Human Fetal Brain-Derived Neuronal Precursor Cell Culture
Human brain tissues collected from elective abortions with the informed consent of mothers, were processed as per the protocols laid down by the Institutional Human Ethics and Stem Cell Research Committee under strict compliance with the guidelines of ICMR, India. Neural Precursor Cells (NPCs) were derived from the telencephalon region of the aborted fetuses of age 10–14 weeks. NPCs were cultured on poly-D-lysine (Sigma-Aldrich, Missouri, USA) coated flasks in Neurobasal Media (Invitrogen, California, USA) supplemented with N2 supplement (Invitrogen, USA), Neural Survival Factor-1 (Lonza, Charles City, IA), 20 ng/mL EGF (Sigma-Aldrich, USA) and 25 ng/mL bFGF (Sigma-Aldrich, USA). NPCs were further assessed for the expression of markers such as Nestin and SOX2 and 99% of the cells were found to be positive. They were also assessed for their ability to form neurospheres and differentiate into Astrocytes and Neurons.
Human Primary Astroglia and Neuronal Culture
NPCs were differentiated into astroglia by replacing the media with Eagle’s Minimal Essential Media (MEM) (Sigma-Aldrich, USA) containing 10% Fetal Bovine Serum (Gibco, California, USA). These cells were cultured for 3 weeks, with half media changes on alternate days, after which they were assessed for the expression of GFAP and Vimentin, for which more than 95% of cells were found immunopositive (Supporting Information Fig. S1). To differentiate NPCs into neurons, EGF and FGF from NPC media were replaced with 10 ng/mL BDNF (Peptrotech, New Jersey, USA) and 10 ng/mL PDGF-AB (Peptrotech, New Jersey, USA). After maintaining them for 3 weeks, with half media changes on alternate days, they were assessed for expression of Tuj-1 and MAP2, for which more than 95% of cells were immunopositive.
[Ca2+]i measurement / Live cell imaging
0.2 million cells were seeded in 35-mm dishes and incubated with 2.5 µM Fluo-4-AM (Invitrogen, USA) in HEPES buffer at 37 0C for 30 min. After 3 washes, the dish was mounted on the stage for live-cell imaging using a spinning disc confocal microscope (Zeiss, Oberkochen, Germany) 10 x objective (Zeiss, Germany) was used for the measurement of fluorescent intensity as a measure of the change in [Ca2+]i. Baseline fluorescence intensity was recorded for the initial 1 minute, thereafter,10 µM ATP (Sigma-Aldrich, USA) was added to the static bath followed by 5 min of recording. Change in fluorescence intensity was calculated by subtracting fluorescence at t = 0 from all the fluorescence intensity values. ZEN software (Zeiss, Germany) was used for data acquisition and analysis. Areas having a significant number of cells (≥ 50) were randomly selected for the recordings after which individual cells were marked as the region of interests and fluorescence intensity was measured across time.
Astrocytes were seeded in a 4-well chamber slide (Nunc, Kamstrupvej, Denmark) at a density of 30,000 cells per well. After 24 h, the cells were fixed using 4% paraformaldehyde, washed thrice with 1X PBS, blocked and permeabilized using 4% BSA containing 0.4% Triton-X 100. The cells were then incubated with antibodies, mouse anti-Coronin 1A (Santa Cruz Biotechnology, Texas, USA, 1:100), moues anti- Ki 67 (Millipore, Billerica, USA, 1:1000), mouse anti-Vimentin (Santa Cruz Biotechnology, USA, 1:1000), overnight at 4 0C or Anti-GFAP (Dako, California, USA, 1:1000) for 1 h at 25 °C. The cells were then washed thrice with 1X PBS and incubated with appropriate fluorophore tagged secondary antibodies (Invitrogen, USA, 1:1000). The wells were washed thrice with 1X PBS and mounted using Hardset mounting media with DAPI (Vector Labs, Burlingam, USA). For each group, a minimum of five images were captured from random fields using the AxioImager Z1 microscope (Zeiss, Germany).
Astroglia knocked down for coronin 1A for 48 h were serum-starved for 2–3 h and then stimulated with 10 µM ATP (Sigma-Aldrich, USA). ATP stimulations were given for either 0, 45, 120, and 300 sec or 5, 10, 30, 60, 120 min, after which the cells were harvested using lysis buffer. The lysates were further processed for protein analysis.
HIV-1 Tat Transfection
Astroglia cultures with 80% confluency were used for transfections with expression vector pcDNA3.1 expressing full-length HIV-1 Tat B, which was a kind gift from Prof. Udaykumar Ranga, Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), India. Transfections were done using Lipofectamine 3000 (Invitrogen, USA) as per the manufacturer’s protocol. After 24 h of transfection, cells were further harvested as per the experimental requisite.
Small Interfering RNA (siRNA) mediated knockdown of coronin 1A
Human primary astrocytes were knocked down using siRNA against coronin 1A. 10 nM coronin 1A siRNA (Dharmacon, Colorado, USA) or control scrambled siRNA (Dharmacon, USA) was transfected using lipofectamine RNAimax (Invitrogen, USA) following manufacturer’s protocol. The cells were either harvested after 48 h of transfection to check the knockdown efficiency or carried over for further treatments or transfections.
The cells were harvested using lysis buffer, which consisted of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 50 mM Sodium fluoride, 1 mM sodium orthovanadate, 1 mM EDTA (pH 8.0), 2% SDS, and protease inhibitor cocktail (Roche, Basel, Switzerland). Estimation of protein concentration was done using 4% copper sulfate and bicinchoninic acid (Sigma-Aldrich, USA). Proteins were resolved on 10–13% SDS- PAGE and transferred onto a nitrocellulose membrane (MDI, Ambala, India). The blots were blocked using 5% skimmed milk in PBS-Tween 20 (PBST) followed by incubation with primary antibodies, mouse anti-Coronin 1A (Santacruz biotech, USA, 1:500), mouse anti-GAPDH (Santacruz biotech, USA, 1:1000), rabbit anti-Phospho-p44/42 MAPK (Cell Signaling Technology, Massachusetts, USA, 1:2000), rabbit anti-p44/42 MAPK (Cell Signaling Technology, USA, 1:2000), rabbit anti-Phospho-PLCγ1 (Cell signaling, USA,1:1000), rabbit anti-PLCγ1 (Cell signaling, USA,1:1000), rabbit anti-GFAP (Dako, USA, 1:20000). Blots were washed thrice with TBS-Tween 20 (TBST) and then incubated with appropriate HRP labeled secondary antibodies (Vector Labs, USA, 1:4000) for 2 h at room temperature. The blots were washed thrice with TBST and then developed using Chemiluminescent Reagent (Millipore, USA) and were imaged using Gel Documentation System (Uvitech, Cambridge, UK). Densitometry of the protein bands was done using ImageJ software (NIH, Maryland, USA).
Quantitative Real-Time PCR
The cells were harvested for RNA isolation using Trizol reagent (Ambion, Texas, USA) following the manufacturer’s protocol. The purified RNA was utilized for synthesizing cDNA using High-capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, California, USA). qPCR was performed using SYBR Green Master Mix (Applied Biosystems, USA) using the following primers: CORO1A forward 5'-CACCAACATCGTCTACCTCTG-3' and reverse 5'-ACTCCTTGGAACTGAACATGG-3', GAPDH forward 5'-CAAGAGCACAAGAGGAAGAGAG-3' and reverse 5'-CTACATGGCAACTGTGAGGAG-3', GFAP forward 5'-ACCTGCAGATTCGAGAAACCAG-3' and reverse 5'-TAATGACCTCTCCATCCCGCATC-3', Vimentin forward 5'- AAGTCCGCACATTCGAGCAA-3' and reverse 5'-CTACCAACTTACAGCTGGGC-3', VCAM1 forward 5'-GGGAAGCCGATCACAGTCAA-3' and reverse 5'-TCCTGTCTGCATCCTCCAGA-3', ICAM1 forward 5'-CCGCAGTCATAATGGGCACT-3' and reverse 5'-GGTTTCATGGGGGTCCCTTT-3', PTGS2 forward 5'-TGTATGAGTGTGGGATTTGACC-3' and reverse 5'-TGTGTTTGGAGTGGGTTTCAG-3', NOS2 forward 5'-CTTTGCCTGTATGCTGATGC-3' and reverse 5'-GCCTCTGATTTTCCTGTCTCTG-3'.
miRNA along with total RNA was isolated using the miRNEASY mini kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. Subsequently, the cDNA was synthesized using the miScript II RT kit (Qiagen, Germany) as per the manufacturer’s protocol. The qPCR assay was done using the miScript SYBR Green PCR kit (Qiagen, Germany) following the manufacturer’s protocol. The qPCR was done in ViiA7™ (Applied Biosystems, USA). The specificity of the primers was confirmed via analyzing the melt curve.
IL-6-release measurement by Flow-cytometry
Astroglia-conditioned-media (ACM) was collected from astroglia knocked down for coronin 1A for 48 h and transfected with HIV-1 Tat for 24 h. The conditioned-media thus collected was then analyzed for the levels of released pro-inflammatory cytokine interleukin-6 (IL-6) by using flow-cytometry based Cytometric bead array (CBA)-human inflammatory cytokines kit (BD Biosciences, San Diego, USA), and following manufacturer’s protocol.
ACM was collected from astroglia knocked down for coronin 1A for 48 h and transfected with HIV-1 Tat for 24 h. The conditioned media thus collected was analyzed for the levels of released glutamate using an enzyme kinetics-based glutamate determination kit (Sigma-Aldrich, USA), following the manufacturer’s protocol.
Assessment of Neuronal Survival by TUNEL Assay
ACM was collected from astroglia knocked down for coronin 1A for 48 h and transfected with HIV-1 Tat for 24 h. Neurons at a density of 20,000 cells/well were seeded in Poly-D-Lysine coated 8 well-chambered slides. The next day the cells were treated with 50% ACM and Neuronal Media for 24 h. Cells were then fixed using 4% paraformaldehyde. Washed thrice with 1x PBS, blocked and permeabilized using 4% BSA containing 0.1% Triton-x 100. Cells undergoing apoptosis were detected using in situ Cell Death Detection Kit, TMR red (Roche, Switzerland), following the manufacturer’s protocol. Cells were washed thrice with 1x PBS, and the slide was mounted using Hardset mounting media with DAPI (Vector Labs, USA). Five images were captured for each group using the AxioImager Z1 microscope (Zeiss, Germany).
miRNA Target Prediction
miRWalk (http://mirwalk.umm.uni-heidelberg.de/) online prediction tool was used to filter miRNAs targeting 3’ UTR of CORO1A.
miRNA Mimic and Inhibitor Transfection
Transfection was done on astroglia, in 80% confluent T25 flasks using RNAi MAX (Invitrogen, USA) as per the manufacturer's protocol. 62.5 nM of Syn-hsa-miR-92b-5p miScript miRNA mimic (Qiagen, Germany) was used for mimic transfection. All-Stars Negative Control siRNA (Qiagen, Germany) was used as mimic control. miRNA inhibition was done using 62.5 nM of miRCURY LNA miRNA inhibitor against has-miR-92b-5p (Qiagen, Germany) and miRCURY LNA miRNA Inhibitor Control (Qiagen, Germany) was used as the inhibitor control. Transfections were done using Opti-MEM media (Invitrogen, USA) for 5 h and replaced with complete MEM. Cells were processed after 48 hours of transfection for RNA and Protein analysis. Effective doses of mimic and inhibitor miRNA were standardized via qPCR and protein studies.
Cloning of CORO1A 3’ UTR and Luciferase Assay
The 3’ UTR sequence of coronin 1A, being very short in length (111 bp), was outsourced in the form of 2 single-stranded nucleotide chains having restriction sites for SpeI and HindIII respectively. Both the chains were annealed, digested, and was cloned into pMIR-Report plasmid between SpeI and HindIII restriction sites. The cloned plasmid was then sequenced to confirm the successful insertion.
CORO1A 3’UTR luciferase reporter plasmid was co-transfected with 12.5 nM of mimic-92b-5p into HeLa cells, using Lipofectamine 3000 as per manufacturer’s protocol. After 24 h the samples were harvested for luciferase detection, using Luciferase Detection Kit (Promega, Wisconsin, USA) following the manufacturer’s protocol, in Tecan SPARK multiplate reader (Tecan, USA). The readings were normalized using total protein content.
Experiments of all kinds were performed independently three to five times; Student’s t-test was used to assess statistical significance between control and experimental groups. P values < 0.05 were considered statistically significant. * represents p < 0.05, **p < 0.005, and ***p < 0.0005.