Epigenetic Promoter DNA Methylation of miR-124 Promotes HIV-1 Tat-Mediated Microglial Activation via MECP2-STAT3 Axis.

Keywords: DNA methylation, epigenetics, MECP2, microglia, miR-124, neuroinflammation

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Introduction

Microglia, the primary brain-resident immune cells, are recognized as versatile effectors and regulators of the CNS in the context of executing both protective as well as pathogenic roles in various neurodegenerative diseases (Grubman et al., 2016; Hambardzumyan et al., 2016; Le et al., 2016; Song and Suk, 2017; Wolf et al., 2017). Under physiological conditions, microglia are dynamically surveying and providing vigilance of the surrounding environment to ensure proper CNS homeostasis (Michell-Robinson et al., 2015; Han et al., 2017; Mosser et al., 2017). Microglia can, however, be quickly activated in response to pathogenic stimuli by interactions with cell debris or microbial products. This activation leads to altered microglia functioning via phagocytosis and/or secretion of a plethora of cytokines and chemokines (Han et al., 2017). Interestingly, microglia are also the key target cells for HIV-1 infection in the CNS. HIV-1 infection leads to microglial activation and the concomitant release of several toxic viral and cellular proteins (Bansal et al., 2000).

HIV-1 Transactivator of transcription (Tat) protein is one of nine HIV-1 proteins actively secreted by HIV-1-infected cells and is an early viral protein expressed following viral entry into the cells. HIV-1 Tat released from the infected cells can be taken up by neighboring noninfected cells, such as the neurons (Bagashev and Sawaya, 2013), thereby affecting both immunological and neurological functioning within the CNS (Falkensammer et al., 2007; Mediouni et al., 2012; Maubert et al., 2015). Moreover, because combination antiretroviral therapy (cART) does not impact levels of HIV-1 Tat and the CNS is often inaccessible to the cART regimens, HIV-1 Tat has been implicated as an underlying mediator of HIV-1-associated neurocognitive disorders. Accumulating evidence indicates that HIV-1 Tat alters microglial functional dynamics by controlling the intracellular signaling cascades that regulate the levels of cAMP, intracellular Ca²⁺, thereby leading to the production of various neurotoxic mediators, such as ROS and proinflammatory cytokines (Minghetti et al., 2004; Bagashev and Sawaya, 2013). Additionally, HIV-1 Tat also elicits other detrimental effects in the CNS, such as neurotoxicity, aberrant cellular activation, and endothelial dysfunction (Maubert et al., 2015).

miRs belong to a class of small noncoding RNAs and function as key regulators of the post-transcriptional expression of various genes. Increasing evidence also points to the role of brain-enriched miRs in regulating gene expression, microglial quiescence, and neuronal activities (Davis et al., 2015; Yu et al., 2015). miR-124 is one such brain-enriched miR that plays crucial roles in neurogenesis, synaptic signal transmission, and glial–neuronal interactions to maintain brain homeostasis. Under basal conditions, miR-124 is abundantly expressed in both microglia and neurons (Sun et al., 2015). Indeed, Ponomarev et al. (2011) showed that increased expression of miR-124 is essential for microglial quiescence and that reduced expression of this miR is closely associated with various neuroinflammatory diseases, such as Parkinson's disease (Kanagaraj et al., 2014), dementia (Gascon et al., 2014), and multiple sclerosis (Ponomarev et al., 2011). Mechanisms mediating downregulated expression of miR-124 in inflammatory as well as other disease conditions include epigenetic changes, such as aberrant DNA hypermethylation in the promoter region of primary miR-124 (Koukos et al., 2013; Murray-Stewart et al., 2016; Roy et al., 2017). Mature miR-124 is processed from three primary miR-124; each of these primary miR-124 contains an established CpG island that can be hypermethylated as shown in some disease states (Koukos et al., 2013; Murray-Stewart et al., 2016; Roy et al., 2017). In the current study, we identified a novel epigenetic/molecular mechanism involved in HIV-1 Tat-mediated microglial activation. Our findings suggest HIV-1 Tat-mediated DNA methylation of the miR-124 promoter leads to its downregulation with a concomitant upregulation of the MECP2-STAT3-IL6 that culminates into microglial activation.

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Materials and Methods

 

Reagents.

Endotoxin-free, HIV-1 recombinant Tat (1032–10) was purchased from ImmunoDX. 5-Aza-2′-deoxycytidine (5-Aza; A3656) was purchased from Sigma-Aldrich. TaqMan miR Reverse Transcription Kit (4366596), TaqMan miR assays for miR-124 (001182), TaqMan miR Control Assay-U6 snRNA (4427975), TaqMan Pri-miRNA Assays for miR-124-1, miR-124-2, and miR-124-3 (4427012), and TaqMan Universal PCR Master Mix, no AmpErase UNG (4324018) were purchased from Applied Biosystems. miRIDIAN miR-124-3p mimic (C-310391-05), miRIDIAN miR-124-3p hairpin inhibitor (IH-310391-07), miRIDIAN miR mimic negative control (CN-001000), and miRIDIAN miR hairpin negative control (IN-001005) were purchased from Dharmacon. 5′-DIG and 3′-DIG-labeled, miRCURY locked nucleic acid detection probe for the miR-124 probe was acquired from Exiqon (619867-360). Lipofectamine 2000 Transfection Reagent (11668019) and Opti-MEM I Reduced Serum Media (31985070) were purchased from Invitrogen. ProLong Gold Antifade Mountant with DAPI (P36935) was obtained from Invitrogen. Antibodies were attained from the following sources: CD11b (Novus catalog #NB110–89474, RRID:AB_1216361), Dnmt1 (Santa Cruz Biotechnology catalog #sc-20701, RRID:AB_2293064), Dnmt3a (Santa Cruz Biotechnology catalog #sc-20703, RRID:AB_2093990), Dnmt3b (Santa Cruz Biotechnology catalog #sc-376043, RRID:AB_10988201), p-MeCP2 (Santa Cruz Biotechnology catalog #sc-136171, RRID:AB_2144015), MeCP2 (Cell Signaling Technology catalog #3456S, RRID:AB_2143849), STAT3 (Cell Signaling Technology catalog #4904, RRID:AB_331269), p-STAT3 (Cell Signaling Technology catalog #9131, RRID:AB_331586), Iba-1 (Wako catalog #019-19741, RRID:AB_839504), Peroxidase-AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories catalog #111-035-003, RRID:AB_2313567), and Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Laboratories catalog #115-035-003, RRID:AB_10015289).

 

Rhesus macaques and Simian immunodeficiency virus (SIV) infection.

Briefly, both sexes of 2- to 3-year-old Indian rhesus macaques (Macaca mulatta) were purchased from the Caribbean Research Primate Center and individually housed in steel holding cages, in two dedicated rooms within the Association for Assessment and Accreditation of Laboratory Animal Care-approved animal facility at the University of Kansas Medical Center and were randomly divided into saline and SIV groups. The monkeys were daily exposed to 12 h light-dark cycles and given laboratory chow and water ad libitum along with daily snacks. All animal protocols were approved by the local animal care committee (Institutional Animal Care and Use Committee) at the University of Kansas in accordance with the Guide for the Care and Use of Laboratory Animals. The SIV group of rhesus macaques were chronically infected with SIVR71/17E for ∼52 weeks and were antiretroviral naive. At necropsy, tissues were harvested following perfusion using PBS under lethal anesthesia. A comprehensive methodology of SIV infection and information on viral load and other parameters related to disease pathogenesis has been described in our previous publications (Bokhari et al., 2011; Hu et al., 2012; Pendyala et al., 2015; Chivero et al., 2017). Archival frozen basal ganglia brain tissues, as well as formalin-fixed-paraffin-embedded basal ganglia brain tissues from saline and SIV groups, were used in this study.

 

Mouse primary microglial culture.

Mouse primary microglial cultures were prepared from 1- to 3-d-old newborn pups of either sex bred from C57B1/6 as specified, under standard conditions as described previously (Skaper et al., 2012) with minor modifications. Mixed cell cultures were maintained in DMEM (Corning Cellgro, 10-013-CV) containing 10% heat-inactivated FBS (Atlanta Biologicals, S11050H), and 10 U/ml penicillin-streptomycin (Invitrogen, 15140122) in a 5% CO₂-humidified incubator at 37°C. Cell culture medium was changed every 5 d; and after the first medium change, macrophage colony-stimulating factor (0.25 ng/ml; Invitrogen, PHC9504) was added to the flasks to promote microglial proliferation. The confluent mixed glial cultures (∼10 d) were then subjected to shaking at 37°C at 220 × g for 2 h to promote microglial detachment from the flasks. The cell medium, comprising the detached microglia cells, was collected from each flask and centrifuged at 1000 × g for 5 min. The collected cells were plated onto 6-well cell culture plates (3 × 10⁵ cells per well) for all ensuing experiments. Microglia purity was evaluated by immunocytochemistry using the antibody specific for Iba-1 and used if >95% pure.

 

miR microarray analysis.

Total RNA was isolated from the frozen basal ganglia specimens of both saline and SIV-infected rhesus macaques using TRIzol (Invitrogen, 15596018) and purified using the miRNeasy mini kit (QIAGEN, 217004) as instructed by the manufacturer. Purified total RNA was used for the Affymetrix microarray and was performed by Asuragen. Heatmaps were generated in R using the heatmap.2 function from the gplots package.

 

TaqMan miR assays for miR-124.

The expression of miR-124 was quantified using TaqMan miR assays as described previously (Guo et al., 2016). Briefly, total RNA was extracted using Quick-RNA MiniPrep Plus (Zymo Research, R1058) as per the manufacturer's protocol and quantified using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Next, the total RNA isolated from each group was reverse-transcribed to synthesize cDNA for individual miR using specific miR primers from the TaqMan miR assays and the TaqMan miR Reverse Transcription kit. Each reverse transcription reaction comprises of 7 μl master mix, 3 μl miR-specific primer (5×), and 5 μl total RNA (10 ng/μl). The tube containing the reaction mixture was briefly centrifuged and positioned onto the thermal cycler for reverse transcription. The reverse transcription product was then diluted 1:10 for the following PCR: TaqMan miR assay (20×) 1 μl, RT reaction product 1.5 μl, TaqMan 2× Universal PCR Master Mix, No AmpErase UNG 10 μl, and nuclease-free water up to 20 μl. Each reaction was performed in triplicate, and six independent experiments were run. TaqMan miR assays were performed using a 7500 Fast Real-Time PCR Systems (Applied Biosystems). The expression level of miR-124 was calculated by normalizing to U6 snRNA.

 

TaqMan primary miR assay for primary miR-124-1, -2, and -3.

Total RNA was column isolated from the control and treated mouse primary microglial cells using Quick-RNA MiniPrep Plus as per the manufacturer's protocol and quantified using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). To determine the expression levels of primary miR-124-1, -2, and -3, reverse transcription and TaqMan qPCR were performed according to the TaqMan Primary miRNA Assay protocols. 18S rRNA was also assessed serving as the endogenous control. PCRs were conducted in six independent triplicates for each sample. Expression of primary miR-124-1, -2, and -3 was normalized to that of 18S rRNA. The relative expression of the primary miR-124 was quantified by the 2^−ΔΔCT method.

 

RNAscope with standard immunohistochemistry.

mRNA expression of IL6 in the basal ganglia of saline and SIV-infected rhesus macaques was determined using a new generation state-of-the-art in situ hybridization (ISH) technology, RNAscope (Advanced Cell Diagnostics). This methodology uses a unique “double Z” probe designed to amplify target-specific signals but not background noise from nonspecific hybridization, thereby enabling the visualization of single transcripts of target RNA within intact cells. RNAscope was performed in the formalin-fixed-paraffin-embedded basal ganglia tissues of saline and SIV-infected rhesus macaques using an RNAscope Fluorescent Multiplex Reagent Kit (Advanced Cell Diagnostics, 320850) and HybEZ Hybridization System (Advanced Cell Diagnostics, 310010) according to the manufacturer's instructions. IL6 mRNA was detected using an RNAscope Made-To-Order Target Probes from Advanced Cell Diagnostics. The RNAscope assay was coupled with standard immunohistochemistry for cell-specific marker CD11b using fluorescent detection.

 

Transient transfection of miR-124 mimic and inhibitor.

Mouse primary microglial cells were seeded into 6-well plates (3 × 10⁵ cells per well) and were transiently transfected with 30 pmol of miR-124 mimic, miR-124 inhibitor, and miR control using Lipofectamine 2000 as described previously (Guo et al., 2016). Following transfection, cells were exposed to HIV-1 Tat (50 ng/ml) for another 24 h, and total RNA and proteins were extracted for further investigation as indicated.

 

Small interfering RNA (siRNA) transfection.

Mouse primary microglial cells were transfected with mouse DNMT1 siRNA (Santa Cruz Biotechnology, sc-35203) or mouse MECP2 siRNA (Santa Cruz Biotechnology, sc-35893) using Lipofectamine 2000 Reagent (Invitrogen, 11668019) as per the manufacturer's instructions. Briefly, the mouse primary microglial cells were seeded in a 6-well plate at a density of 3 × 10⁵ cells per well at 37°C in a humidified, 5% CO₂ incubator. At 70% confluence, the culture medium was replaced with 1 ml of Opti-MEM I Reduced Serum Medium (Invitrogen, 31985070). Meanwhile, Lipofectamine 2000 Reagent (3 μl/ml) and 120 pmol/ml of individually targeted siRNA were incubated separately with Opti-MEM I Reduced Serum Medium for 5 min at room temperature. Subsequently, the Lipofectamine 2000 mix was added to the individually targeted siRNA mix; this mixture was set aside for 20 min, after which the combined mixture was added to the cells. Scrambled siRNA mixture was also prepared similarly. The culture plate was then shaken gently for 5 s and incubated for 24 h at 37°C in a 5% CO₂ incubator (humidified). Knockdown efficiencies were determined by Western blotting.

 

qPCR.

qPCR experiments were performed according to the protocol described previously (Guo et al., 2016; Periyasamy et al., 2016). Briefly, total RNA was extracted using Quick-RNA MiniPrep Plus (Zymo Research, R1058) as per the manufacturer's protocol and quantified using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Reverse transcription reactions were performed using a Verso cDNA kit (AB-1453/B; Thermo Fisher Scientific), as per the manufacturer's instructions. qPCRs were completed using SYBR Green ROX qPCR Mastermix (QIAGEN, 330510). The 96-well plates were placed into an Applied Biosystems 7500 Fast Real-Time PCR Systems for a program running. Each reaction was performed in triplicate, and six independent experiments were run. GAPDH was used as a housekeeping control for the normalization, and the fold change in expression was obtained by the 2^−ΔΔCT method.

 

Western blotting.

Western blotting was performed using standard procedures as described previously (Guo et al., 2016; Periyasamy et al., 2016). Briefly, the control and treated microglial cells were harvested and lysed using the 200 μl of RIPA buffer (Cell Signaling Technology, 9806). Lysates were centrifuged at 12000 × g for 10 min at 4°C, and the protein content of the supernatant was determined by a BCA assay using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23227) as per the manufacturer's instructions; 10 μg of soluble proteins was resolved in a 10% SDS-PAGE, followed by blotting onto a PVDF membrane (Millipore, IPVH00010). Then, the membranes were blocked with 5% nonfat dry milk (in 1× TTBS buffer) for 1 h at room temperature followed by overnight incubation with the indicated primary antibodies at 4°C. After washing 3 times, the membranes were incubated with a secondary antibody for 1 h at room temperature. Next, the protein signals were visualized using Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, 34078). Each band intensity was normalized to the internal control, β-actin (Sigma-Aldrich catalog #A1978, RRID:AB_476692), and the data were presented as a relative fold change by using ImageJ analysis software (Schneider et al., 2012).

 

miR-124 ISH and CD11b immunostaining.

miR-124 ISH was performed using the methodology described previously (Guo et al., 2016). Briefly, mouse primary microglial cells were seeded into 24-well plate containing sterile glass coverslips (11 mm; 5 × 10⁴ cells per well) at 37°C in a humidified, 5% CO₂ incubator for 24 h. Overnight serum-starved mouse primary astrocytes were then exposed with HIV-1 Tat (50 ng/ml) for the next 24 h. Later, the cells were rinsed twice with 1 × PBS at room temperature and fixed with 4% PFA followed by prehybridization with the hybridization buffer (50% formamide, 10 mm Tris-HCl, pH 8.0, 200 μg/mg yeast tRNA, 1× Denhardt's solution, 600 mm NaCl, 0.25% SDS, 1 mm EDTA, 10% dextran sulfate) at 37°C for 1 h. In the meanwhile, locked nucleic acid-modified miR-124, labeled at both the 5′ and 3′ ends with digoxigenin, was diluted to a final concentration of 2 pm in hybridization buffer (heated to 65°C for 5 min) and individually hybridized with the sections at 37°C overnight. The slides were then washed twice in the hybridization buffer (without probe) at 37°C, followed by washing 3 times in 2× SSC and twice in 0.2× SSC at 42°C. The slides were then blocked with 1% BSA, 3% normal goat serum in 1 × PBS for 1 h; after that, slides were incubated with anti-digoxigenin conjugated with HRP and anti-Iba-1 antibodies overnight at 4°C. Following this, the slides were washed twice with 1 × PBS and incubated with AlexaFluor-488-conjugated goat anti-rabbit IgG (H+L) antibody for 1 h at room temperature. This was followed by 2 washes with 1 × PBS and signal amplification using TSA Cy5 kit (PerkinElmer). The coverslips were then mounted on glass slides with ProLong Gold Antifade Reagent with DAPI. Fluorescence images were taken on an Observer using a Z1 inverted microscope (Carl Zeiss), and the acquired images were analyzed using the AxioVs 40 version 4.8.0.0 software (Carl Zeiss).

 

Bisulfite-converted genomic DNA sequencing.

Bisulfite-converted genomic DNA sequencing was performed using the protocol described previously with slight modifications (Palsamy et al., 2012, 2014a,b,c; Guo et al., 2016). Briefly, genomic DNA extracted from mouse primary microglial cells was exposed to bisulfite conversion by EZ DNA Methylation-Direct Kit (Zymo Research). The bisulfite-modified DNA was amplified by bisulfite sequencing PCR using Platinum PCR SuperMix High Fidelity (Invitrogen) with primers specific to mouse primary miR-124-1, primary miR-124-2, and primary miR-124-3 promoter regions. Subsequently, the amplified PCR products were purified by gel extraction with Zymoclean Gel DNA recovery kit (Zymo Research) followed by cloning into pCR4-TOPO vectors using the TOPO TA Cloning kit (Invitrogen). The recombinant plasmids were transformed into One Shot TOP10 chemically competent Escherichia coli (Invitrogen) using the conventional chemical transformation method. Plasmid DNA was isolated from ∼10 independent clones of each amplicon with PureLink Quick Plasmid Miniprep Kit (Invitrogen) and then sequenced (High-Throughput DNA Sequencing and Genotyping Core Facility, University of Nebraska Medical Center, Omaha, NE) to determine the status of CpG methylation. Only the clones with an insert containing >99.5% bisulfite conversion (i.e., nonmethylated cytosine residues to thymine) were included in this study. The sequence data of each clone were analyzed for methylation in the miR-124 promoter by BISMA software (http://services.ibc.uni-stuttgart.de/BDPC/BISMA/) using default threshold settings.

 

Dual-luciferase reporter assay.

Mouse primary microglial cells were seeded into 96-well plates and cotransfected with target plasmids, such as pmirGLO-MECP2 3′UTR-miR-124-target or pmirGLO-MECP2 3′UTR-miR-124-target-mutant and miR-124 mimic/miR control in a molar ratio 10:1. The luciferase activity was determined 24 h after transfection, and the reporter assay was performed according to the manufacturer's protocol (Promega). Renilla luciferase activity was normalized to firefly luciferase and was expressed as a percentage of the control.

 

Ago2 immunoprecipitation.

Mouse primary microglial cells were plated onto 6-well plates and transfected with either miR mimic control or miR-124 mimic for 24 h. After treatment, cells were washed in the cold 1 × PBS, scraped, and then lysed with a buffer containing 0.5% NP40, 150 mm KCL, 25 mm Tris-glycine, pH 7.5, 2 mm EDTA, 0.5 mm DTT, and inhibitors of RNases, proteases, and phosphatases; 10% of total lysate was removed and kept as the input samples, and the remainder used for immunoprecipitation. A total of 10 μg of anti-Ago2 (Sigma-Aldrich catalog #SAB4200085, RRID:AB_10600719) or anti-FLAG (Sigma-Aldrich catalog #F1804, RRID:AB_262044) antibodies were kept overnight with protein A/G agarose beads (Thermo Fisher Scientific, 20423) at 4°C. Precleared lysates were then incubated with the appropriate antibody-bound beads, and the immunoprecipitated proteins were washed and incubated with DNase I (Invitrogen, 18068015) followed by digestion with proteinase K (Zymo Research, D3001-2) for 15 min. RNA extraction was then performed using Quick-RNA MiniPrep Plus (Zymo Research, R1058) and quantified using the NanoDropTM 2000 spectrophotometer (Thermo Fisher Scientific). Total as well as Ago2-immunoprecipitated RNA samples were then used for determining the binding targets of miR-124, such as MECP2 and STAT3 by qPCR.

 

Experimental design and statistical analyses.

All the data were expressed as mean ± SEM, and appropriate statistical significance was chosen based on the experimental strategy using GraphPad Prism version 6.01. The precise statistical analyses and experimental designs, including tests performed, exact p values, and sample sizes, are provided with the results describing each figure, or within the legend of each figure. Nonparametric Kruskal–Wallis one-way ANOVA followed by Dunn's post hoc test was used to determine the statistical significance between multiple groups, and Wilcoxon matched-pairs signed rank test was used to compare between two groups. For the in vivo experiments, unpaired Student's t test was used to compare between two groups. In all cases, values were statistically significant when p < 0.05.

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Results

SIV infection downregulates miR-124 in the basal ganglia of rhesus macaques

Based on the premise that miRs play pivotal roles in brain functioning by regulating gene expression of various CNS cells, we first sought to examine the miR expression profiles in the basal ganglia of saline (n = 4) and SIV-infected rhesus macaques (n = 6) using the Affymetrix microarray platform. Our microarray data showed that SIV infection significantly altered the expression profiles of various miRs, including 84 downregulated miRs, and 21 upregulated miRs in the basal ganglia of rhesus macaques compared with the saline group. Interestingly, most of the miRs altered during SIV infection were found to be associated with neuroinflammation (Fig. 1A). Of these, miR-124 was of interest to us as it was significantly downregulated in the basal ganglia of SIV-infected rhesus macaques compared with the saline group (Fig. 1B; p = 0.0477, unpaired Student's t test). Next, we wanted to validate the downregulation of miR-124 in the basal ganglia of SIV-infected rhesus macaques, by assessing the expression of mature miR-124 using qPCR. As represented in Figure 1C, the expression of mature miR-124 was significantly downregulated in the basal ganglia of SIV-infected rhesus macaques compared with the saline group (p = 0.0001, unpaired Student's t test). Downregulation of miR-124 was not region-specific because other brain regions, such as the cortex, also exhibited a similar downregulation of miR-124 in the SIV-infected rhesus macaques (data not shown). Collectively, these results demonstrated that SIV infection significantly decreased the expression levels of mature miR-124 in the basal ganglia of rhesus macaques