All animal experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Hanks balanced salt solution (HBSS) and penicillin-streptomycin solution were from Mediatech Cellgro (Manassas, VA, USA). Alexa Fluor® 488 phalloidin, 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) AM, 2’,7’-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluoreescein (BCECF) AM, calcein AM, 4',6-diamidino-2-phenylindole (DAPI), Dulbecco’s Modified Eagle Medium (DMEM), DMEM-F12, fetal bovine serum (FBS), Fluo-4 AM, Fura-Red AM, goat anti-mouse Alexa Fluor® 488-conjugated IgG, goat anti-rabbit Alexa Fluor® 546-conjugated IgG, Lipofectamine® RNAiMAX Transfection Reagent, Opti-MEM®, sodium-binding benzofuran isophthalate (SBFI) AM, To-pro-3 iodide and Versene were from Invitrogen (Carlsbad, CA, USA). Bradykinin, Cytochalasin D, gramicidin, monensin and nigericin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-NHE-1 monoclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-ezrin polyclonal antibody, rabbit anti-ezrin/radixin/moesin polyclonal antibody, rabbit anti-phospho-ezrin (Thr567) / radixin (Thr564) / moesin (Thr558) polyclonal antibody, and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody were purchased from Cell Signaling (Danvers, MA, USA). Rabbit anti-Na+/Ca2+ exchanger isoform 1 (NCX-1) polyclonal antibody was purchased from Swant (Marly, Switzerland). HOE 642 was a kind gift from Aventis Pharma (Frankfurt, Germany). SEA0400 was a kind gift from Taisho Pharmaceutical Co. Ltd. (Omiya, Saitama, Japan).
Primary microglial culture
Mixed primary glial cultures were established from mouse brains (Black Swiss; Taconic Farms, Inc., Hudson, NY, USA) as described previously [15
] with minor modifications. Briefly, dissociated cells from brain tissues of 1-3 day-old mice (male and female) were collected. The cell suspension was centrifuged and resuspended in the DMEM-F12 complete medium (containing 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin). The cells were seeded in poly-D-lysine-coated culture flasks and maintained at 37°C with 5% CO2
+ 95% air in an incubator (Thermo Scientific; Model: 3110) and refed three times a week. Following 10-14 days of incubation, microglia were removed from the astroglial layer by shaking the flasks on an orbital shaker (Labnet; Model: Orbit P4) for 2 h at 250 g. The microglial pellets were resuspended in the complete DMEM medium and seeded on the poly-D-lysine coated glass coverslips in 6-well plates (4-5 × 105
cells/well) or on 35 mm glass bottom dishes (4-5 × 105
cells/dish). Experiments were performed on day 1-3 after seeding.
exchanger isoform 1 heterozygous (NCX-1+/-
) mouse colony in SV129/Black Swiss background was maintained as described previously [16
]. For NCX-1+/-
cultures, tail biopsy was obtained from each pup and polymerase chain reaction (PCR) analysis of DNA was performed to determine the genotype of each culture as described previously [16
BV2 microglial culture
Murine BV2 microglial cells were cultured in T25 flasks in the complete DMEM-F12 medium. Cell cultures were maintained at 37°C with 5% CO2 + 95% air and subcultured (1:4) every 2 days by gently removing the cells with Versene. Cells were seeded on poly-D-lysine coated glass coverslips in 6-well plates (105 cells/well) or on 35 mm glass bottom dishes (105 cells/dish). Experiments were performed on day 1 after seeding. Cultures of passage 18 or lower were used in this study.
Cell motility measurement
Microglial cells seeded on 35 mm glass bottom dishes were placed in a stage top incubator at 37°C with 5% CO2 + 95% air (model: TIZ, Tokai Hit; Shizuoka-ken, Japan). Motility of microglia was monitored under the 40 × oil immersion objective lens with a time-lapse video microscope system (the Nikon TiE 300 inverted epifluorescence microscope with perfect focus) and MetaMorph software (Molecular Devices, Sunnyvale, CA, USA). Time-lapse DIC images were acquired in 1-min intervals for 60 min either under serum-free DMEM control condition or in the presence of BK, HOE 642, SEA 0400, or BAPTA AM. Images were analyzed by ImageJ software (National Institute of Health, USA) and cell tracking was performed using the Manual Tracking plugin. Total distance traveled was determined by tracking the movement of the cell gravity center, and its coordinates were used to calculate the distances.
In some studies, BV2 or primary microglia were incubated with 0.5 µM calcein AM for 30 min at 37°C. Calcein fluorescence in a cell was monitored under a 40 × objective lens using a FITC filter set (excitation 480 nm, emission 535 nm, Chroma Technology, Rockingham, VT, USA).
Image analysis for microglial lamellipodial morphological changes
Analysis for morphological changes of lamellipodia in microglia was performed using the ImageJ software. To measure fluctuations in lamellipodial area through time, the lamellipodia was traced manually every 2 min and area calculated with the region of interest (ROI) manager. Lamellipodial length was defined as the furthest point of lamellipodia to cell body and quantified using the Straight Line tool. Lamellipodial persistence was defined as the time of lamellipodial extension along the moving direction before it retracts, and was calculated using the kymograph plugin as described before [12
]. Protrusion rate was calculated as lamellipodial length divided by persistence (µm/min). Lamellipodial change was calculated by dividing the mean lamellipodia area over time by the corresponding persistence (µm2
/min). A minimum of five cells were analyzed from each experiment and an average calculated.
measurement in microglia was performed as described previously [17
]. Briefly, microglial cultures grown on coverslips were incubated with 0.5-2.5 µM BCECF AM at 37 °C for 30 min. The coverslips were placed in a temperature-controlled (37 °C) open-bath imaging chamber containing HCO3-
-free HEPES-MEM [17
]. On the stage of the Nikon TiE 300 inverted epifluorescence microscope, the cells were visualized with a 40 × oil immersion objective lens and excited every 20 s at 440 and 490 nm, and the emission fluorescence at 535 nm was recorded. Images were collected using a Princeton Instruments MicroMax CCD camera and analyzed with MetaFluor image-processing software (Molecular Devices, Sunnyvale, CA, USA) as described previously [17
Intracellular Ca2+ measurement
) elevation was detected by ratiometric imaging of Fluo-4 and Fura-Red fluorescent signals as described before [18
]. Cells grown on coverslips were incubated with 7.5 µM Fluo-4 AM and Fura-Red AM (with 0.01% pluronic acid and 0.2% DMSO) in the HEPES-MEM buffer at room temperature for 30 min. Cells were washed and then incubated at 37°C for another 30 min to allow deesterification prior to the measurement. Under the similar setting described in the pHi
measurement, cells were excited at 490 nm and the emission fluorescence at both 530 nm (Fluo-4) and 660 nm (Fure-Red) wavelengths collected. Images were collected every 5 min for 40-70 min and analyzed with the MetaFlour image-processing software. The changes of [Ca2+
in single cells were determined by calculating fluorescence intensity ratios of Fluo-4/Fura-Red.
Intracellular Na+ measurement
) was measured in primary microglia with the fluorescent dye SBFI AM as described previously [19
]. Cultured microglia grown on coverslips were loaded with 10 µM SBFI AM plus 0.02% pluronic acid at 37°C for 60 min. Using the Nikon TiE 300 inverted epifluorescence microscope and a 40 × oil immersion lens, cells were excited at 345 and 385 nm, and the emission fluorescence at 510 nm was recorded. Images were collected every 5 min for 35 min to determine Na+i
levels, and the 345/385 ratios were analyzed with the MetaFluor image-processing software. Absolute [Na+
was determined for each cell as described before [19
Chemotaxis of microglia was tested using a 24-well microchemotaxis Boyden chamber (8 µm pore size; BD Falcon, Sparks, MD, USA) as described before [5
]. Microglial cells (105
/mL) in 100 µL of serum-free DMEM with or without 1 µM HOE 642 were added to the upper wells, and the lower wells contained either 700 µL serum-free DMEM or DMEM plus 300 nM BK. The chamber was incubated at 37°C and 5% CO2
in the incubator for 5 h. Cells remaining on the upper surface of the membrane were removed with cotton-tipped swabs. The membrane was rinsed with PBS and the migrated cells on the bottom surface of the membrane were fixed with 4% paraformaldehyde (PFA) and subjected to DAPI staining (2 µg/mL in PBS). Samples were excited at 358 nm with a xenon lamp and the emission fluorescence at 461 nm recorded with a Princeton Instruments MicroMax CCD camera attached to the Nikon TiE300 microscope using the MetaMorph software. Images of 5 random fields (220 µm × 165 µm) under the 40 × objective lens were captured. Migrated cells in all 5 fields were averaged to give a mean cell count for each experiment. Migration rate was expressed as percentage of control.
Actin staining and quantification
Cells were fixed in 3.7% methanol-free formaldehyde solution in PBS for 10 min at room temperature. After rinsing in PBS, cells were extracted with 0.1% Triton X-100 in PBS for 5 min. After washing, cells were incubated with 1 unit of Alexa Fluor® 488 phalloidin in PBS/1% BSA (200 µL/coverslip) for 20 min. Cells were rinsed and incubated with To-pro-3 iodide (1:1000 in staining solution) for 15 min. Fluorescence images were captured with a Leica DMIRE2 inverted confocal laser-scanning microscope under the 40 × oil immersion objective lens (Leica Software, Mannheim, Germany). Samples were excited at 488 nm (argon/krypton) and 633 nm. The emission fluorescence was recorded at 512-548 nm and 650-750 nm, respectively. Actin quantification was performed using the ImageJ software on phalloidin-staining images. The lamellipodial area of each cell was traced with the ROI manager, and phalloidin fluorescent intensity was measured in each ROI. A minimum of five cells were analyzed from each experiment and an average intensity was calculated.
Immunofluorescence staining and image analysis
Microglial cells grown on coverslips were fixed in 4% PFA in PBS. For Cytochalasin D (Cyt.D) treatment, 100 nM Cyt.D was added into the medium 20 h prior to fixation. After rinsing in PBS for 15 min, cells were incubated with a blocking solution (PBS/0.3% Triton X-100/5% goat serum) for 60 min. Cells were incubated with monoclonal mouse anti-NHE-1 antibody (1:100) and polyclonal rabbit anti-NCX-1 antibody (1:200) or polyclonal rabbit anti-ezrin antibody (1:200) diluted in the antibody diluting solution (PBS/0.3% Triton X-100/1% BSA) at 4°C overnight. After rinsing in PBS, cells were incubated with goat anti-mouse secondary antibody IgG conjugated to Alexa Fluor® 488 and goat anti-rabbit secondary antibody IgG conjugated to Alexa Fluor® 546 (1:200 dilution) for 2 h. Cells were then rinsed and incubate with To-pro-3 iodide (1:1000 in antibody diluting solution) for 15 min. Fluorescence images were captured with the Leica DMIRE2 inverted confocal laser-scanning microscope as described before [15
]. Co-localization of NHE-1 and ezrin was analyzed using ImageJ software with the Colocalization Indices plug-in [20
]. The Pearson’s correlation coefficient (CC) and overlap coefficient (OC) were used as indices of the frequency of co-localization between NHE-1 and ezrin as described previously [20
]. For quantitative analysis, single optical sections were taken randomly from five to seven different regions on each coverslip. Cell roundness was calculated as 4π × cell area/(cell perimeter)2
, with smaller value indicating more elongated morphology [21
RNA interference (RNAi) knockdown of NHE-1
Knockdown of NHE-1 protein expression in BV2 microglial cells was induced by the double-strand small interfering RNAs (siRNAs). SiRNA targeting NHE-1 (Sequence #1) was prepared as 3’-overhanged form (forward, 5’-CCACAAUUUGACCAACUUAtt-3’; reverse, 5’-UAAGUUGGUCAAAUUGUGGtc-3’; Invitrogen, Carlsbad, CA, USA). Stealth RNAi Negative Control (low GC content; Cat. No 12935-200; Invitrogen) was used as a control. Lipofectamine® RNAiMAX/siRNA complexes were formed in serum-free Opti-MEM® at 25°C for 5 min and added to 6-well plate. 250 µL of complexes in Opti-MEM containing 50 pmol siRNA and 7.5 µL Lipofectamine® RNAiMAX was added into each well. 105 of BV2 cells in DMEM-F12 supplemented with 10% FBS was then added into each well to make a final volume of 2.5 mL per well and incubated at 37°C. Cultures were used at 48 h after transfection.
As shown in Figure S1
, Sequence #1 showed excellent efficiency in selective knockdown of NHE-1 protein and did not affect expression of 2 other proteins NCX-1 and tERM, which are closely related in regulation of microglial migration. In contrast, Sequence #2 (forward, 5’-CGAAGAGAUCCACACACAGtt-3’
; reverse, 5’-CUGUGUGUGGAUCUCUUCG
tt-3’. Ambion) had minimum effects on reducing NHE-1 expression but downregulatied NCX-1 protein significantly (Figure S1
). In light of the high efficiency and selectivity of NHE-1 siRNA sequence #1, we have used it in all experiments of this study.
Immunoprecipitation and immunoblotting
BV2 cells were lysed in ice-cold lysis buffer (0.025 M Tris, 0.15 M NaCl, 0.001 M EDTA, 1% NP-40, 5% glycerol, pH 7.4) containing protease inhibitors and phosphatase inhibitor cocktail, and centrifuged at 11000 g for 10 min at 4°C. The supernatant was collected and protein content was determined by the bicinchoninic acid method. Protein samples (40-100 µg protein lysate) and pre-stained molecular mass markers (Bio-Rad) were denatured and separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, immunoprecipitation was conducted to examine interactions of NHE-1 and ezrin using the Pierce® Classic IP Kit (ThermoScientific, Rockford, IL, USA). Protein lysate samples (0.5 mg protein) were incubated with 20 µL of rabbit polyclonal antibody against ezrin at 4°C overnight. Immunocomplexes were mixed with 20 µL protein A/G beads (50% slurry) in a Pierce spin column and incubated for 2 h. The immunocomplexes were washed and dissociated from beads with the Laemmli sample buffer and heated at 95°C for 10 min. The resolved proteins and prestained molecular mass markers were separated on 10% SDS-PAGE, as described before [17
]. The blots were incubated with rabbit anti-ezrin/radixin/moesin polyclonal antibody (1:1000), rabbit anti-phospho-ezrin (Thr567)/radixin (Thr564)/moesin (Thr558) polyclonal antibody (1:200), rabbit anti-GAPDH monoclonal antibody (1:5000), or rabbit anti-NHE-1 polyclonal antibody (1:500) overnight at 4°C. After rinsing, the blots were incubated with goat anti-rabbit or goat anti-mouse horseradish peroxidase-conjugated secondary IgG (1:2000) for 1 h. Bound antibody was visualized using the enhanced chemiluminescence assay (ThermoScientific, Rockford, IL, USA). Densitometric measurement of each protein band was performed using the Gel Analysis Tool in ImageJ.
Data are expressed as the mean ± SEM. Comparisons between groups were made by Student’s t-test or one-way ANOVA using the Bonferroni post-hoc test in the case of multiple comparisons (SigmaStat, Systat Software, Point Richmond, CA, USA), unless otherwise indicated. p < 0.05 was considered statistically significant. n values represent the number of independent cultures.