Clinical Samples
The frozen hippocampus of AD patients with APOE4/E4 carriers (N = 9) and no-cognitive impairment (NCI) with APOE3/E3 carriers (N = 7) were collected from the University of Southern California (USC) Alzheimer Disease Research Center (ADRC) Neuropathology core, which was approved by USC’s Institutional Review Board (IRB) protocol (HS-16-00888). The frozen inferior frontal lobe (Brodmann area 10) of the individuals with NCI and the APOE3/E3 carriers (N = 12) and APOE3/E4 carriers (N = 10), and persons with AD patients and the APOE3/E3 (N = 12) and APOE3/E4 genotypes (N = 10) were obtained from the Rush Alzheimer's Disease Center (RADC) at the Rush University Medical Center. Rush Memory and Aging Project was approved by an Institutional Review Board (IRB) of Rush University Medical Center.
Animals
ApoE3-TR and ApoE4-TR mice were a generous gift from Dr. Patrick Sullivan, in which the endogenous mouse ApoE was replaced by either human APOE3 or APOE4, were created by gene targeting as described previously [54]. All experiments were performed on age-matched male animals (8 months of age) and were approved by the USC Animal Care Committee. Every effort was made to reduce animal stress and to minimize animal usage. The mice were anesthetized with isoflurane and perfused with PBS. The brains were split in half for further analysis.
Cell cultures
Primary astrocytes were obtained from C57JB6, ApoE3-TR and ApoE4-TR mice pups, and cultured as described previously[55]. Briefly, cerebral cortices from each 1 to 3 day-old neonatal mouse were dissected in ice-cold Hanks’ Balanced Salt Solution (HBSS) (Corning, 21-021-CV), and digested with 0.25% trypsin for 20 min at 37°C. Trypsinization was stopped by addition of 2-fold volume of DMEM (Corning, 10–013) with 10% fetal bovine serum (FBS) (Omega Scientific, FB-12) and 1% antibiotic-antimycotic (Anti-anti) (Thermo Fisher, 15240062). The cells were dispersed into a single-cell level by repeated pipetting and filtered through 100µ m cell strainers (VWR, 10199-658). After filtering, cells were centrifuged for 5 min at 1000 rpm and resuspended in culture medium supplemented with 10% FBS and antibiotics. Then, cells were seeded in a 75 cm2 flask and cultured at 37°C in 5% CO2. The medium was changed on the next day and then replaced every 3 days. These mixed glia cultures reached confluence after 7–10 days. Then, the cells were shaken at 250 rpm for 16 h at 37°C to remove microglia and oligodendrocyte progenitor cells. The remaining cells were harvested by digestion with trypsin. At this stage the culture contains 95% astrocytes and was used for further experiments.
Immortalized mouse astrocytes derived from human ApoE3 and ApoE4 knock-in mice [56] were gifts from Dr. David Holtzman and grown in DMEM/F12 (Corning, MT10090CV) containing 10% FBS, 1 mM sodium pyruvate (Thermo Fisher, 11360070), 1 mM geneticin (Thermo Fisher, 10131-035) and 1% anti-anti.
Cell lysate and brain homogenate preparation
The immortalized or primary astrocyte were lysed with 1x RIPA buffer (Cell Signaling Technology, CST 9806) containing protease inhibitor cocktail (Sigma, P8340) and phosphatase inhibitor cocktail (Sigma, P0044), followed by centrifugation at 14,000 gs for 10 min at 4 °C. The supernatant was collected for further analysis.
The mouse cerebral cortex, human hippocampus and inferior frontal cortex were weighed, then RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail was added as 1:30 (w/v). Then, the tissue was homogenized using a 2 mL glass dounce tissue grinder, followed by centrifugation with 14,000 gs for 10 min at 4 °C. The supernatant was collected, and the concentration was measured by BCA kit.
cPLA2 protein enrichment
To detect the phosphorylated cPLA2 in mouse cortex homogenates, cPLA2 protein was enriched by immunoprecipitation. For each mouse sample, 5 µg of cPLA2 antibody (Santa Cruz Biotechnology, sc-376618) was conjugated to 50 µL Dynabeads Protein G (Thermo Scientific, 10003D) for 1 hr at room temperature, then 500 µg total protein in 500 µL RIPA was added to the cPLA2-beads complex and incubated with rotation overnight at 4 °C. The beads were washed with 0.1% PBST 3 times by rotation for 5 min. After washing, 30 µL of 1x sample buffer (Bio-Rad, 1610747) was added to the beads and heated for 10 minutes at 100 °C. The supernatant was collected by magnetic force and used for the further Western-blot assay.
Western-blot
The cell lysates, cortex homogenate and enriched cPLA2 proteins were separated by 4–15% mini-precast protein gels (Bio-Rad, 4561086) under reducing conditions and then transferred onto nitrocellulose membranes (Bio-Rad, 1704270). After transferring, membranes were blocked with 5% fat-free milk (Bio-Rad, 1706404) in TBST for 1 h at room temperature, followed by overnight incubation with primary antibody in 5% BSA at 4 °C. Then, the membranes were incubated with HRP conjugated secondary antibody for 1 h at room temperature. Chemiluminescent HRP substrate (Millipore, WBKLS0500) was used for detection. Fujifilm LAS-4000 imager system was used to capture images and the densitometric quantification was done by Gel Quant NET software.
The following antibodies and dilution factors were used: cPLA2 antibody (Santa Cruz Biotechnology, sc-376618) (1:200), phospho-cPLA2 (Ser505) antibody (CST, 53044) (1:1000), phospho-ERK1/2 antibody (CST, 4370) (1:1000), ERK1/2 antibody (CST, 4595) (1:1000), p38 antibody (CST, 9212) (1:1000), phospho-p38 antibody (CST, 4511) (1:1000), GFAP antibody (CST, 12389) (1:1000), Iba-1 antibody (GeneTex, GTX100042) (1:1000), iNOS antibody (CST, 13120) (1:1000), β-actin antibody (CST, 3700) (1:1000), β-tubulin antibody (CST, 2146) (1:1000), HRP-linked anti-mouse IgG (CST, 7076) (1:2000), HRP-linked anti-rabbit IgG (CST, 7074) (1:2000).
qPCR
The cells and brain specimens were harvested, and RNA was extracted using an RNA extraction kit (Thermo Fisher, K0731). Synthesis of cDNA was done using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, 4368814). qPCR was performed using the PowerUp SYBR Green Master Mix (Thermo Fisher, A25742). The following primers were synthesized by Integrated DNA Technologies. The cPLA2 sense (5'-CTGCAAGGCCGAGTGACA-3') and antisense (5'-TTCGCCCACTTCTCTGCAA-3'); mouse Tnfα sense (5'-GCCTCTTCTCATTCCTGCTTG-3') and antisense (5'-CTGATGAGAGGGAGGCCATT-3'); mouse Il1β sense (5'-GCAACTGTTCCTGAACTCAACT-3') and antisense (5'-ATCTTTTGGGGTCCGTCAACT-3'); mouse Il6 sense (5'-TAGTCCTTCCTACCCCAATTTCC-3') and antisense (5'-TTGGTCCTTAGCCACTCCTTC-3'); mouse Ccl2 sense (5'-GTCCCTGTCATGCTTCTGG-3') and antisense (5'-GCTCTCCAGCCTACTCATTG-3'); mouse Mip1α sense (5'- TGAAACCAGCAGCCTTTGCTC-3') and antisense (5'-AGGCATTCAGTTCCAGGTCAGTG-3'); mouse Mip2 sense (5'-ATCCAGAGCTTGAGTGTGACGC-3') and antisense (5'- AAGGCAAACTTTTTGACCGCC-3'); mouse β-actin sense (5'-ACCTTCTACAATGAGCTGCG-3') and antisense (5'-CTGGATGGCTACGTACATGG-3'); human TNFα sense (5'-ACTTTGGAGTGATCGGCC-3') and antisense (5'-GCTTGAGGGTTTGCTACAAC-3'); human IL1β sense (5'-ATGCACCTGTACGATCACTG-3') and antisense (5'-ACAAAGGACATGGAGAACACC-3');
human IL6 sense (5'-CCACTCACCTCTTCAGAACG-3') and antisense (5'-CATCTTTGGAAGGTTCAGGTTG-3'); human CCL2 sense (5'- TGTCCCAAAGAAGCTGTGATC-3') and antisense (5'-ATTCTTGGGTTGTGGAGTGAG-3'); human GAPDH sense (5'-ACATCGCTCAGACACCATG-3') and antisense (5'-TGTAGTTGAGGTCAATGAAGGG-3')
AA and DHA efflux assays
To investigate arachidonic acid (AA) and docosahexaenoic acid (DHA) release by cPLA2 and iPLA2 activation respectively, we performed an AA and DHA efflux assay as described previously [2]. ApoE3 and ApoE4 primary astrocytes were seeded at 5000 cells/well in 96-well plates. After 24 h the culture medium was changed with serum-free DMEM containing fatty acid-free BSA (5 mg/mL) (Sigma, A9647) and 3H-AA (1 µCi/mL) or 14C-DHA (1 µCi/mL) (Moravek) for 24 h. Then, the cells were washed twice with 100 µL of DMEM and 100 µL of DMEM containing BSA (5 mg/mL) was added. After 30 minutes, the medium was removed and 100 µL of ATP (100 µM) in DMEM without BSA was added. After 15 minutes, cell culture medium was collected and transferred to scintillation vials filled with 3 mL of scintillation cocktail. The cells were solubilized in 90 µL of NaOH (0.5N) for 5 minutes and neutralized with 60 µL PBS, and then transferred to scintillation vials filled with 3 mL of scintillation cocktail. After mixing rigorously, the vials were counted in a Beckman LS6500 liquid scintillation counter (Beckman Coulter). The efflux of AA and DHA were assessed by the ratio of cholesterol in the medium to total cholesterol (medium and cell lysate). The change of AA and DHA efflux was calculated by subtracting the levels of AA and DHA in the ATP treated group to ATP non-treated group for each genotype. WT primary astrocytes were plated and labelled with 3H-AA (1 µCi/mL) or 14C-DHA (1 µCi/mL) as described above. Then, the cells were washed twice with 100 µL of DMEM. After wash, 10 µL of DMEM containing BSA and 0.2 µM recombinant ApoE3 or ApoE4 protein were added. After 24 h, the medium was removed and 100 µL of ATP (100 µM) in DMEM without BSA was added. The AA and DHA efflux were measured as described above after 15 minutes.
cPLA2 activity assay
cPLA2 activity was detected by cPLA2 activity assay kit (Cayman Chemical, 765021). The mouse cortex was homogenized into HEPES buffer (50 mM, pH 7.4, containing 1 mM EDTA) as 1:10 (w/v) and supernatant was collected after centrifuged and used for cPLA2 activity detection.
LTB4 and PGE2 measurement
For the LTB4 an PGE2 measurement in the human brain samples, brain tissue was weighed, then PBS containing 1 mM EDTA, 10 µM indomethacin (Cox inhibitor, Sigma I8280) and 10 µM NDGA (Lox inhibitor, Sigma 479975) as 1:10 (w/v) were added. Then, the tissue was homogenized using a 2 mL glass dounce tissue grinder, followed by centrifugation with 8,000 xg for 10 minutes at 4 °C. The supernatant was collected, and the concentration was measured by a BCA kit. LTB4 and PGE2 levels were detected by the assay kit (LTB4 ELISA Kit, Cayman Chemical, 10009292; PGE2 ELISA Kit, Cayman Chemical, 500141).
For the LTB4 measurement in the cells, ApoE3 and ApoE4 primary astrocytes were seeded in 24-wells plate with the intensity of 100,000 cells per well. Forty-eight hours later, cells were pre-treated with cPLA2 inhibitor-Pyrrophenone (500 nM, Sigma, 5305380001) in the DMEM culture medium without FBS but containing N2 supplement for 30 minutes, followed by the treatment with vehicle or TNFα (10 ng/mL) (R&D Systems, 210-TA-005) plus IFNγ (100 ng/mL) (Sigma, SRP3058) together for 18 hours. Then, the culture media and cell lysate were collected. LTB4 levels were measured in 4-fold concentrated medium by the assay kit.
ROS measurement
ROS were detected by the DCFDA cellular ROS detection assay kit (Abcam, ab113851). ApoE3 and ApoE4 primary astrocyte were seeded in dark, clear bottom 96-wells plate with the intensity of 20,000 cells per well. Forty-eight hours later, cells were pre-treated with cPLA2 inhibitor (1 µM) in the DMEM culture medium without FBS but containing N2 supplement for 30 minutes, followed by the treatment with vehicle or TNFα (10 ng/mL) plus IFNγ (100 ng/mL) together for 24 hours. After removing the media and washing plate once with 1x assay buffer, the cells were stained with DCFDA solution (100 µL/well) for 45 minutes at 37 °C in the dark. Then, the DCFDA solution was removed and the 1x assay buffer (100 µL/well) was added into the plate. ROS levels were measured using a fluorescent plate reader at Excision/Emission = 485/585 nm.
Statistical analysis
Descriptive results are presented as the mean ± SD. Data were analyzed using Student’s unpaired t-test or ANOVA. The cPLA2 phosphorylation was compared in APOE groups using linear regression model, adjusting for age, sex, and Braak stage. Non-parametric tests were used for non-normally distributed data. Statistical significance was present at p < 0.05. Statistical program R, version 3.5 was used.