Research Articles, Cellular/Molecular

Reduction of Ether-Type Glycerophospholipids, Plasmalogens, by NF-κB Signal Leading to Microglial Activation

Md. Shamim Hossain, Yuichi Abe, Fatma Ali, Mohammed Youssef, Masanori Honsho, Yukio Fujiki and Toshihiko Katafuchi
Journal of Neuroscience 12 April 2017, 37 (15) 4074-4092; DOI: https://doi.org/10.1523/JNEUROSCI.3941-15.2017

Abstract

Neuroinflammation characterized by activation of glial cells is observed in various neurodegenerative diseases including Alzheimer's disease (AD). Although the reduction of ether-type glycerophospholipids, plasmalogens (Pls), in the brain is reported in AD patients, the mechanism of the reduction and its impact on neuroinflammation remained elusive. In the present study, we found for the first time that various inflammatory stimuli reduced Pls levels in murine glial cells via NF-κB activation, which then downregulated a Pls-synthesizing enzyme, glycerone phosphate O-acyltransferase (Gnpat) through increased c-Myc recruitment onto the Gnpat promoter. We also found that systemic injection of lipopolysaccharide, aging, and chronic restraint stress reduced brain Pls contents that were associated with glial NF-κB activation, an increase in c-Myc expression, and downregulation of Gnpat in the mouse cortex and hippocampus. More interestingly, the reduction of Pls contents in the murine cortex itself could increase the activated phenotype of microglial cells and the expression of proinflammatory cytokines, suggesting further acceleration of neuroinflammation by reduction of brain Pls. A similar mechanism of Gnpat reduction was also found in human cell lines, triple-transgenic AD mouse brain, and postmortem human AD brain tissues. These findings suggest a novel mechanism of neuroinflammation that may explain prolonged progression of AD and help us to explore preventive and therapeutic strategies to treat neurodegenerative diseases.

SIGNIFICANCE STATEMENT Ether-type glycerophospholipids, plasmalogens (Pls), are reduced in the brain of Alzheimer disease (AD) patients. We found that inflammatory stimuli reduced Pls contents by downregulation of the Pls-synthesizing enzyme glycerone phosphate O-acyltransferase (Gnpat) through NF-κB-mediated recruitment of c-Myc onto the Gnpat promoter in both murine and human cell lines. Murine brains after systemic lipopolysaccharide, chronic stress, and aging, as well as triple-transgenic AD mice and postmortem human AD brain tissues all showed increased c-Myc and reduced Gnpat expression. Interestingly, knockdown of Gnpat itself activated NF-κB in glial cell lines and microglia in mouse cortex. Our findings provide a new insight into the mechanism of neuroinflammation and may help to develop a novel therapeutic approach for neurodegenerative diseases such as AD.

Introduction

Plasmalogens (Pls) are unique glycerophospholipids containing a vinyl ether bond at the sn-1 position of the glycerol moiety; the well known diacylglycerophospholipids such as phosphatidyl-ethanolamine, phosphatidyl-choline, and phosphatidyl-inositol have an ester bond at the same site. Pls are not only structural membrane components and reservoirs for second messengers, but are also involved in membrane fusion, ion transport, and cholesterol efflux (Farooqui and Horrocks, 2001). In addition, because the vinyl ether bond at the sn-1 makes Pls more susceptible to oxidative stress than corresponding ester-bonded glycerophospholipids, Pls act as antioxidants and protect cells from oxidative stress (Braverman and Moser, 2012). We have reported previously that Pls can suppress the lipopolysaccharide (LPS)-induced accumulation of β-amyloid protein (Aβ) and activation of glial cells in the hippocampus (Ifuku et al., 2012) and prevent neuronal cell death through the activation of AKT and ERK signaling (Hossain et al., 2013). More recently, Pls-synthesizing enzyme (glycerone phosphate O-acyltransferase, GNPAT) knock-out mice have been reported to show a reduction of AKT signaling in Schwann cells, resulting in defects in myelination (da Silva et al., 2014).

Although the accumulating evidence suggests that reduction of Pls, but not ester-type diacylglycerophospohlipids, in the brain of Alzheimer's disease (AD) patients can be a risk factor for this disease (Ginsberg et al., 1995; Guan et al., 1999; Han et al., 2001; Goodenowe et al., 2007; Hartmann et al., 2007; Grimm et al., 2011; Maeba et al., 2016), the mechanism behind the reduction of Pls remains elusive. It has been reported that inflammatory stimuli, stress, and aging activate NF-κB in glial and neuronal cells, leading to neuroinflammation (O'Neill and Kaltschmidt, 1997; Mattson and Camandola, 2001; Chinta et al., 2013), which is known to be associated with neurodegenerative diseases including AD (Frank-Cannon et al., 2009; Amor et al., 2010). Here, we demonstrate that inflammatory signals, aging, and stress reduce the Pls levels in glial cells by the downregulation of Gnpat, which is mediated by the NF-κB-induced increase in recruitment of c-Myc onto the promoter region of the Gnpat gene. Furthermore, local knockdown of Gnpat alone can give rise to microglial activation both in vitro and in vivo accompanied by the enhanced expression of proinflammatory cytokines. More interestingly, the reduction of Gnpat and increase in c-Myc were also observed in the glial cells of postmortem AD human brains and in brain tissues from an AD mouse model. These findings suggest for the first time that the reduction of Pls, which is associated with inflammatory signals, aging, and stress, can be one of the causes behind the neuroinflammation process of the brain, which are mostly associated with the various neurodegenerative diseases including AD.

Materials and Methods

Animal, cell lines, and reagents.

All of the animal experiments were conducted in accordance with the guidelines provided by the Committee on the Ethics of Animal Experiments, Kyushu University and performed in accordance with the Guidelines provided by the National Institutes of Health regarding the care and use of animals for experimental procedures. All efforts were made to minimize animals' suffering and the number of animals used for the study.

Male C57BJ6 mice (8 weeks or 16 months old as the aged group) were used for the in vivo study. For the chronic restraint stress (CRS) experiments, 7 adult male mice in each group were subjected to being immobilized in a tube for 2 h/d for 10 d. On the last day, 2 h after the stress, mice were killed for analysis. We used 18-month-old homozygous 3Tg-AD male mice, which had two familial AD-related gene mutations (APPKM670/671NL/PS1M146V) and a tau gene mutation (TauP301L) (Oddo et al., 2003) and control mice of the same genetic background for the Western blotting and immunohistochemistry analysis (Billings et al., 2005). Mouse neuroblastoma-derived cells [Neuro2A (N2A), Riken Cell Bank catalog #RCB2639, RRID: CVCL_0470], p53 knock-out mice derived astrocyte cell lines (A1, Japanese Collection of Research Bioresources IFO catalog #IFO50519, RRID:CVCL_U223), the microglial cell lines (MG6, RCB catalog #RCB2403, RRID: CVCL_8732), and the human embryonic kidney-derived cell lines (Hek293-T, RCB catalog #RCB2202, RRID:CVCL_0063) were from Health Science Research Resources Bank. The human neuroblastoma-derived cell line SH-SY5Y was the gift of Dr. Yoshinori Katakura, Department of Bioscience and Biotechnology, Kyushu University. Cells were maintained in DMEM containing 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen), 50 μg/ml penicillin, 50 μg/ml streptomycin (Invitrogen), and glucose at 37°C in 5% CO2 as described previously (Hossain et al., 2013). Primary hippocampal neurons were prepared from embryonic day 18 (E18) mice. After dissection of anesthetized pregnant mice, meninges of the embryo were removed carefully. The hippocampi were cleared with the surrounding cortex and dissolved in trypsin solution containing PBS, bovine serum albumin (BSA), and glucose at 37°C for 15 min. FBS was used to neutralize trypsin activity. The hippocampi were then dissociated in neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen) by appropriate pipetting using different pore-sized Pasteur pipettes. The dissociated neurons were then cultured on poly-d-lysine-coated glass coverslips (30,000 cells/15 mm coverslip) with neurobasal medium in a 5% CO2 humidified incubator. On d in vitro 3(DIV3), 90% of cultured medium was replaced with B27-free neurobasal medium. Cytosine arabinoside (Ara-C) from Sigma-Aldrich was added on DIV3 primary neurons at a concentration of 1 μm to inhibit microglial proliferation. More than 95% pure primary hippocampal pyramidal neuronal cells (on D21) were used as primary neurons (Hossain et al., 2013). Primary microglia (>90% pure) and astrocytes (>85% pure) were collected according to our previous report (Ifuku et al., 2014) from the hippocampal tissue of newborn mice. Purity data are available upon request. LPS, polyriboinosinic:polyribocytidylic acid (Poly I:C), and recombinant interleukin-1β (IL-1β) were from Sigma-Aldrich.

Real-time PCR analyses.

Total RNA was extracted from cells and tissues using TRIZOL reagents (Life Technologies) following standard protocols. cDNAs were prepared from the purified total RNA using ReverTra Ace qPCR RT Kit (Toyobo). Real-time PCR was performed by SYBR Premix Ex Taq (RR420Q; Takara) following the manufacturer's protocol. The quantifications were performed by 7500 Real Time PCR System software version 2.0 (Applied Biosystems). The specific primers used to amplify the each mouse gene from the cDNA were as follows: Gnpat, forward 5′-TCCTGGGAATGAGAGTGGTC-3′ and reverse 5′-TTCAGAGAATACGGCCCAGT-3′; IL-1β, forward 5′-AAAGCTCTCCACCTCAATGG-3′ and reverse 5′-AGGCCACAGGTATTTTGTCG-3′; Mcp-1, forward 5′-AGGTCCCTGTCATGCTTCTG-3′ and reverse 5′-TGGGATCATCTTGCTGGTGA-3′; TNF-α, forward 5′-AAGCCTGTAGCCCACGTCGT-3 and reverse 5′-AGGTACAACCCATCGGCTGG-3′; Igf-1, forward 5′-AGCAACTGTGTAGAGGTGGT-3 and reverse 5′-AGGGTGTGTCTAATGCAGCT-3′; P2Y12 receptor, forward 5′-AGTATTCCCGGAGACACTC-3 and reverse 5′-AGAAGGTGGTATTGGCTGAG-3′; Tgf-β, forward 5′-CTCTCCACCTGCAAGACCAT-3′ and reverse 5′-GCGAGCCTTAGTTTGGACAG-3′; c-Myc, forward 5′-AGATCAGCAACAACCGCAAG-3′ and reverse 5′-GTTCCTCCTCTGACGTTCCA-3′; Mycn, forward 5′-AACAACCAAGGCGGTAACCAC-3′ and reverse 5′-GAACACAGCGCTTGAGGATC-3′; Tlr4, forward 5′-TCCCTGATGACATTCCTTCTT-3′ and reverse 5′-TGAGCCACATTGAGTTTCTTTA-3′; Tlr3, forward 5′-ACTTGCTATCTTGGATGC-3′ and reverse 5′-AGTTCTTCACTTCGCAAC-3′; p65, forward 5′-CACCGGATTGAAGAGAAGCG-3′ and reverse 5′-AGTTGAGTTTCGGGTAGGCA-3′; β-Actin, forward 5′-CACTGTGCCCATCTACGA-3′ and reverse 5′-CAGGATTCCATACCCAAG-3′.

Western blotting assays.

For whole lysates, cells were washed in ice-cold PBS and lysed using RIPA buffer (1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS dissolved in 1× TBS) mixed with the complete protease inhibitor cocktail tablets (Roche). After 30 min on ice with the lysis buffer, cells were subjected to brief sonication at 4°C, followed by centrifugation at 15,000 rpm for 10 min to remove insoluble cell debris. For subcellular fractionation assays, cells were suspended in mild buffer (0.5% NP40, 1 mm EDTA, and 10 mm Tris-HCl), followed by centrifugation at 15,000 rpm, and the supernatant (cytoplasmic fraction) was collected. The precipitate was then washed three times in the same mild buffer and dissolved with RIPA buffer to collect the nuclear fraction. Protein concentration was measured by the BCA protein assay kit (Thermo Scientific) and a total of 20 μg of protein was loaded for analysis by SDS-PAGE. After the protein transfer, nitrocellulose membranes (Bio-Rad) were blocked with Tris-buffered saline containing 5% Difco skim milk (BD Biosciences) and 0.1% Tween 20 for 1 h at room temperature. Membranes were then incubated at 4°C overnight with antibodies against GNPAT (Abcam catalog #ab75060 RRID:AB_2232364), c-MYC (Santa Cruz Biotechnology catalog #sc-764 RRID:AB_631276), Mycn (Calbiochem catalog #GTX16898 RRID:AB_422464), p65 (Cell Signaling Technology catalog #6956 also 6956S RRID:AB_10828935), Lamin A (BD Biosciences catalog #612162 RRID:AB_399533), Flag tag (MBL catalog #M185–3L RRID:AB_11123930), and β-Actin (Cell Signaling Technology catalog #sc-47778 RRID:AB_626632). After the addition of the primary antibodies, membranes were washed and then incubated with horseraplate peroxidase-coupled goat anti-mouse or anti-rabbit IgG secondary antibody (Cell Signaling Technology) at room temperature for 2 h. Specific signals from the transferred protein were then visualized by SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) using a LAS4000 biomolecular imager. ImageJ software was used to quantify the signals.

Liquid chromatography (LC), electron spray ionization (ESI), and -tandem mass spectrometry (MS) assays.

Extraction of the total lipids was performed using the method of Bligh and Dyer (Bligh and Dyer, 1959; Abe et al., 2014). Briefly, the cells and tissues were dissolved in PBS, followed by sonication in ice-cold water. After determining the protein concentration, an equal amount (50 μg) of protein was dissolved in methanol/chloroform/water (v/v/v: 2:1:0.8) and 50 pmol internal standard (1-heptadecanoylsn-glycero-3-phosphocholine, 1, 2-didodecanoyl-sn-glycero-3-phosphocholine and 1, 2-didodecanoyl-sn-glycero-3-phosphoethanolamine). After 5 min, 1 ml each of water and chloroform was added and the mixture was centrifuged to collect the lower organic phase. Next, 1 ml of chloroform was added to reextract the lipids. The collected organic phase was then evaporated under a nitrogen stream and suspended in pure methanol. The LC-MS assay was performed using a 4000 Q-TRAP quadrupole linear ion trap hybrid mass Q8 spectrometer (AB Sciex) with the ACQUITY UPLC System (Waters). Samples were injected to an ACQUIRY UPLC BEH C18 column and then subjected directly to ESI-MS/MS analysis. A 10 μl aliquot of each sample was directly introduced by autosampler injector and the samples were separated by step gradient elution with mobile phase A (acetonitrile:methanol:water at 2:2:1 (v/v/v), 0.1% formic acid and 0.028% ammonium) and mobile phase B (isopropanol, 0.1% formic acid and 0.028% ammonium) at the following ratios: 100:0 (for 0–5 min), 95:5 (5–20 min), 70:30 (20–21 min), 50:50 (21–45 min), 50:50 (45–100 min), and 100:0 (100–120 min). The flow rate (70 μl/min at 30°C), source temperature (200°C), declustering potential (60), and collision cell exit potential (15) were kept constant. Ethanolamine Pls (PlsEtns) with alkenyl p16:0, p18:0, and p18:1 at the sn-1 position was detected by precursor ion scan of m/z 364, 392, and m/z 390, respectively, in positive ion mode. The data were analyzed and quantified using Analyst software (AB Sciex).

Cloning of promoter constructs and luciferase reporter assays.

Genomic DNA near the first exon of the mouse GNPAT gene (−1000/+226) and the human GNPAT gene (−966/+288) were amplified from mouse embryonic genomic DNA and human-derived cell line (Hek-293T) genomic DNA, respectively, by PCR using LA-Taq polymerase (Takara). The PCR products were then cloned into the pGEM-T Easy Vector System 1 (Promega) and sequenced. By using the T-vector as a template DNA, the luciferase constructs were cloned into the EcoR1-digested pBV-Luc vector (Addgene plasmid 16539; He et al., 1999) with the Infusion HD cloning kit (Takara). The following primers were used for mouse Gnpat promoters, mGnpat-700FW: 5-CGTCGATATCGAATTAAGTGTTGTCGGTGG-3, mGnpat-150FW: 5-CGTCGATATCGAATTACCCAGGTCGTAGAG-3 and mGnpat +226RV: TTATATACCCGAATTGGAGGAGCTAGGAAC-3. For the human GNPAT promoters hGNPAT −400 forward: 5′-CGTCGATATCGAATTTACAAGCAAGTAGGATGGTC-3′ hGNPAT −150 forward: 5′-CGTCGATATCGAATTCCAAAGTCGTAAAGGTTCTGG-3′ and hGNPAT +183 reverse: 5′-TTATATACCCGAATTCGGTCTGGGATTCTTTGCTAAGCC-3′ For infusion ligation, the underlined sequences were for ligation into the EcoR1-digested pBV-Luc vectors. Two Myc sites (+70 and +121)-deleted mouse promoter constructs were performed by PCR amplification of the whole vector using the primers 5-AGATGGCGGTGCCCGGGGT(CCCGTG)CAGTGGCCGTACGGAGTGCC-3 and 5-TGGGCAGACGGGAGCCGC(CCCGTG)CGAAAGAACCGCCCCCAG-3. The underlined Myc binding sequences were deleted. The cells were seeded in the 96-well plate (2000 cells/well), followed by the transfection of Gnpat promoter constructs (100 ng) with or without the expressing plasmids p65 (Addgene plasmid 20012; Sanjabi et al., 2005) and p50 (Addgene plasmid 20018) with Fugene-HD (Promega) and viruses derived from the vectors [c-MYC (Addgene plasmid 20324; Brambrink et al., 2008) and N-MYC (Addgene plasmid 15951; Blelloch et al., 2007). After the 48 h transfection and infection period with lentiviruses (as described in the “Preparation of sh-RNA lentiviruses and in vivo injection into mouse brain” section), luciferase activities were measured by the luciferase assay kit (Promega).

ChIP assays.

ChIP assays were conducted as described previously (Hossain et al., 2008; Hossain et al., 2012). In brief, cells or tissues were cross-linked with 1% formaldehyde in PBS for 10 min at 37°C. ChIP was performed following the protocol of Millipore's ChIP Assay Kit. Briefly, the cross-linked chromatin was sonicated to an average length of 200–300 nt and cleaned with protein A-agarose beads pretreated with shared salmon sperm DNA. The chromatin fragments were then mmunoprecipitated with rabbit anti-c-MYC (sc-764; Santa Cruz Biotechnology) and mouse anti-Mycn (NCM II 100; Calbiochem) antibodies conjugated with protein A-agarose. After washing, the immunoprecipitates were eluted with 200 μl of elution buffer (1% SDS and 0.1 m NaHCO3). Cross-links were reversed by heating at 65°C for 5 h and the samples were treated with proteinase K at 45°C for 1.5 h. The precipitated DNA and control input DNA was purified using the extraction kit (K210012; Invitrogen). Amplification of the DNA was performed using conventional PCR with rTaq DNA polymerase (R001A; Takara) by the primers forward: 5′-CCTGGTTAAGATGGCGGTGC-3′ and reverse: 5′-GGTCTGCTACACGGCTGTA-3′ for the Myc binding sites onto the mouse Gnpat promoter region (+43 to +171); forward: 5′-ATTTGGCCGTATTGGGCGCCT-3′ and reverse: 5′-CTTCCCATTCTCGGCCTTGACTGT-3′ for the input control of mice Gapdh genomic region; forward-250: 5′-CCTTTAGCCTCAAAGCAGAGG-3′ and reverse-130: 5′-CCAGAACCTTTACGACTTTGG-3′ for the MYC binding sites (−178 of the first exon) onto the human GNPAT promoter region; and forward: 5′-GTTTTACGGGCGCACGTAGCTCA-3′ and reverse: 5′-CTGGCGACGCAAAAGAAGATG-3′ for the input control of human GAPDH promoter region.

Preparation of sh-RNA lentiviruses and in vivo injection into mouse brain.

The sh-RNA sequences were cloned into the pLL3.7 lentiviral vector following the protocol provided on the Addgene website (plasmid 11795; Rubinson et al., 2003). The target sequences were as follows: sh-Gnpat #1 (5′-GTCCCAATTAGCATCAGT-3′), sh-Gnpat #2 (5′-GGCTCAATCGGAACACGT-3′), sh-c-Myc (5′-CACAACGTCTTGGAACGT-3′) and sh-Luc (5′-CTTACGCTGAGTACTTCGAG-3′). For the viral constructs, Hek-293T cells were transfected with the cloned pLL3.7 vectors along with the vectors pMD2.G (Addgene plasmid 12259), pRSV-Rev (Addgene plasmid 12253), and pMDLg/pRRE (Addgene plasmid 12251). The cell supernatant was centrifuged at 24,000 rpm for 3 h at 4°C and the viral pellet was dissolved in PBS (1% BSA). After determining the viral titer in N2A cells, 5 × 105 transduction units were injected into the cortical region of mouse brain using a stereotaxic apparatus (injection rate: 0.05 μl/min). The stereotaxic coordination of the cortex was: A 0.67 mm, L 1.80 mm, and H 1.65 mm. After the injection, the wound was sutured and antibiotics were applied. Two weeks after the injection, the mouse brains were analyzed using immunohistochemistry.

Immunohistochemical staining.

To prepare the cryosections, mice were perfused intracardially with PBS followed by 4% PFA solution. The brains were then collected in 4% PFA solution and kept overnight at 4°C, followed by embedded in optimal cutting temperature compound. The brains were then stored at −80°C before making thin sections (30 μm thick). Sections were collected in ice-cold PBS and treated to 3 n HCl for 30 min, followed by extensive washing with PBS on a shaker. After 1 h of treatment with a blocking solution (0.2% Triton X-100 + 5% BSA dissolved in PBS), tissue slices were incubated with the antibodies dissolved in the same blocking solution overnight at 4°C on a shaker. The following antibody dilutions were used: Iba-1 (1:10,000; Sigma-Aldrich), goat Iba-1 Cy3 conjugated GFAP (1:4000; Sigma-Aldrich catalog #C9205 RRID:AB_476889), NeuN (1:1000; Millipore), c-MYC (1:500; Santa Cruz Biotechnology catalog #sc-764 RRID:AB_631276), p65 (1:1000; Cell Signaling Technology catalog #6956 also 6956S RRID:AB_10828935), and GNPAT (1:500; Abcam catalog #ab75060 RRID:AB_2232364). After brief washing in PBS, the brain slices were incubated with fluorochrome-conjugated secondary antibodies (1:1000 in blocking solution; Alexa Fluor 488/568; Life Technologies) for 3 h at room temperature. DAPI was used for nuclear staining. For the amyloid β staining of 3Tg mice brain tissue, the standard DAB staining protocols provided with the kits (Vectastain ABC kit PK-6200 and ImmPACT DAB kit SK-4105 of Vectro) for the amyloid β polyclonal antibody (Abcam catalog #ab2539 RRID:AB_303141) were used. To detect the glial expression of c-MYC and GNPAT,paraffin tissue panels of the human postmortem brains from BioChain (catalog #T8236446Alz) were used. Photographs of all the stained samples were taken with a KEYENCE (BZ-9000) fluorescence microscope.

Statistics.

To analyze the statistical significance between experimental groups, one-way ANOVA followed by a post hoc test (Bonferroni's test) were performed. The data are presented as mean ± SEM. p < 0.05 was considered significant.

Results

Inflammatory stimuli reduce Pls levels through the activation of NF-κB in glial cells

To understand the critical process of Pls reduction in the brain, we challenged microglial (MG6) and astrocyte (A1) cell lines with major inflammatory stimuli, LPS, Poly I:C, and IL-1β and found a significant reduction in both mRNA and protein expression of a Pls-synthesizing enzyme, Gnpat (Fig. 1A–C). Consistent with these results, LC-MS assays showed a significant reduction of PlsEtn, which was more abundant than choline Pls in the brain (Farooqui and Horrocks, 2001), in the MG6 (Fig. 1D) and A1 (Fig. 1E) cells after these stimuli (p < 0.01). We also observed a reduction of Gnpat mRNA and of total Pls in primary microglia and astrocytes after exposure to LPS, Poly I:C, and IL-1β (Fig. 1F,G). There was also a reduction of GNPAT protein expression by inflammatory agents in the primary microglial and astrocytes (Fig. 1H). However, Gnpat mRNA and Pls content failed to decrease in the primary neurons and neuronal cell line N2A cells by these stimuli (Fig. 1I).

Figure 1.
Figure 1.

Downregulation of Gnpat by LPS, Poly I:C, and IL-1β in glial cells but not in neuronal cells. A, Real-time PCR data showing the reduction of Gnpat mRNA expression in MG6 and A1 cells treated with LPS (10 μg/ml), Poly I:C (10 μg/ml), and IL-1β (20 ng/ml) for 6 h. B, Western blot data show the reduction of Gnpat protein by treatment with inflammatory stimuli as in the same condition of A. C, Quantification of the Western blot images in B. D, E, LC-MS analysis showing the reduction of PlsEtn species in MG6 (D) and A1 (E) cells after inflammatory treatments for 12 h. F, G, In similar experimental conditions to A, the LPS-Poly I:C- and IL-1β-induced decreases in Gnpat mRNA expression and total PlsEtn were also observed in mice primary microglial and astrocytes. H, Reduction of protein expression of Gnpat in the primary microglial and astrocytes by similar treatment with ainflammatory agents as in A. I, Similar treatments with LPS, Poly I:C, and IL-1β failed to reduce Gnpat expression in mice primary neurons and N2A cells (top) and Pls-Etn content in N2A cells (bottom). The data represent four independent experiments. **p < 0.01; ***p < 0.001.

The activation of NF-κB is often characterized by nuclear localization of its major subunits, p65 or p50, in the brain (O'Neill and Kaltschmidt, 1997; Mattson and Camandola, 2001). Therefore, we investigated activation in our experimental conditions and confirmed that all of the inflammatory stimuli used increased the nuclear translocation of p65 in the MG6 (Fig. 2A) and A1 (2B) cells. Pretreatment with an NF-κB inhibitor, PDTC (Zhou et al., 1999), inhibited nuclear localization of p65 (Fig. 2C,D). In addition, the LPS-, Poly I:C-and IL-1β-induced reduction of Gnpat expression was attenuated by pretreatment with PDTC (Fig. 2E,F). Similar results as found in the cell lines were also obtained with primary microglia (Fig. 2G) and astrocytes (Fig. 2H), showing that PDTC treatment reduced the increase in nuclear p65 protein by inflammatory stimuli. However, inflammatory stimuli did not increase nuclear p65 in either the neuronal cell line (N2A) or in the primary neuronal culture (Fig. 2I). These findings suggest that Gnpat expression is reduced by inflammatory signals both in glial cell lines and primary cultured glia, but not neuronal cells, through activation of the NF-κB pathway.

Figure 2.
Figure 2.

NF-κB activation-induced downregulation of GNPAT in glial cells. A, B, Increase in the nuclear localization of p65 by LPS, Poly I:C, and IL-1β treatments (arrowheads) in MG6 (A) and A1 (B) cells. Scale bar, 20 μm. ***p < 0.001. C, D, Western blot data with the nuclear fractionation samples of MG6 cells (C) and A1 cells (D) pretreated with an NF-κB inhibitor, PDTC (50 μg/ml), for 30 min followed by LPS, Poly I:C, and IL-1β treatments for 2 h. E, F, Real-time PCR data showing the attenuation of inflammatory stimuli-induced downregulation of Gnpat by pretreatment with PDTC (50 μg/ml, 30 min) in MG6 (E) and A1 (F) cells. G, H, Treatments with LPS, Poly I:C, and IL-1β also increased nuclear p65 expression in primary microglia and astrocytes that were reduced by the PDTC treatments. I, Nuclear p65 was unchanged by the same treatments in N2A (top) and mouse primary neurons (bottom). **p < 0.01; ***p < 0.001 (n = 4).

NF-κB-mediated recruitment of c-Myc onto the promoter region downregulates Gnpat expression

To identify the possible transcription factors regulating Gnpat expression under the NF-κB pathway, we cloned the Gnpat promoter regions and identified the maximum promoter activity in glial and neuronal cells from the core promoter (−150 to +226) of the first exon (Fig. 3A). This core promoter activity (Fig. 3B) was significantly reduced in the glial cells after exposure to LPS, Poly I:C, and IL-1β, but not in neuronal cells (Fig. 3C–E). The reason that neuronal cells did not respond to these proinflammatory stimuli might have been the low expression of Toll-like receptor 4 (Tlr4), Tlr3, and IL-1R1, receptors for LPS, Poly I:C, and IL-1β, respectively (Fig. 3F–H). Although there was a significant amount of IL-1R1 in the primary neurons, they did not respond to IL-1β to reduce Gnpat expression (Fig. 1G). This could be explained by the evidence that IL-1β failed to activate NF-κB in primary neurons (Fig. 2I, bottom), which is consistent with other reports (Nadjar et al., 2003; Srinivasan et al., 2004; Allan et al., 2005).

Figure 3.
Figure 3.

Gnpat promoter activity and endogenous expression of Tlr4, Tlr3, and IL-R1 in neurons and glial cells. A, Two possible promoter regions upstream of the Gnpat first exon were cloned into the luciferase vector. B, Promoter activity of the luciferase vectors was examined in the indicated cells 48 h after transfection (100 ng vectors/2000 cells/well in 96 well platees). C–E, Forty-two hours after Gnpat core promoter (−150 to +226) luciferase vector transfection, the indicated cells were treated with the same dose of LPS, Poly I:C, and IL-1β as in Figure 1 for additional 6 h, followed by the luciferase assays. F, G, Real-time PCR data showing the increased expression of Tlr4 (F) and Tlr3 (G) among glial cells compared with neuronal cells. H, Expression of IL-1R was higher in the glial cell lines compared with N2A, but the primary neurons had abandoned expression for IL-1R. **p < 0.01; ***p < 0.001 (n = 5).

The core Gnpat promoter sequence analysis revealed two possible Myc protein-binding sites within the 5′-untranslated region (Fig. 4A). ChIP assays showed that both c-Myc and Mycn were recruited onto these sites in the glial and neuronal cells (Fig. 4B,C). Specificity of the antibodies (c-Myc and Mycn) used for the ChIP assays were confirmed by immunoprecipitation assays (Fig. 4D,E). Appropriate positive and negative control experiments were performed to optimize the ChIP assays (data not shown). We also examined the effects of MYC and NF-κB proteins on Gnpat promoter activity. The Gnpat promoter for luciferase reporter assay revealed that the promoter activity was significantly decreased by overexpression of p65/p50 proteins (Fig. 4F) and c-Myc or Mycn (Fig. 4G) in the N2A cells. N2A cells were used in this experiment because these cells showed a higher transfection efficiency than glial cells.

Figure 4.
Figure 4.

Recruitment of NF-κB and Myc proteins onto the Gnpat promoter. A, Two possible Myc protein-binding sequences onto the mouse Gnpat promoter region are shown in red. B, C, ChIP assays show the basal recruitment of c-Myc (B) and Mycn (C) onto the Gnpat promoter (+43 to +171) in the indicated cultured cells. D, E, Immunoprecipitation (IP) data showing specific pull-down of c-Myc (D) and Mycn (E) by the antibodies. F, Promoter assays show the reduction of Gnpat promoter activity 48 h after NF-κB (p65 + p55)-expressing lentiviruses (1 × 105 transduction units/2000 cells/well of 96-well plate) infection. N2A cells were previously transfected with the luciferase promoter vectors (100 ng/2000 cells/well in 96-well plate). G, Luciferase promoter assays showing that overexpression of c-Myc and Mycn by lentiviral vectors (100 ng each) reduced the Gnpat promoter activity.

Interestingly, treatment with LPS, Poly I:C, and IL-1β increased the recruitment of c-MYC onto the promoter region of Gnpat (+43 to +171) in the microglia (Fig. 5A, left) and astrocytes (Fig. 5A, right) but reduced Mycn recruitment in these cells (Fig. 5B). This can be explained by the evidence that all of these stimuli increase c-Myc expression (Fig. 5C) but reduce Mycn (Fig. 5D) in glial cells. Most notably, an NF-κB inhibitor, PDTC, significantly attenuated inflammatory stimuli-induced c-Myc induction and Mycn reduction (Fig. 5C,D), confirming the previous findings that NF-κB activation increased expression of c-MYC (La Rosa et al., 1994; Kozlowski and Weimer, 2012). In addition, exposure to inflammatory stimuli did not induce c-Myc in neuronal cells (data not shown), indicating that this induction could be limited to glial cells. Real-time PCR revealed that basal levels of GNPAT mRNA in primary glial cells were less than those in the primary neurons (Fig. 5E), possibly due to the higher expression of p65 (Fig. 5F) and c-Myc in the primary glial cells compared with primary neurons (Fig. 5G).

Figure 5.
Figure 5.

Differential regulation of c-Myc and Mycn by inflammatory stimuli. A, B, ChIP data showing increased c-MYC retreatment (A) and decreased Mycn recruitment (B) onto the Gnpat promoter in the MG6 (left in A and B) and A1 cells (right) by similar treatments with LPS, Poly I:C, and IL-1β as in Figure 1. C, D, Inflammatory stimuli increased c-Myc (C) and decreased Mycn (D) expression in MG6 (left) and A1 (right) cells, which were attenuated by the pretreatment with an NF-κB inhibitor, PDTC. E–G, Real-time PCR data showing the endogenous expression of GNPAT (E), p65 (F), and c-Myc and Mycn (G) in mouse primary cultured cells. **p < 0.01; ***p < 0.001 (n = 5).

Consistent with the promoter activity assay, the overexpression of p65/p50 increased c-Myc mRNA (Fig. 6A, left) and protein (Fig. 6A, right) levels and reduced Gnpat expression in N2A cells (Fig. 6B). These findings indicate that, although inflammatory stimuli failed to reduce Gnpat and activate NF-κB in neuronal cells, alternative activation of NF-κB and MYC overexpression can lead to the reduction of Gnpat in the neuronal cells. Furthermore, overexpression of c-Myc and Mycn reduced Gnpat expression in microglial cells (Fig. 6C), astrocytes, and neuronal cells (data not shown), suggesting that increased expression of either NF-κB or Myc proteins could reduce the Gnpat transcription in any of these cells. To confirm that the reduction of Gnpat expression was due to the Myc proteins, we deleted one of the two Myc-binding consensus sequences (red “x” in Fig. 6D, left) from the promoter and found that neither p65/p50 nor c-Myc overexpression reduced the mutated Gnpat promoter activity (Fig. 6D, middle and right). Furthermore, knockdown of c-Myc by lentiviruses encoding the short hairpin RNA against c-Myc in microglial cells (Fig. 6E) significantly increased the basal Gnpat expression (Fig. 6F) and attenuated Gnpat downregulation by inflammatory stimuli (Fig. 6G).

Figure 6.
Figure 6.

Suppression of Gnpat expression by NF-κB-induced c-Myc. A, Transfection of p65- and p50-expressing plasmids (1 μg each) in N2A cells (1 × 105 cells/well of 6-well plate) for 48 h increased c-Myc mRNA (left) and protein (right) expression. B, In the same experimental condition, p65 and p50 overexpression reduced Gnpat expression. C, MG6 cells (1 × 105 cells/well of 6 well platees) infected with c-Myc- and Mycn-expressing lentiviruses for 48 h showing the reduction of Gnpat expression compared with control lentivirus infection. D, Luciferase assays in N2A cells showing that the reduction of Gnpat promoter activity by NF-κB (p65 plus p50) (middle panel, third row) and c-Myc (right panel) was canceled by the deletion of Myc-binding sequences from the Gnpat promoter (first and second rows). The data represent six independent experiments. **p < 0.01; ***p < 0.001. E, Western blot data showing the reduction of c-Myc protein expression in MG6 cells infected with the c-Myc sh-RNA-delivering lentivirus for 48 h. F, Gnpat mRNA expression was increased in the c-Myc knockdown cells. G, Reduction of Gnpat expression induced by 6 h treatments with LPS (10 μg/ml), Poly I:C (10 μg/ml), and IL-1β (20 ng/ml) was blocked by c-Myc knockdown in MG6 cells. *p < 0.05; **p < 0.01 (n = 4).

Pls and Gnpat expression are reduced in inflammatory/stress model mice

We then sought to clarify the role of Pls using in vivo model of neuroinflammation. When mice were intraperitoneally injected with LPS over a period of 7 d, the level of PlsEtn in the hippocampus was significantly reduced and the expression of IL-1β mRNA increased (Fig. 7A,B), as we reported previously (Ifuku et al., 2012). LPS injections significantly reduced hippocampal Gnpat expression (Fig. 7C) and increased nuclear localization of p65 in the Iba-1+ microglia and GFAP+-astrocytes, but not NeuN+-neurons (Fig. 7D,E). In addition to the LPS model, aged mice (16 months old) and mice subjected to CRS also showed reduction of Gnpat expression and PlsEtn content with the increase in IL-1β expression in the hippocampus (Fig. 8A,B). More consistently, we also noticed that in both the aged and CRS groups of mice, p65 nuclear localization was prominent in glial cells (Fig. 8C–E). However, in contrast to LPS injection (Fig. 7E), aged and CRS mice showed small but significant increases in nuclear p65 immunoreactivity in neuronal cells (Fig. 8D,E).

Figure 7.
Figure 7.

LPS-induced suppression of Gnpat and nuclear localization of p65. A, B, Total PlsEtn content was reduced (A) and expression of IL-1β mRNA was increased (B) in the hippocampus of mice injected with LPS (250 μg/kg/d, i.p., for 7 d). C, LPS injection reduced expression of Gnpat in the hippocampus. D, Increase in nuclear localization of p65 (arrowheads) in the cortical Iba-1+ microglia (top) and GFAP+ astrocytes (middle), but not in NeuN+-neuronal cells (bottom) by LPS injections. E, Quantification of the nuclear p65 signals showing the significant increase in nuclear localization only in the glial cells of mouse cortex. The data represent 5 mice in each group and 10 slices from a single mouse. Scale bar, 20 μm. *p < 0.05; **p < 0.01; **p < 0.001.

Figure 8.
Figure 8.

Reduction of Gnpat and PlsEtn in aged mice and mice subjected to CRS. A, mRNA expression for Gnpat and relative PlsEtn content were decreased, whereas IL-1β expression was increased, in the hippocampal tissues of aged mice (n = 6). B, Similar changes in Gnpat, PlsEtn content, and IL-1β in the hippocampus of mice subjected to CRS were observed. C, Immunohistochemical data showing increases in nuclear localization of p65 (arrowheads) in Iba-1+ microglia (top) and GFAP+ astrocytes in the cortex of aged mice and CRS mice compared with the respective control groups. Scale bar, 20 μm. D, E, Quantification of data in C showing marked increases in nuclear p65 in glial cells and small but significant increases in neurons of aged (D) and CRS (E) mice. **p < 0.01 (n = 5).

c-Myc expression is increased in neuroinflammation models

To address the question of whether the increased c-Myc recruitment in cultured cells can be also found in brain tissues of LPS, aged, and CRS mice, we examined hippocampal tissues and observed an increased recruitment onto the Gnpat promoter region using the ChIP assay (Fig. 9A). In the same mice, expression of c-Myc was also increased in the cortex (Fig. 9B). Because glial cells expressed only small amount of Mycn compared with c-Myc (Fig. 5G), it was likely that the reduction of Gnpat expression was caused mainly by c-Myc induction during inflammatory stimuli. LPS injection induced activation of microglia and astrocytes, as indicated by amoeboid/hypertrophic changes (Fig. 9C, top, D, left), with an increase in c-Myc (Fig. 9C,D, middle) and a decrease in Gnpat (Fig. 9C, bottom, D, right) in the cortex. Western blot analysis of the tissues also showed an increase in c-MYC and a decrease in Gnpat proteins (Fig. 9E). The increases in c-Myc and the decreases in Gnpat were observed in the activated Iba-1+ microglia and GFAP+ astrocytes, but not in neurons (Fig. 9F–H), which might be consistent with the findings that systemic LPS did not activate NF-κB in neuronal cells (Fig. 7E).

Figure 9.
Figure 9.

Increases in c-Myc expression in the mouse neuroinflammation model. A, ChIP assays showing increased recruitment of c-Myc onto the Gnpat promoter in the hippocampal tissues from mice injected with LPS (250 μg/kg/d, i.p., for 7 d), aged mice (16 months), and mice subjected to CRS compared with the saline-injected, adult (2 months), and sham groups, respectively. B, c-Myc expression was increased in the cortex of LPS, aged, and CRS mice. C, Immunohistochemical data for Iba-1, GFAP, c-Myc, and Gnpat expression in the cortex of LPS-injected mice and the saline group. Scale bar, 50 μm. D, Quantification of data in C showing increase in amoeboid Iba-1+ microglial cells and c-Myc and Gnpat expressions in LPS-injected mice (n = 5). E, Protein expression of Gnpat, c-Myc, and β-actin (loading control) in the cortical tissue of three representative mice from the LPS and saline groups. Right panel shows the relative changes in the protein expression. F, Immunohistochemical data showing coexpression of c-Myc with the NeuN, Iba-1, and GFAP in the LPS and saline groups. Scale bar, 50 μm. G, Quantification of data in F showing increases in c-Myc protein in glial cells but not in neurons. H, Quantification data of Gnpat costained with the Iba-1, GFAP, and NeuN. The image data for this graph are available upon request. All of the image quantification data were derived from 5 mice in each group. More than 7 slices of each mouse brain were examined. **p < 0.01; ***p < 0.001.

Knockdown of GNPAT induces glial activation in vivo

Microglial activation is often described as the increased conversion from ramified (resting) to amoeboid (activated) morphology after exposure to proinflammatory cytokines (Kettenmann et al., 2011; Kozlowski and Weimer, 2012). To assess the possible impact of the reduction of Gnpat in glial cells, we performed an intracortical injection of lentiviruses encoding sh-Gnpat (#1 or #2). Two weeks after the injection, Western blotting assays showed significant decreases in Gnpat proteins, along with a reduction of p-AKT and p-ERK (Fig. 10A,B). The decreases in p-Akt and p-Erk seem to be consistent with our previous report showing Pls-induced activation of Akt and Erk signaling (Hossain et al., 2013). LC-MS analysis also showed a significant reduction of Pls content in the infected brain regions (Fig. 10C). Interestingly, a significant increase in activated (amoeboid) microglia was found in the sh-Gnpat-infected brain region, which was further associated with the decrease in resting (ramified) microglia compared with the control sh-Luciferase infected area (Fig. 10D–F). In support of the morphological changes, we also observed an increase in proinflammatory cytokines (IL-1β and Mcp-1) and a decrease in anti-inflammatory genes (Igf-1 and Tgf-β) expression in the infected brain regions (Fig. 10G–J). Consistent with the in vivo study, knockdown of Gnpat induced by sh-Gnpat #1 in cultured microglial cells showed enhanced gene expression of IL-1β and Mcp-1 and decreased expression of Igf-1 and Tgf-β (Fig. 10K–N).

Figure 10.
Figure 10.

Glial activation induced by knockdown of Gnpat. A, B, Western blot data (A) and summary (B) of infected cortical tissues showing a decrease in the Gnpat, p-Akt, and p-Erk proteins by injection of lentiviruses encoding sh-RNA against Gnpat (sh-Gnpat #1 and #2) compared with the control sh-Luc-infected area. C, LC-MS data showing the reduction of total PlsEtn in the infected area of the knockdown mice (n = 4). D, Two weeks after intracranial injection of 0.5 μl of sh-Gnpat #1 (5 × 105 transduction units) in the cortex, immunohistochemistry was performed with Iba-1 antibody (red). Amoeboid (arrowhead) and ramified (arrow) Iba-1-positive microglia were counted in the GFP-positive infected area (green) and uninfected areas from >20 brain slices from 5 mice of each group. Scale bar, 20 μm. E, F, In the sh-Gnpat-infected area, the number of amoeboid microglia increased (E), whereas the number of ramified microglia decreased (F). G–J, In the same experimental condition of D, real-time PCR was performed using the RNA extracted from the lentivirus injection sites to determine the expression of mRNAs for IL-1β (G), Mcp-1 (H), Igf-1 (I), and Tgf-β (J). K–N, The microglial (MG6) cells were infected with sh-Gnpat for 72 h followed by real-time PCR to determine the expression of mRNAs for IL-1β (K), Mcp-1 (L), Igf-1 (M), and Tgf-β (N). *p < 0.05; **p < 0.01; ***p < 0.001 (n = 5).

Knockdown of Gnpat induces glial activation in vitro

To support the in vivo findings that reduction of Gnpat can lead to the microglial activation in the murine cortex, we knocked down Gnpat in the microglial cell culture and determined NF-κB activation. Nuclear localization of p65 is a common marker of NF-κB activation associated with the microglial activation and neuroinflammation, which was also observed in the AD brain tissue (Chen et al., 2012). Surprisingly, Gnpat knockdown significantly increased the nuclear localization of p65 protein in the microglial cells and also in the primary microglial cultures (Fig. 11A–D). Consistent with the increased proinflammatory gene expression in in vivo brain tissue, we also observed an enhancement of the LPS-induced expression of the IL-1β and Mcp-1 genes (Fig. 11E,F).

Figure 11.
Figure 11.

Nuclear localization of p65 is increased by the knockdown of Gnpat. A, B, Western blot data (A) and summary (B) of lentivirus-infected MG6 cells and the primary microglial cells tissues showing a significant increase in nuclear p65 protein expression by sh-Gnpat compared with control sh-Luc. The nuclear fractions were collected after 72 h of the lentivirus infections. The data represent 5 independent experiments. **p < 0.01 (n = 5). C, D, Merged images of immunocytochemistry (C) and summary (D) showing a significant increase in nuclear p65 in primary microglial cells by sh-Gnpat. Cells having strong nuclear p65 staining were counted from a total of 200 GFP-positive cells randomly chosen from 5 observation areas of each experimental slide. **p < 0.01 (n = 5). E, F, mRNA expression of IL-1β (E) and Mcp-1 (F) was increased more significantly among the sh-Gnpat-infected primary microglial cells by LPS treatment (1 μg/ml for 24 h) compared with the control sh-Luc group. **p < 0.01 (n = 5).

Expression of GNPAT is also reduced in human cell lines by NF-κB and c-MYC

To determine whether GNPAT reduction by inflammatory signals can also be seen in human cells, we overexpressed p65 and p50 proteins in human SH-SY5Y and Hek293-T cells. Overexpression of NF-κB proteins reduced the protein and mRNA expression of GNPAT in those cells (Fig. 12A,B). Consistent with the murine cells, NF-κB overexpression caused the increase in c-MYC protein expression in SH-SY5Y cells (Fig. 12A). We also identified a c-MYC-binding consensus sequences onto the human GNPAT promoter region (Fig. 12C). ChIP analysis showed that c-MYC protein was recruited onto this promoter site of both the human cell lines Hek293-T and SH-SY5Y (Fig. 12D). Overexpression of NF-κB proteins increased the c-MYC protein recruitments onto this promoter site in these cell lines (Fig. 12E), which suggested that this increased recruitment could be associated with the reduction of GNPAT expression that we observed in the murine cells. This was further supported by the finding that the promoter construct lacking this c-MYC-binding site did not respond to the overexpression of NF-κB proteins in SH-SY5Y cells (Fig. 12F). These data suggest that NF-κB activation can also reduce the expression of GNPAT in human cells as in murine cells.

Figure 12.
Figure 12.

Overexpression of p65 and p50 reduces the expression of GNPAT in human cell lines. A, Western blot data showing an increase in c-MYC and decrease in GNPAT protein expression by the transient transfection of p65 and p50 expressing plasmids in SH-SY5Y cells for 48 h. B, Real-time PCR data performed after 48 h of overexpression showing the decrease in GNPAT expression by the overexpression of p65 and p50 in the cells. C, Position of c-MYC-binding sites and the primer locations for the ChIP assays onto the human GNPAT promoter. The number (+1) indicates the transcription start site located within the first exon. D, E, ChIP assays showing the recruitment of c-MYC proteins onto the binding sites of GNPAT promoter in the steady state (D) and after the overexpression of p65 and p50 proteins in the cells for 48 h (E). F, Schematic representing the cloned human GNPAT promoter regions in the luciferase vector. The short form of the promoter (−150 to +183) lacks the putative c-MYC-binding site (C). Promoter assays showed that the overexpression of p65 and p50 proteins could not reduce the promoter activity of the human GNPAT promoter lacking the c-MYC binding site (n = 5).

AD mice model and postmortem AD brain tissues also show an increase in c-Myc and a reduction of Gnpat expression in glial cells

Finally, we investigated whether the reduction of Pls content was associated with the reduction of Gnpat expression via NF-κB and c-Myc signaling in the AD brain. It has been shown previously that there was a significant reduction of total Pls in the brain tissue of amyloid precursor protein (APP)-transgenic AD model mice compared with control mice (Han et al., 2001). In the present study, we used a triple-transgenic (3Tg) AD mouse model (Oddo et al., 2003) and found a significant reduction of total PlsEtn in the 18-month-old 3Tg mice compared with control mice of the same age (Fig. 13A). We then determined protein expression in the nuclear fractions of hippocampal and cortex tissues and found an increased amount of nuclear p65 protein in the 3Tg AD model mice brain compared with the control mice (Fig. 13B). Total tissue lysates showed the significant increase in c-Myc and decrease in Gnpat expression in the 3Tg mice hippocampus and cortex tissues (Fig. 13B–D) (p < 0.01). We then examined the brain tissue sections by immunohistochemistry and observed a significant increase in c-Myc and a decrease in Gnpat proteins in the Iba-1 positive glial cells of 3Tg mice (Fig. 13E–H). To prove that these 3Tg mice have a significant amount of Aβ deposition in the cortex, we performed immunostaining with the specific antibody against Aβ (Fig. 13I). We also confirmed that there was an increase in c-MYC and a decrease in GNPAT proteins in the Iba-1-positive glial cells of human postmortem AD brain tissues compared with that from healthy control subjects (Fig. 13J,K). These data suggest that the similar changes in c-MYC and GNPAT proteins can be observed in the AD brains of both mice and humans and this may be correlated with the reduction of brain Pls.

Figure 13.
Figure 13.

Increased c-MYC and reduced GNPAT expression in an AD mouse model and in human postmortem AD brain tissue. A, LC-MS analysis showing a decrease in PlsEtn in 18-month-old 3Tg mice cortex compared with that of the same age control mice cortex *p < 0.05 (n = 5). B–D, Western blot data (B) showing increased nuclear p65 and c-Myc expression and reduced Gnpat expression in 18-month-old 3Tg mice brain tissues. The quantification data show an significant increase in c-Myc protein (C) and a decrease in Gnpat protein expression (D) in 18-month-old 3Tg mice brain tissues. **p < 0.01 (n = 5). E, F, Double staining of Iba-1 with c-Myc (E) and Gnpat (F) in cortex tissue slices of 18-month-old control and 3Tg mice. Scale bar, 50 μm. G, H, Image quantification data showing the relative number of strongly stained Iba-1 cells by c-Myc (G) and Gnpat (H) antibodies in the control and 3Tg mice brain cortex as shown in E and F. These quantification data represent 5 mice in each group and 10 randomly chosen cortical areas from each mice cortex. *p < 0.05 (n = 5). I, DAB staining showing strong Aβ expression in 18-month-old 3Tg brain cortex compared with control mice of same age. Scale bar, 50 μm. The white arrow indicates Aβ expression inside the cells and the yellow arrow indicates extracellular Aβ. The data represent 3 individual mice in each group. J, K, Immunohistochemistry of postmortem precentral brain tissue showing higher c-MYC expression (J) and lower GNPAT expression (K) among Iba-1-positive cells in pathologically diagnosed AD brain compared with the non-AD brain. Statistical significance was calculated from the 10 randomly chosen observation sites (a total of about 200 Iba-1-positive cells) from the same slide. The data represent the same tissue of three individual slides of the same lot. Scale bar, 50 μm. *p < 0.05; **p < 0.01 (n = 3).

Discussion

It is well known that the human brain contains a limited number of neuronal cells compared with the huge numbers of the glial cells, suggesting that brain glial cells are the major reservoir for brain Pls. Pls contain a saturated fatty acid with a vinyl-ether bond at the sn-1 position and the sn-2 position is enriched in polyunsaturated fatty acids such as arachidonic acid and docosahexaenoic acid, which are released by Pls-selective phospholipase A2 (PLA2) (Farooqui, 2010). Pls are found in all mammalian tissues, especially 20–30% of the glycerophospholipids in the heart and brain, in which most of the brain Pls are PlsEtn (Farooqui and Horrocks, 2001). Pls are biosynthesized by two key enzymes in the peroxisome, GNPAT and alkylglycerone phosphate synthase (AGPS), and then transferred to the endoplasmic reticulum for the final step of the biosynthesis (Farooqui and Horrocks, 2001; Braverman and Moser, 2012).

It is known that mutation or reduction of the Pls-synthesizing enzymes GNPAT and AGPS, as well as the defects in the peroxisome function, can give rise to the reduction of Pls content in the nervous system, making the brain susceptible to the damage seen in the diseases such as Zellweger syndrome, X-linked adrenoleukodystrophy, and rhizomelic chondrodysplasia punctate (Farooqui and Horrocks, 2001; Brites et al., 2009; Braverman and Moser, 2012; Noguchi et al., 2014). Gnpat knock-out mice exhibit dwarfism, defects in eye development, male infertility, optic nerve hypoplasia, reduction in natural killer cell activity, and demyelination of Schwann cells (Brites et al., 2011; Saab et al., 2014; da Silva et al., 2014). Some of the knock-out mice die prematurely, suggesting that the Pls contents are important for normal cellular activity. It has also been reported that AD patients show a reduction of brain and blood PlsEtn levels (Ginsberg et al., 1995; Guan et al., 1999; Han et al., 2001). Consistent with these data in humans, we found a reduction of PlsEtn in the brains of the AD mice model and confirmed a possible mechanistic insight behind this reduction for the first time. More surprisingly, the similar mechanism of murine cells was also observed in the human cell lines and in the human AD postmortem brain. The mechanisms of the neuroinflammation-induced reduction of Pls could be explained as follows. First, due to the Pls-specific vinyl-ether bond at the sn-1 position, Pls may be targeted by oxidants including reactive oxygen/nitrogen species (Yavin and Gatt, 1972; Khaselev and Murphy, 1999; Engelmann, 2004), resulting in the reduction of Pls contents. It has been shown that Pls have antioxidant properties and oxidative stress preferentially oxidizes PlsEtn over phosphatidyl-ethanolamine (Reiss et al., 1997; Zoeller et al., 2002). Second, Pls may be degraded by Pls-selective PLA2, which is activated by, for example, ceramide, produced under inflammatory conditions (Latorre et al., 2003; Farooqui, 2010), contributing to the loss of Pls in the brain. Finally, it has been suggested that the Pls biosynthesis in the AD patients is decreased since the high amount of Aβ and the Aβ-induced ROS/RNS may impair peroxisomal function (Grimm et al., 2011). However, the exact molecular mechanisms of the downregulation of Pls de novo synthesis have been remained elusive.

Our results demonstrate for the first time that expression of a Pls-synthesizing enzyme, Gnpat, is downregulated by inflammatory stimuli, aging, and stress in glial cells, leading to a reduction of Pls levels. Only LPS treatment reduced AGPS expression, not treatment with Poly I:C or IL-1β. We therefore focused only the downregulation of Gnpat expression by inflammatory stimuli in our present study. The promoter studies revealed that the downregulation of Gnpat was caused by the NF-κB-mediated increase in recruitment of c-Myc onto the promoter of Gnpat gene. Therefore, the induction of c-Myc might be one of the reasons for the reduction of Pls contents in the murine brain. Interestingly, in our study, we found that the two binding sites of c-Myc on the Gnpat promoter region were equally important, but the reason for this was still elusive. It might be possible that the c-Myc recruitments could form a common complex with other transcriptional factors necessary to reduce the transcription from the Gnpat promoter. However, future experiments are necessary to address this issue. The speculation that the inflammatory signals induce c-Myc to cause pathological changes is supported by the finding that c-Myc expression is positively associated with the activation of NF-κB (Ji et al., 1994; La Rosa et al., 1994), which is often observed in the pathology of neuroinflammation (O'Neill and Kaltschmidt, 1997; Mattson and Camandola, 2001). We also found in the present study that the expression of c-Myc was enhanced by activation of NF-κB in the cell lines and in the murine brain, working as a transcriptional repressor. This may be consistent with previous reports showing that c-Myc can function as both the transcriptional activator and repressor depending on the target genes and association with the other cotranscription factors (Gartel and Shchors, 2003; Xu et al., 2009; Lüscher and Vervoorts, 2012). As was the case in murine cells, we also noticed a similar effect of NF-κB on c-MYC and GNPAT in the human cell lines. Furthermore, we found a reduction of GNPAT and increased expression of c-MYC in the human AD postmortem brain. Interestingly, we found that the downregulation of Gnpat itself caused activated phenotype of microglial cells, as well as an enhancement of proinflammatory cytokine production (Fig. 10). The mechanism of the glial activation caused by decrease in Pls in the present study is not known. However, we observed that the knockdown of Gnpat induced a significant activation of NF-κB in the glial cells, which raised the possibility that a reduction of Pls in the AD brain may not be an artifact, but rather could accelerate neuroinflammation, resulting in the prolonged/chronic progression of AD.

We have reported previously that application of Pls activates Akt and Erk signaling to inhibit neuronal apoptosis (Hossain et al., 2013). It has also been shown that extracellular addition of Pls enhances natural killer (NK) cell activity, whereas the reduction of endogenous Pls induces the death of NK cells (Ni et al., 2014). These findings support a hypothesis that the reduction of Pls may be one of the causes of neuronal damages leading to memory loss in the AD brain. It has been shown that the lipid rafts present in the cellular membrane, which are known to play an important role in signal transduction through the localization, trafficking, and regulation of G-protein coupled receptor (GPCR)-related molecules (Allen et al., 2007), are rich in Pls compared with the nonlipid rafts region (Rodemer et al., 2003). Therefore, it is possible that reduction of Pls content can affect signal transduction cascade. This is supported by our recent findings that Pls can activate some GPCRs, which are believed to function in the rafts (Hossain et al., 2016). However, further studies are required to demonstrate how Pls reduction activates NF-κB to induce neuroinflammation.

Bottelbergs et al. (2012) reported that there was not a significant increase in the expression of proinflammtory cytokines TNFα, TLR2, and C1q in Gnpat knock-out mice and concluded that there was no change in the inflammation status. However, they did not mention the major proinflammtory cytokines IL-1β and Mcp-1 in their experiments. In addition, there was a tendency toward increased expression of TNFα and TLR2 in the Gnpat knock-out mice in that study. Based on our extensive experimental data, the reduction of Pls induced by knockdown of Gnpat resulted in the increased expression of IL-1β and Mcp-1 in microglial cells, which was also associated with the nuclear localization of the p65 protein.

Our cumulative evidence suggests that levels of the unique glycerophospholipid, Pls, are commonly reduced by inflammatory stimuli, aging, and stress, which are pathological causes of glial activation and neuronal cell death. Further study will be needed to understand how Pls is necessary for maintaining the cellular signaling in the brain with the aim of finding possible therapeutic strategies to prevent the neuronal damage associated with various neurodegenerative diseases.

Footnotes

  • This work was supported by the Japan Society for the Promotion of Science (KAKENHI Grant 26460320 to T.K. and Grant-in-Aid for Young Scientists Wakate B 16K19007 to M.S.H.). We thank Stephen Smale for kindly providing the p50 expressing vectors through the Addgene plasmid sharing service, Rhodri E. Jones (Kyushu University) and Ako Niwase for help with English, and the Research Support Center, Graduate School of Medical Sciences, Kyushu University, for technical assistance.

  • The authors declare no competing financial interests.

  • Correspondence should be addressed to Toshihiko Katafuchi, M.D., Ph.D., Department of Integrative Physiology, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. kataf{at}physiol.med.kyushu-u.ac.jp

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Reduction of Ether-Type Glycerophospholipids, Plasmalogens, by NF-κB Signal Leading to Microglial Activation
Md. Shamim Hossain, Yuichi Abe, Fatma Ali, Mohammed Youssef, Masanori Honsho, Yukio Fujiki, Toshihiko Katafuchi
Journal of Neuroscience 12 April 2017, 37 (15) 4074-4092; DOI: 10.1523/JNEUROSCI.3941-15.2017
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