Conceived and designed the experiments: YO YY HK KI TF HB. Performed the experiments: YO YY HK YS TF. Analyzed the data: YO YY HK KI YS TF HB. Wrote the paper: YO YY HK KI TF HB.
The authors have no financial relationships and no conflicts of interest relevant to this article.
Phospholipase D4 (PLD4) is a recently identified protein that is mainly expressed in the ionized calcium binding adapter molecule 1 (Iba1)-positive microglia in the early postnatal mouse cerebellar white matter. Unlike PLD1 and PLD2, PLD4 exhibits no enzymatic activity for conversion of phosphatidylcholine into choline and phosphatidic acid, and its function is completely unknown. In the present study, we examined the distribution of PLD4 in mouse cerebellar white matter during development and under pathological conditions. Immunohistochemical analysis revealed that PLD4 expression was associated with microglial activation under such two different circumstances. A primary cultured microglia and microglial cell line (MG6) showed that PLD4 was mainly present in the nucleus, except the nucleolus, and expression of PLD4 was upregulated by lipopolysaccharide (LPS) stimulation. In the analysis of phagocytosis of LPS-stimulated microglia, PLD4 was co-localized with phagosomes that contained BioParticles. Inhibition of PLD4 expression using PLD4 specific small interfering RNA (siRNA) in MG6 cells significantly reduced the ratio of phagocytotic cell numbers. These results suggest that the increased PLD4 in the activation process is involved in phagocytosis of activated microglia in the developmental stages and pathological conditions of white matter.
Phospholipase D4 (PLD4) is a member of the recently defined non-classical PLD family, which is characterized by two conserved HKD motifs (His-x-Lys-xxxx-Asp) in the C-terminal region
The expression of PLD4 is strictly regulated in mouse brain development. By RT-PCR analysis, PLD4 mRNA was first detected in mouse cerebellum at postnatal day 0 (P0), increased with age and peaked at P7, and then rapidly decreased to adult levels by P21
In the present study, we investigated the role of PLD4 in microglia. We analyzed the distribution of PLD4 mRNA in mouse cerebellar white matter, during development and under pathological conditions, to determine whether PLD4 expression was associated with microglial activation. The function of PLD4 was examined using a primary cultured microglia and microglial cell line; both of which were derived from C57BL/6J mouse brain. Our results demonstrated that PLD4 expression was closely associated with microglial activation, and inhibition of its expression by siRNA led to a significant decrease in phagocytotic cells. This suggests that this protein is involved in phagocytosis of microglia in the central nervous system (CNS) under physiological and pathological conditions.
C57BL/6J mice were purchased from Japan SLC (Hamamatsu, Japan) and sacrificed at postnatal day (P) 0, 3, 5, 7, 10 and 21. The transgenic mouse line that contained two copies of mouse myelin proteolipid protein (PLP) gene
For microglial culture, a mixed glial culture was prepared from cerebral cortices of 1-day-old C57BL/6J mice according to the method of Giulian and Baker (1986)
MG6 cell line is a c-myc-immortalized cell line of mouse microglia
To determine the microglial activation levels during lipopolysaccharide (LPS) stimulation, the amount of tumor necrosis factor (TNF)-α in the culture medium was measured by mouse TNF-α instant ELISA kit, according to the manufacturer's instructions (Bender Med Systems, Vienna, Austria).
The following antibodies were used for immunohistological and immunocytochemical studies: rabbit polyclonal anti-Iba1 antibody (1∶400) (Wako, Osaka, Japan); rat monoclonal anti-myelin basic protein (MBP) (1∶100) antibody (Chemicon/Millipore, Billerica, MA, USA); rabbit polyclonal anti-PLD4 C-terminal antibody (1∶200), which was produced by immunization with 16 amino acid residues (from amino acid 488 to 503, YAMDLDRQVPSQDCVW) of PLD4
PLD4 cDNA (nucleotides 103–1613 corresponding to Genbank NM_178911) was cloned in pcDNA3 vector and used to prepare the probes. Digoxigenin-labeled antisense riboprobes were prepared using DIG RNA labeling kit (Roche, Basel, Switzerland). Paraffin sections of cerebella were prepared from P0–P21 C57BL/6J mice. The sections (6 µm) were treated with proteinase K (P0, 2 min; P3, 5 min; P5, 10 min; P7 and P10, 15 min; P21, 20 min), as described previously
The paraffin sections (6 µm) were boiled in citrate buffer (pH 6.0) for 1 min in a microwave oven for heat-induced antigen retrieval. The sections were incubated for 1 h in 0.01 M phosphate-buffered saline (PBS) that contained 0.3% Triton X-100 and 10% goat serum (PBS-TGS), and then overnight at 4°C with primary antibodies diluted in PBS-TGS. After rinsing, the sections were incubated with biotinylated secondary antibodies for 30 min at room temperature (RT). They were incubated with the ABC reagent (1∶50) (Vector Laboratories) for 30 min at RT, and immunoreactions were visualized using 0.005% H2O2 in 3,3′-diaminobenzidine/50 mM Tris buffer for 10 min at RT. Images were captured by light microscopy (Axio Scope Imaging System; Carl Zeiss, Oberkochen, Germany). For quantification, the cells were counted in three different regions (gray matter, proximal and distal white matter). Distal white matter indicated subcortical white matter. PLD4-positive cell numbers were obtained from six individual sections from mice at each age. The areas were measured by ImageGauge v4.23 (Fujifilm Tokyo Japan).
Primary microglia were collected by gentle shaking by hand, and were transferred directly onto poly-L-lysine coated 13-mm cover slips overnight. A total of 105 MG6 cells were grown on 13 mm cover slips overnight. The cells were treated with LPS (500 ng/ml) or vehicle (PBS) for 24 h. The cells were fixed with 4% paraformaldehyde on ice for 30 min, and preincubated for 1 h in PBS-TGS. The cells were incubated overnight at 4°C with primary antibodies diluted in PBS-TGS. The cells were then incubated with Alexa 350-, 488-, or 594-conjugated secondary antibodies for 1 h at RT. Images were captured by confocal microscopy (Olympus, Tokyo, Japan). Intensity of PLD4 signal per µm2 of each nucleus was measured by FV10-ASW v3.0 (Olympus).
The MG6 cells were plated at 2.5×105 cells on a 100-mm Petri dish. After cells became confluent, the plate was washed with PBS, and homogenization buffer that contained 0.32 M sucrose, 5 mM Tris–HCl, pH7.5, 0.75 µM aprotinin, 1 µM leupeptin, 1 µM pepstatin, 0.4 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM dithiothreitol (DTT) was added. MG6 cells were harvested by a cell scraper, and lysed by syringe with sequential passes through 21 G, 23 G and 26 G needles. For nuclear fractionation, hypotonic homogenization buffer that contained 0.5% Nonidet-P40, 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.75 µM aprotinin, 1 µM leupeptin, 1 µM pepstatin, 0.4 mM PMSF, and 1 mM DTT was used. The nuclear fraction was collected as the pellet by centrifugation of whole cell homogenate at 8,000 rpm (MX-200; Tomy, Tokyo, Japan) for 15 min. The protein concentration was determined by the bicinchoninic acid method (Pierce/Thermo Scientific, Rockford, IL, USA). For deglycosylation, the samples were incubated at 95°C for 5 min in the presence of 0.5% sodium dodecyl sulfate (SDS) and 100 mM 2-ME, and cooled on ice for 3 min. Triton X-100 were added to a final concentration of 2.5%, and incubated with 0.25 U/30 µg protein of Peptide: N-glycosidase F (PNGase F) (Roche) for 16 h at 30°C.
Sample preparation, SDS-polyacrylamide gel electrophoresis (PAGE), and immunoblot analysis were performed as previously described
The commercially available double-stranded siRNA oligonucleotide against PLD4 gene was purchased from Invitrogen. The MG6 cells were transfected by 100 nM siRNA against PLD4 or control siRNA (Invitrogen) for 48 h with the PrimaPort siRNA transfection reagent (Credia-Japan, Kyoto, Japan), according to the manufacturer's protocol. The sequences of the double strand PLD4-siRNA sets were as follows:
sense siRNA;
anti-sense siRNA;
Total RNA was extracted from MG6 cells grown in six-well tissue culture plates (7×104 cells/well) by TRIzol Plus RNA Purification Kit (Invitrogen). Isolated total RNA was then reverse transcribed with TaKaRa RNA LA PCR™ Kit (AMV) Ver.1.1 (Takara Bio Inc., Shiga, Japan). PLD4 and control glyceraldehyde-3-phoshate dehydrogenase (GAPDH) cDNAs were amplified by the following specific primer sets:
PLD4 forward;
PLD4 reverse;
GAPDH forward:
GAPDH reverse;
Primary microglia were transferred directly onto cover slips and MG6 cells were plated at 105 cells/well in six-well tissue culture plates that contained 1.5 ml DMEM/10% FBS. The cells were treated with LPS (500 ng/ml) or vehicle for 24 h. For simple phagocytosis assay, Alexa-594-conjugated
For siRNA treatment, MG6 cells were plated at 7×104 cells/well in six-well tissue culture plates. The cells were treated with PLD4-siRNA (100 nM), control-siRNA (100 nM) or vehicle for 48 h by PrimaPort siRNA transfection reagent. After rinsing, Alexa-488-conjugated
MG6 cells were plated at 5,000 cells/well in a 96-well microplate that contained 200 µl DMEM/10% FBS. They were treated with PLD4-siRNA (100 nM), control-siRNA (100 nM) or vehicle for 48 h. The following day, the cells were quantitated using a CCK-8 cell counting kit (Dojindo Laboratories, Kumamoto, Japan). After 2 h incubation with the reagent, absorbance at 460 nm was determined using a microplate reader (Tecan, Männedorf, Switzerland). The measured absorbance at 0 h after adding LPS or PBS was used as a standard value.
In our previous study, PLD4 mRNA was expressed in activated microglia in 7-day-old cerebellar white matter
Distribution of PLD4-mRNA-positive cells was examined by
Microglial activation occurs under various pathological conditions in adult brain. In order to clarify whether expression of PLD4 is induced in adult brains under pathological conditions that activate microglia, we investigated the expression of PLD4 in demyelinating lesions in the
Sections prepared from 4.5-month-old PLPtg/− (D–F) and wild type (A–C) mouse cerebella were immunostained with antibodies against Iba1 (A, D), MBP (B, E), and PLD4 (C, F). In wild type, weak signals of Iba1 (A) and PLD4 (B) were found in white matter. In contrast, the Iba1-positive (D) and PLD4-positive (F) strong signals were observed in the proximal cerebellar white matter of PLPtg/− mice, where abnormal MBP-positive signals indicated demyelinated lesions (E). The enlarged images of the squares in A–F are shown in G–L, respectively. The results indicate that PLD4 is upregulated in activated microglia in demyelinating conditions in cerebellar white matter. Arrowheads in L show strong PLD4 signals in the cytoplasm. Scale bars; 100 µm in F for A–F and 20 µm in L for G–L.
To determine the subcellular localization of PLD4 in microglia, we used a primary microglia derived from cerebral cortices of 1-day-old C57BL/6J mice. Primary microglia were cultured with or without LPS for 24 h, and immunocytochemical analysis was performed using anti-PLD4 and anti-CD11b antibodies. Only weak PLD4-positive signals (green) were detected in nuclei excluding the nucleolus of the vehicle-treated CD11b-positive (red) microglia (
Primary microglial cells (A, B) and MG6 cells (C, D) were treated with LPS (500 ng/ml) (B, D) or vehicle (A, C). (A and B) Primary cultured microglia were identified by CD11b-positive signals (red). The nuclei were weakly positive for PLD4 in vehicle-treated microglia (A), whereas strong PLD4 signals were found in the nuclei of LPS-stimulated microglia (B). (C and D) In MG6 cells, PLD4-positive signals (green) were detected in the nucleus, except the nucleoli (C). The signal intensities of PLD4 in the nuclei were markedly increased in LPS-stimulated MG6 cells (D). The Z-stack image of the nucleus was obtained using confocal microscopy. PLD4-positive signals (green) were detected in the internal part of the nucleus (blue) in MG6 cells (D). Blue signals indicated DAPI-stained nuclei. Scale bars, 20 µm. Intensity of nuclear PLD4 signals in LPS-treated primary microglia (E) and MG6 cells (F) was calculated against that of vehicle-treated controls. The data were presented as mean ± SE of four experiments. Asterisks in E and F indicate P<0.01 (Mann-Whitney's U test).
As shown in
Primary cultured microglia (A, B) and MG6 cells (E, F) were treated with LPS (500 ng/ml) (B, F) or vehicle (A, E) and were incubated with BioParticles (red). The cells were immunostained with anti-PLD4 antibody (green). LPS treatment stimulated phagocytosis, and these cells contained more BioParticles (B, F). PLD4-positive signals were co-localized with BioParticles in phagosomes (yellow in A, B, E, F), whereas they were mainly found in the nuclei of the cells without BioParticles as shown in
To establish whether PLD4 was implicated in early or late endosomes, primary microglial cells were labeled with transferrin (early endosome marker) or lysotracker (late endosome maker), and PLD4 localization was examined after phagocytosis of BioParticles. Most PLD4-positive signals were found in transferrin-containing phagosomes (
Increased level of PLD4 was confirmed by western blot using activated MG6 cells. After treatments of MG6 with LPS (500 ng/ml) or vehicle for 24 h, the secretion levels of TNF-α in the culture medium were 2,069±245 pg/ml in LPS-stimulated cells and 28±10 pg/ml in vehicle-treated control cells, which indicated that MG6 cells were activated. Western blot analysis of these cell homogenates exhibited multiple PLD4-related bands of 70–80 kDa (
MG6 cells were treated with LPS (500 ng/ml) or vehicle (PBS) for 24 hrs. (A) The Western blot analysis (15 µg proteins, 10.5% SDS-PAGE) using anti-PLD4 antibody revealed that the levels of PLD4-related bands (70–80 kDa) were increased in LPS-stimulated MG6 cells (left) compared with those in control cells (right). (B) Whole cell homogenates (W), nuclear fractions (N), and supernatants (C) were prepared. Mouse spleen homogenate (S) was prepared as a positive control. Each sample was deglycosylated by peptide-N-glycosidase F (PNGase F). Western blot analysis (15 µg proteins, 10.5% SDS-PAGE) using PLD4 antibody revealed that PLD4-positive bands were found in the nuclear fraction and supernatants. After PNGase F treatment, PLD4-positive band sizes were changed (48 kDa). (C) Intensity of each band was measured and relative increase of PLD4 by LPS-stimulation in each fraction was expressed graphically. The data are presented as mean ± SE of four experiments. Asterisk and double asterisk in C indicate P<0.01 and P<0.05 by Mann-Whitney's U test, respectively. Levels of PLD4 were increased in all LPS-stimulated MG6 cell samples compared with those in control cell samples.
The nuclear localization of PLD4 was also confirmed by western blot analyses of the fractions prepared from MG6 cells with or without LPS stimulation (
Involvement of PLD4 in phagocytosis was examined by measuring phagocytotic activity under PLD4 knockdown conditions using RNA interference (RNAi). First, we searched for an appropriate RNAi condition, by which PLD4-siRNA showed the highest efficiency of inhibition of PLD4 expression in the MG6 cells. MG6 cells were transfected by PLD4- or control-siRNA for 48 h and PLD4 mRNA expression levels were examined. RT-PCR showed that MG6 cells silenced by 100 nM PLD4-siRNA exhibited a significantly higher rate of inhibition compared with cells treated by control-siRNA (
(A) MG6 cells were transfected with PLD4-siRNA or control (cont)-siRNA (100 nM each) for 48 h. Total RNA was isolated from these cells and analyzed by RT-PCR for PLD4 (above) and GAPDH (below) mRNA expression. Expression of PLD4 mRNA was efficiently reduced by 100 nM PLD4 siRNA treatment. The immunofluorescence staining of PLD4 (green) revealed that PLD4 was downregulated in PLD4-siRNA-treated cells (left), compared with Cont-siRNA (right) by 100 nM siRNA treatment. Scale bars, 20 µm. (B) MG6 cells were transfected with siRNA for 24 h, and LPS (500 ng/ml) or PBS (vehicle) were added to the medium. After 24 h, secretion of TNF-α was measured by ELISA. Measurement of PLD4-siRNA-treated cells was used as a standard value. Secretion of TNF-α was not significant in the siRNA-treated groups (n = 4). (C) LPS-stimulated (dark gray bars) or vehicle-treated (gray bars) MG6 cells were treated with or without siRNA for 48 h. Measurement at 0 h before addition of LPS or PBS was used as a standard value. Proliferation was examined by cell counting kit (n = 4). (D) MG6 cells were transfected with PLD4- or control (cont)-siRNA for 48 h. Vehicle-treated cells were used as a control. Cells were incubated with BioParticles. BioParticle-containing cells were analyzed by FACS, and phagocytic activity was calculated by dividing these cell numbers by the total. The graph shows the percentage of phagocytic activity of each siRNA-treated cells compared with that of the control cells. The data are presented as mean ± SE of five experiments. Asterisks in C and double asterisk in D indicate P<0.01 and P<0.05 by Mann-Whitney's U test, respectively.
We demonstrated that one of the novel PLD family members containing the transmembrane domain, PLD4, was expressed in activated microglia found in developing cerebellum, as well as in demyelinated white matter lesions. Primary cultured microglia and the microglial cell line MG6 showed that PLD4 immuno-signals were mainly present in the nucleus, apart from the nucleolus, and were upregulated by LPS stimulation. PLD4 immunoreactivity accumulated in phagosomes when BioParticles were added to primary microglial cultures (
PLD4 was located in the nucleus during the resting state. After LPS stimulation, expression of PLD4 was increased. In the phagocytotic state eating BioParticles, PLD4 accumulated in the early phagosomes.
Microglia are cells of myeloid origin that are distributed diffusely throughout the brain. Microglia under normal conditions monitor the status of the local surroundings, whereas under pathological conditions, they migrate to the lesion, release a wide range of soluble factors that include cytotoxins, neurotrophins and immunomodulatory factors, and clear cellular debris by phagocytosis
In the adult CNS, microglia thought to have at least three clearly identifiable states, resting, reactive but non-phagocytic, and phagocytic
The distribution was different from our previous study of PLD4-transfected cells. When PLD4 is exogenously overexpressed in HEK293 or HeLa cells, the immunoreactivity is mainly observed in the Golgi complex and endoplasmic reticulum
The classical PLDs are involved in phagocytosis of macrophages
The authors thank Dr. Schuichi Koizumi (University of Yamanashi, Yamanashi, Japan) for his helpful discussion and technical advice.