pathway opens astroglial Cx43 hemichannels [41
]. By making use of a combination of selective inhibitors, we observed that gp120-induced Etd uptake embraces the activation of IL-1β/TNF-α and p38 MAPK, being this consistent with the fact that gp120 stimulates p38 MAPK in astrocytes [75
]. Downstream signaling of IL-1β/TNF-α and p38 MAPK leads to the expression of iNOS [76
] and, in consequence, increases the amounts of NO [77
]. Despite that NO is essential for synaptic transmission and plasticity [78
], its high production has been linked to astroglial-mediated neurotoxicity [79
] and the opening of astroglial Cx43 hemichannels via NO-mediated S-nitrosylation of Cx43. Conformity with this, we found that gp120 dramatically augmented NO production in astrocytes, this effect being moderately abrogated by inhibition of Cx43 hemichannels or Panx1 channels. The latter harmonizes with previous studies describing that activation of Cx43 hemichannels/Panx1 channels and NO production are reciprocal processes occurring on glial cells exposed to proinflammatory conditions [44
Prior data indicate that gp120 may induce the release of several “danger” signals from glial cells, including ATP [81
], the latter molecule being recently proposed as a biomarker of HIV-1-mediated cognitive impairment [82
]. With this in mind, two significant elements underscore the importance of ATP signaling on the gp120-induced astrocyte changes in our system. On the one hand, we found that suppression of both P2X7
receptors greatly abrogated the Etd uptake evoked by gp120. At the other end, the activation of Panx1 channels was crucial for the release of ATP in gp120-stimulated astrocytes. Previous evidence has highlighted that ATP causes its release through hemichannels or pannexons, leading to the subsequent stimulation of purinergic receptors [44
]. We conjecture that ATP release may serve as a downstream mechanism that results in the opening of Cx43 hemichannel and/or Panx1 channels, where P2Y1
receptor-dependent rise in [Ca2+
could be fundamental, as already reported [44
]. Indeed, a mild rise in [Ca2+
up to 500 nM significantly reinforces the function of Cx43 hemichannels [42
], while analogous responses seem to occur in Panx1 channels [45
]. In this line, we observed that BAPTA,
strongly reduced the gp120-induced Etd uptake in astrocytes. This is consistent with recent works showing that gp120 increases [Ca2+
in astrocytes [84
] and that purinergic receptors are critical for HIV infection and gp120-mediated signaling [85
]. Alongside this, given that Cx43 hemichannels are permeable to Ca2+
] and one could infer the same for Panx1 channels [58
], they potentially may contribute to sustaining [Ca2+
-dependent pathways linked to ATP release (see below).
Both the P2Y receptor-dependent release of internal Ca2+
and its extracellular influx via P2X receptors are emblematic astrocyte [Ca2+
responses evoked by ATP [88
]. In this study, we observed that although gp120 had no consequences on basal levels of [Ca2+
, it caused a drastic augment in ATP-induced Ca2+
responses, particularly, concerning the signal amplitude, integrated area under the curve and sustained signal. Worthy of note, the blockade of Cx43 hemichannels or Panx1 channels strongly antagonized the increased ATP-mediated [Ca2+
responses triggered by gp120. With this in mind, we speculated that the release of ATP and/or its derivates (e.g., ADP) from astrocytes might spread the gp120-mediated signaling to neighboring cells, resulting in Ca2+
responses that may impair the function and eventually the survival of glial cells and neurons. In such circumstances, the opening of Cx43 hemichannels and Panx1 channels could be crucial, whereas the signaling of purinergic receptors likely will be counteracted by 1) diffusion of ATP towards distant areas; 2) desensitization of P2Y1
receptors, 3) degradation of extracellular ATP via exonucleases and 4) self-inhibition of Panx1 channels by the direct action of ATP [89
Although in vitro culture preparations are useful to dissect cellular mechanisms, they not always recapitulate the processes that take place in vivo. As a result of the use of acute brain slices, we corroborated in a much comprehensive model the stimulatory action of gp120 on Cx43 hemichannel/Panx1 channel function observed in astrocyte cultures. It is relevant to mention that gp120 exposure quickly augmented in a transient form the opening of Panx1 channels overall in the CA1 region, but only reproduce this effect on Cx43 hemichannel function in the stratum oriens. One plausible explanation is the existence of local stimuli (e.g., elevation in extracellular K+
) affecting specifically Panx1 channels rather than Cx43 hemichannels in the CA1 region of the hippocampus. Alternatively, the differential influence of gp120 on channel opening may rely on the unique topological organization of hippocampal area CA1 astrocytes and pyramidal neurons [91
]. For instance, astrocytes near to the stratum pyramidale are arranged in circuits that stay parallel to this layer, while astrocytes in the stratum radiatum constitute circular circuits [92
Similarly, although the number of astrocytes remains unchanged between young and middle-aged mice, their quantity declines with the age in the stratum oriens, whereas the opposite occurs in the stratum lacunosum-moleculare [93
]. In the same line, the electrophysiological features, cell–cell coupling, antigen profiles and [Ca2+
responses of astrocytes at the hippocampus are diverse depending on their location in this brain region [91
]. The heterogeneity of neuronal types (e.g., pyramidal, baskets, etc.) and their projections (e.g., inhibitory and excitatory) is another factor that turns even more complex in these analyses. Likely, this diversity may account for the gp120-induced mediated heterogeneity in channel responses in the regions analyzed. Further research is required to unveil the mechanisms of these differential effects.
Overall, our findings support the idea that gp120-induced hemichannel/pannexon activation could occur rapidly upon HIV-1 brain invasion and being consequences of this process: i) the increase in ATP release, ii) the augment of NO production and iii) the rise in ATP-mediated [Ca2+]i, dynamics. We propose a novel mechanism by which gp120 could disturb the astrocyte function implicating the successive activation of inflammatory cascades that in consequence enhance the activation of astroglial hemichannels and pannexons. The molecular mechanisms behind this phenomenon could serve as pharmacological targets for exploring new therapies aiming to tackle the pathogenesis and progression of HAND.
4. Materials and Methods
4.1. Reagents and Antibodies
Dulbecco’s Modified Eagle Medium (DMEM), water, L-N6, SB203580, MRS2179, oxidized ATP (oATP), HEPES, anti-GFAP monoclonal antibody, A74003, Cx43 rabbit polyclonal antibody (SAB4501174), ethidium (Etd) bromide and probenecid (Prob) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Cx43 monoclonal antibody (610061) was obtained from BD Biosciences (Franklin Lakes, NJ, USA). Penicillin, BAPTA-AM, FURA-2AM, diamidino-2-phenylindole (DAPI), goat anti-mouse Alexa Fluor 488, streptomycin, DAF-FM diacetate, goat anti-mouse Alexa Fluor 488/555 and goat anti-rabbit Alexa Fluor 488/555 were from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA). Normal goat serum (NGS) was purchased from Zymed (San Francisco, CA, USA). A soluble form of the TNF-α receptor (sTNF-αR1) and a recombinant receptor antagonist for IL-1β (IL-1ra) were from R&D Systems (Minneapolis, MN, USA). The mimetic peptides gap19 (KQIEIKKFK, intracellular loop domain of Cx43), gap19I130A (KQAEIKKFK, negative control), Tat-L2 (YGRKKRRQRRR-DGANVDMHLKQIEIKKFKYGIEEHGK, second intracellular loop domain of Cx43), Tat-L2H126K/I130N (YGRKKRRQRRR-DGANVDMKLKQNEIKKFKYGIEEHGK, negative control) and 10panx1 (WRQAAFVDSY, first extracellular loop domain of Panx1) were obtained from Genscript (New Jersey, NJ, USA). The HIV-1 BaL gp120 recombinant protein recombinant protein (Cat#4961) was obtained from the NIH AIDS Reagent Program, Division of AIDS, NIAID and NIH.
C57BL/6 (PUC/The Jackson Laboratory) male mice of 2-month-old were housed in cages at temperature- (24 °C) and humidity-controlled ambient under a 12 h light/dark cycle (lights on 8:00 AM), with ad libitum access to food and water. Animal protocols were conducted following the guideline and approved protocol for care and use of experimental animals of the Bioethics Committee of the Pontificia Universidad Católica de Chile (PUC; n°: 150806013, 6 June 2016).
4.3. Cell Cultures
Primary cultures of cortical astrocytes were obtained from cortices of postnatal day 2 mice as previously reported [44
]. After dissection of cortices, meninges were carefully peeled off and tissue was mechanically dissociated in Ca2+
free Hank’s balanced salt solution (CM-HBSS) with 0.25% trypsin and 1% DNase. Cells were seeded onto 60-mm plastic dishes (Corning, NY, USA) or onto glass coverslips (Fisher Scientific, Waltham, MA, USA) placed inside 16-mm 24-well plastic plates (Corning, NY, USA) in DMEM, supplemented with streptomycin (5 µg/mL), penicillin (5 U/mL) and 10% FBS. Cells were grown at 37 °C in a 5% CO2
/95% air atmosphere at nearly 100% relative humidity. Of cytosine-arabinoside 1 µM was added for 3 days after 8–10 days in vitro to eliminate proliferating microglia. Medium was changed twice a week and cultures were used after 3 weeks.
Astrocytes were treated for 0, 1, 24, 48 or 72 h with 1, 5, 10 or 20 ng/mL of HIV-1 BaL gp120 (from now on referred as to “gp120”). To obtain conditioned media (CM) from astrocytes, cells (2 × 106 cells in 35 mm dishes) were treated with 10 ng/mL gp120 for 24 h and supernatants filtered (0.22 µm), and stored at −20 °C before used for experiments. Different antagonists were preincubated 1 h prior and co-incubated with 10 ng/mL gp120 before experiments: mimetic peptides against Cx43 hemichannels (Tat-L2 and gap19, 100 µM) and Panx1 channels (10panx1, 100 µM), Prob (Panx1 channel blocker, 500 µM), sTNF-αR1 (soluble form of the receptor that binds TNF-α), IL-1ra (IL-1β receptor endogenous blocker), SB203580 (p38 MAP kinase inhibitor, 1 µM), L-N6 (iNOS inhibitor, 1 µM), BAPTA-AM (intracellular Ca2+ chelator, 10 µM), oATP (general P2X receptor blocker, 200 µM), MRS2179 (P2Y1 receptor blocker, 1 µM) and A740003 (P2X7 receptor blocker, 200 nM).
4.5. Dye Uptake and Time-lapse Fluorescence Imaging
Astrocytes plated on glass coverslips were washed twice in Hank’s balanced salt solution and bathed at room temperature with Locke’s solution (154 mM NaCl, 5.4 mM KCl, 2.3 mM CaCl2, 5 mM HEPES, pH 7.4) containing 5 µM Etd. Cells were then visualized with an Olympus BX 51W1I upright microscope with a 40× water immersion objective for time-lapse imaging. Images were captured using a Retiga 1300I fast-cooled monochromatic digital camera (12-bit; Qimaging, Burnaby, BC, Canada) controlled by imaging Metafluor software (Universal Imaging, Downingtown, PA) every 30 s (exposure time = 0.5 s; excitation and emission wavelengths were 528 nm 598 nm, respectively). The fluorescence intensity recorded from ≥30 regions of interest (representing at least 30 cells per cultured coverslip) was defined with the following formula: corrected total cell Etd fluorescence = integrated density – ((area of the selected cell) × (mean fluorescence of background readings)). The Etd uptake rate represent the mean slope of the relationship over a given time interval (ΔF/ΔT). To examine variations in the slope, regression lines were fitted using Microsoft Excel, and mean slope values were analyzed employing GraphPad Prism software and expressed as AU/min. In each independent experiment three replicates were performed. In some experiments, cultured astrocytes were preincubated with gap19 (100 µM), Tat-L2 (100 µM), 10panx1 (100 µM) or probenecid (500 µM) for 15 min before and during the Etd uptake.
4.6. Dye Coupling
Astrocytes plated on glass coverslips were iontophoretically microinjected with a glass micropipette filled with 75 mM Etd in recording medium (HCO3--free F-12 medium buffered with 10 mM HEPES, pH 7.2) containing 200 μM La3+. This blocker was used to prevent cell leakage of the microinjected Etd via hemichannels, which could underestimate the transfer of Etd to neighboring cells. Astrocytes were visualized through a Nikon inverted microscope equipped with epifluorescence illumination (Xenon arc lamp) and Nikon B filter to Etd (excitation wavelength 528 nm; emission wavelength above 598 nm) and XF34 filter to DiI fluorescence (Omega Optical, Inc., Brattleboro, VT, USA). Photomicrographs were captured employing a CCD monochrome camera (CFW-1310M; Scion; Frederick, MD, USA). Five minutes after dye injection, the coupling incidence was calculated as the percentage of injections that resulted in Etd transfer from the injected cell to more than one neighboring cell, whereas the coupling index was calculated as the mean number of cells to which the Etd spread. Etd coupling was tested by microinjecting a minimum of 10 cells per experiment.
4.7. Immunofluorescence and Confocal Microscopy
Astrocytes plated on glass coverslips were with 2% paraformaldehyde (PFA) for 30 min fixed at room temperature. After washing three times with PBS, they were rinsed three times with (5 min each) 0.1 M PBS-glycine, and then incubated with 0.1% Triton X-100 in PBS containing 10% NGS for 30 min. Then astrocytes were incubated with an anti-GFAP monoclonal antibody (BD Biosciences, 1:400) or anti-Cx43 polyclonal antibody (SIGMA, 1:400) diluted in 0.1% Triton X-100 in PBS with 2% NGS at 4 °C overnight. After five rinses in 0.1% Triton X-100 in PBS, cells were incubated with goat anti-mouse IgG Alexa Fluor 355 (1:1000) or goat anti-rabbit IgG Alexa Fluor 488 (1:1000) at room temperature for 50 min. After washing, coverslips were mounted in DAKO fluorescent mounting medium and examined with an Olympus BX 51W1I upright microscope with a 40× water immersion. Nuclei were stained with DAPI or Hoechst 33342.
4.8. [Ca2+]i and NO Imaging
Astrocytes plated on glass coverslips were loaded with 5 µM Fura-2-AM or 5 µM DAF-FM in DMEM without serum at 37 °C for 45 min and then washed three times in Locke’s solution followed by de-esterification at 37 °C for 15 min. The experimental protocol for [Ca2+]i and nitric oxide (NO) imaging involved data acquisition every 5 s (emission at 510 and 515 nm, respectively) at 340/380-nm and 495 excitation wavelengths, respectively, using the same microscope and acquisition mentioned above for Etd uptake. The FURA-2AM ratio was obtained after dividing the 340-nm by the 380-nm fluorescence image on a pixel-by-pixel base (R = F340 nm/F380 nm).
4.9. Measurement of IL-1β, TNF-α and ATP Concentration
Extracellular amounts of IL-1β, TNF-α and ATP were measured in CM of astrocytes. Samples were centrifuged at 14.000× g
for 40 min and then supernatants were collected and protein content analyzed through the bicinchoninic acid assay (BCA) technique. The amounts of IL-1β and TNF-α were determined by sandwich ELISA, as stated by the manufacturer (eBioscience, San Diego, CA, USA), whereas ATP levels were determined using a luciferin/luciferase bioluminescence assay kit (Sigma-Aldrich) as previously reported [44
4.10. Acute Brain Slices
Coronal slices (300 µm) from anesthetized mice with isoflurane were obtained using a vibratome (Leica, VT1000GS; Leica, Wetzlar, Germany) in ice-cold slicing solution containing (in mM): sucrose (222); KCl (2.6); NaHCO3 (27); NaHPO4 (1.5); glucose (10); MgSO4 (7); CaCl2 (0.5) and ascorbate (0.1), bubbled with 95% O2/5% CO2, pH 7.4. Then, the slices were transferred at room temperature (20–22 °C) to a holding chamber in ice-cold artificial cerebral spinal fluid (ACSF) containing (in mM): NaCl (125), KCl (2.5), glucose (25), NaHCO3 (25), NaH2PO4 (1.25), CaCl2 (2) and MgCl2 (1), bubbled with 95% O2/5% CO2, pH 7.4, for a stabilization period of 60 min before dye uptake experiments.
4.11. Dye Uptake in Acute Brain Slices and Confocal Microscopy
Acute brain slices were incubated with 25 µM Etd for 10 min in a chamber filled with ACSF and bubbled with 95% O2/5% CO2, pH 7.4. Afterward, the slices were washed three times (5 min each) with ACSF, and fixed at room temperature with 4% paraformaldehyde for 60 min, rinsed once with 0.1 mM glycine in phosphate-buffered saline (PBS) for 5 min and then twice with PBS for 10 min with gentle agitation. Then, the slices were incubated two times for 30 min each with a blocking solution (PBS, gelatin 0.2%, Triton-X 100 1%) at room temperature. Further, the slices were incubated overnight at 4 °C with an anti-GFAP monoclonal antibody (1:500, SIGMA) to detect astrocytes. Later, the slices were washed three times (10 min each) with a blocking solution and then incubated for 2 h at room temperature with goat anti-mouse Alexa Fluor 488 (1:1000) antibody and Hoechst 33342. Afterward, the slices were washed three times (10 min each) in PBS and then mounted in Fluoromount, cover-slipped and examined in a confocal laser-scanning microscope (Eclipse Ti-E C2, Nikon, Japan). Stacks of consecutive confocal images were taken with 40× objective at 100 nm intervals were acquired sequentially with three lasers (in nm: 408, 488 and 543), and Z projections were reconstructed using Nikon confocal software (NIS-elements) and ImageJ software. Etd uptake was calculated with the same formula mentioned for cell cultures. At least six cells per field were selected from at least three fields in each brain slice.
4.12. Data Analysis and Statistics
For each data group, results were expressed as mean ± standard error (SEM); n refers to the number of independent experiments. Detailed statistical results were included in the figure legends. Statistical analyses were performed using GraphPad Prism (version 7, GraphPad Software, La Jolla, CA, USA). Normality and equal variances were assessed by the Shapiro–Wilk normality test and Brown–Forsythe test, respectively. Unless otherwise stated, data that passed these tests were analyzed by unpaired t-test in case of comparing two groups, whereas in case of multiple comparisons, data were analyzed by a one or two-way analysis of variance (ANOVA) followed, in the case of the significance, by a Tukey’s post-hoc test. When data were heteroscedastic as well as not normal and with unequal variances, we used the Mann–Whitney test in the case of comparing two groups, whereas multiple comparisons data were analyzed by Kruskal–Wallis test followed, in the case of the significance, by a Dunn’s post-hoc test. A probability of p < 0.05 was considered statistically significant.