Calcium-dependent cytosolic phospholipase A2 activation is implicated in neuroinammation and oxidative stress associated with ApoE4

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 signicance was present at p < 0.05. Statistical program R, version 3.5 was used.


Background
The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the stereo-speci cally numbered (sn-2) ester bond of substrate phospholipids in the cell membrane, to produce a free fatty acid and a lysophospholipid [1]. Calcium-independent PLA2 (iPLA2) has a greater a nity for releasing docosahexaenoic acid (DHA, 22:6 n-3), which acts as a signaling molecule during neurotransmission and as a precursor of anti-in ammatory and antioxidant resolvins [2,3]. Calcium-dependent cytosolic phospholipase A2 (cPLA2) releases arachidonic acid (AA, 20:4 n-6), which plays important functions in storing energy, as a second messenger in neurotransmission, and as a the precursor of eicosanoids [4,5]. Free AA can be oxidized by cyclooxygenase (COX) or lipoxygenase (LOX) to produce prostaglandins or leukotrienes, which are potent mediators of in ammation [1,6]. In astrocytes, cPLA2 interacts with mitochondrial antiviral-signaling protein (MAVS) to boost nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB)-driven in ammatory responses [7]. In microglia, cPLA2 and AA metabolic pathways contribute to reactive oxygen species (ROS) and nitric oxide (NO) production during cell activation [8]. cPLA2 activity depends on its phosphorylation, which is regulated by mitogen-activated protein kinase (MAPK) pathways [9,10].
A lower amount of Aβ oligomers and the absence of markers of glial activation in both astrocytic and microglia distinguish the brains of individuals with greater brain Aβ plaques and tangles but resilience to AD dementia from those with dementia [11]. cPLA2 activation is one of the pathways that activates microglia and astrocytes in the brain. The cPLA2 gene, protein levels, and its phosphorylated form are increased around plaques of AD brains compared to healthy controls [12][13][14]. In AD animal models, increased activation of cPLA2 is observed in the hippocampus of human amyloid precursor protein (hAPP) transgenic mice [14]. The activation of cPLA2 by Aβ oligomers is believed to contribute to a dysregulation of fatty acid metabolism, promoting neurodegeneration [15,16]. Overexpression of p25 in neurons increases the expression of cPLA2 leading to lysophosphatidylcholine (LPC) secretion, and to the activation of astrocytes and production of pro-in ammatory cytokines [17]. Conversely, cPLA2 de ciency in AD mouse models ameliorates the memory impairment and hyperactivated glial cells observed in AD mouse models [14,18]. Knocking out cPLA2 in microglia decreases lipopolysaccharide (LPS) induced oxidative stress and in ammatory response [8].
Carrying the APOE4 allele is the strongest genetic risk factor for late-onset AD. The ApoE4 protein seems to have proin ammatory and/or reduced anti-in ammatory functions, which could exacerbate AD pathology. This was clearly demonstrated in the Framingham cohort where participants with APOE4 and elevated plasma C-reactive protein (CRP) levels had a greater risk of developing late-onset of AD than age and sex-matched APOE2 and APOE3 carriers [19]. In brains of participants with AD, APOE4 is associated with greater levels of lipid peroxidation, eicosanoids and oxidative stresses markers [20], but the mechanisms for these observations are not clear. Here, we hypothesized that ApoE4 activates cPLA2 to enhance AA release and eicosanoid levels, leading to an enhanced in ammatory and oxidative stress response. Accordingly, we examined cPLA2 expression and activation in mouse primary astrocytes, mouse and human brain samples that differed by APOE genotype and determined the cellular effects of cPLA2 inhibition on measurements of neuroin ammation and oxidative stress.

Results
1. cPLA2 and phosphorylated cPLA2 are increased in ApoE4 mouse primary astrocytes We previously found that DHA/AA ratio in cerebrospinal uid (CSF) is lower in APOE4/E4 carriers compared to APOE3/E3 carriers [21,22]. Since astrocytic cPLA2 and iPLA2 enzymes are important determinants of brain AA and DHA metabolism [2,23], the expression and activity of these enzymes were examined in primary astrocytes from ApoE-TR mice. ApoE4 astrocytes had greater mRNA and protein levels of cPLA2 and phosphorylated cPLA2 compared with ApoE3 astrocytes (Fig. 1A, B). In contrast, iPLA2 mRNA and protein levels did not differ between ApoE4 and ApoE3 primary astrocytes (Fig.1C, D).
These measures were also signi cantly greater in ApoE4 immortalized astrocytes compared to ApoE3 (Fig. S1). To further explore the activity of cPLA2 and iPLA2, the e ux of 3 H-AA or 14 C-DHA from ApoE3 and ApoE4 primary astrocyte cells to media with or without ATP stimulation for 15 min was examined. 3 H-AA e ux was signi cantly greater in stimulated ApoE4 compared to ApoE3 primary astrocytes (Fig.   1E), whereas 14 C-DHA e ux showed no difference between ApoE4 and ApoE3 (Fig. 1F). To con rm the effect of the ApoE protein, cultured primary astrocytes from C57BL/6 mice were labeled with 3 H-AA or 14 C-DHA and then treated with 0.2 µM rApoE3 or rApoE4 proteins for 24h under similar conditions to primary astrocytes cultured from ApoE-TR mice. 3 H-AA e ux was greater after rApoE4 than rApoE3 treatment (Fig. 1G), whereas DHA e ux did not differ between rApoE4 and rApoE3 treatments (Fig. 1H). Taken together, these results con rmed that cPLA2 expression and activity were greater in ApoE4 compared to ApoE3 astrocytes.

Phosphorylated cPLA2 and cPLA2 activity are increased in APOE4 mouse brains
To investigate the effect of the ApoE isoforms on cPLA2 in vivo, mRNA, total protein, and phosphorylated protein levels of cPLA2 were measured in the cerebral cortex from 8-month-old of ApoE3-TR and ApoE4-TR mice. There was no difference in cortical cPLA2 mRNA levels between ApoE3-TR and ApoE4-TR mice ( Fig. 2A). Since phosphorylated cPLA2 levels were too low to detect in total brain homogenates, cPLA2 was enriched by immunoprecipitation with a cPLA2 antibody using 500 µg of cortical homogenate, and total and phosphorylated cPLA2 were measured by immunoblotting. Total cPLA2 levels did not differ between ApoE3-TR and ApoE4-TR mouse cortex (Fig. 2B). However, phosphorylated cPLA2 was signi cantly increased in ApoE4-TR mouse cortex compared to ApoE3-TR mouse cortex (Fig. 2B).
1. p38 MAPK but not ERK1/2 is increased in ApoE4 mouse primary astrocytes Phosphorylation of cPLA2 is regulated by MAPK pathways, including p38 MAPK and ERK1/2 MAPK [10,24,25]. We tested the phosphorylation of p38 and ERK1/2 in primary astrocytes and in mouse cortex from ApoE3 or ApoE4-TR mice by immunoblot using antibodies against total and phosphorylated proteins. Total p38 and ERK1/2 proteins did not differ between ApoE3 and ApoE4 primary astrocytes (Fig.  3A). Interestingly, only phosphorylated p38, but not phosphorylated ERK1/2, was signi cantly greater in ApoE4 primary astrocyte compared with ApoE3 primary astrocytes (Fig. 3A). This nding suggests that the greater phosphorylation of cPLA2 observed in ApoE4 is driven by p38 MAPK activation, but not the ERK1/2 MAPK pathway. In agreement, greater p38 phosphorylation but not of ERK1/2 was evident in the cerebral cortex of 8-months old ApoE4-TR mice compared to ApoE3-TR mice (Fig. 3B).
To determine where these ndings can be demonstrated in human brains, we rst compared the total and phosphorylated forms of cPLA2 in human hippocampus samples from individuals with no cognitive impairment (NCI) and homozygous APOE3 (APOE3/E3) carriers with individuals with AD and homozygous APOE4 (APOE4/E4). Characteristics of brain samples tested are summarized in Table 1.
After enrichment of cPLA2 protein, phosphorylated and total protein levels of cPLA2 were detected by Western Blot. There was no difference in total cPLA2 protein between APOE3/E3 and APOE4/E4 human hippocampus, while phosphorylated cPLA2 trended toward an increase in APOE4/E4 human hippocampus compared to that of APOE3/E3 carriers. However, this difference did not reach statistical signi cance, likely a result of the small size of samples of the human brain samples (Fig. 4A). Moreover, this comparison was limited by virtue of a comparison that included both disease state and APOE genotype. To overcome this limitation, we compared phosphorylated and total cPLA2 in the inferior frontal cortex of persons with similar clinical diagnosis but with different APOE genotypes. After a similar enrichment of cPLA2 from the cortex, phosphorylated and total cPLA2 were measured by Western Blot. In the NCI group, total cPLA2 did not signi cantly differ between the APOE3/E3 and APOE3/E4 carriers, while the phosphorylated cPLA2 level showed a trend increase in APOE3/E4 carriers compared to APOE3/E3 carriers (Fig. 4B). In patients with AD, phosphorylated cPLA2 levels were signi cantly greater in APOE3/E4 carriers compared with APOE3/E3 carriers (p=0.039), while the total cPLA2 levels did not differ between the two groups ( Fig. 4C). Greater cPLA2 phosphorylation in APOE3/4 group was not affected by sex, age or Braak stage.
3. p38 MAPK is increased in APOE4 human brain samples Previous results from mouse astrocyte and cortex showed increased p38 activation in ApoE4-TR compared to ApoE3-TR mice. In agreement, greater levels of phosphorylated p38 in the hippocampus of the APOE4/E4 AD group were observed compared to the APOE3/E3 NCI group (Fig. 5A). Phosphorylated and total p38 levels did not differ between NCI APOE3/E3 and NCI APOE3/E4 groups (Fig. 5B), while total p38 level was signi cantly greater in the AD APOE3/E4 group compared with the AD APOE3/E3 group (Fig. 5C). These results support that the greater activation of cPLA2 in ApoE4 might be regulated by the p38 MAPK pathway and is most prominent in persons with AD.

LTB4 levels is increased in APOE4 human brain samples
AA is released by cPLA2 hydrolysis of membrane phospholipids, which then can be rapidly oxidized by COX or LOX enzymes to prostaglandins or leukotrienes (LTB4 and PGE2), potent mediators of in ammation and signal transduction [2]. To test the effect of the greater cPLA2 phosphorylation in APOE4 AD brains, PGE2 and LTB4 levels were assayed in the brain homogenates from the inferior frontal cortex. LTB4 levels were signi cantly greater in the AD APOE3/4 group compared with the AD APOE3/3 group (p=0.01) (Fig. 6A), while PGE2 levels did not differ between the two groups (Fig. 6B). The greater LTB4 levels in APOE3/E4 group were also not affected by sex, age or Braak stage. No signi cant differences were found in either LTB4 or PGE2 levels between the NCI APOE3/4 and NCI APOE3/4 groups ( Fig. 6C and D). The expression of 5-LOX and COX-2 did not differ between the AD APOE3/3 and AD APOE3/4 groups (Fig. 6E). These results indicate that ApoE4's activation of cPLA2 in AD selectively increases LTB4 levels in the AD brain.

The NF-kB in ammasome is not induced in the APOE4 brain
It is not clear whether APOE4 can induce neuroin ammation via activation of the NF-kB in ammasome in vivo, and whether cPLA2 is involved in this pathway. Although we found greater TNFa mRNA levels in ApoE4 than in ApoE3 astrocytes, IL1b, IL6 and Ccl2 did not differ between ApoE3 and ApoE4 astrocytes (Fig. 7A). In addition, none of these cytokines and chemokines differed by genotype among the mouse brain ( Fig. 7B) or the human brain samples (Fig. 7D), including glial brillary acid protein (GFAP) for astrocytes and ionized calcium binding adaptor molecule 1 (Iba1) for microglia ( Fig. 7D-E). These results indicate that neuroin ammation with APOE4 does not favor the NF-kB in ammatory response pathway.

cPLA2 is involved in the ApoE4 mediated up-regulation of LTB4 and ROS
To explore whether inhibition of cPLA2 mitigates the downstream effects of LTB4 production on ROS and iNOS, ApoE3 and ApoE4 primary astrocytes were treated with the cPLA2 inhibitor pyrrophenone ( Fig 8A). Treatment with pyrrophenone reduced LTB4 levels in both ApoE3 and ApoE4 astrocytes, but to a greater extent with ApoE4 astrocytes (Fig. 8B). Furthermore, cPLA2 inhibition signi cantly decreased ROS and iNOS expression in both ApoE3 and ApoE4 primary astrocytes (Fig. 8C). These results indicated that greater cPLA2 activity is also involved in mediating the greater levels of iNOS and ROS in the ApoE4 group and can be reduced with cPLA2 inhibition.

Discussion
Despite multiple past observations associating APOE4 with greater neuroin ammatory and oxidative stress response than APOE2 or APOE3 (Table 2), the underlying mechanisms are not clearly understood.
Here, we identify a plausible mechanism where APOE4 induces greater activation of the MAPK p38-cPLA2 system, leading to greater release of AA, LTB4, iNOS and generation of ROS in astrocytes. The increase in LTB4 in APOE4 was corroborated in human brain samples matched by disease state. Inhibition of cPLA2 activity lowered the greater measurements of neuroin ammation associated with APOE4, reinforcing the candidacy of cPLA2 as a therapeutic target for mitigating the increase in AD risk conferred by carrying APOE4. Vitek et al. [26] Microglia derived from ApoE4-TR mice demonstrate increased NO production, increased NOS2 mRNA levels, and greater TNFα, IL-6, IL12 levels compared to microglia from ApoE3-TR mice.
Colton et al. [27] Signi cantly more NO was produced in primary microglia and macrophages from ApoE4-TR mice compared to ApoE3-TR mice.
Guo et al [28] Addition of exogenous ApoE4 induced greater IL1β than apoE3 in rat mixed glial cells.
Shi et al [30] Higher TNFα, IL1β and IL1α levels were observed in primary microglia from ApoE4-TR mice stimulated with LPS than ApoE2 and ApoE3.
Tai et al [31] Greater astrogliosis and microgliosis, higher levels of IL1β in E4FAD mice compared with E3FAD and E2FAD mice.
Ophir et al [33] The expression of in ammation-related genes (NF-κB response elements) following intracerebroventricular injection of LPS was signi cantly higher and more prolonged in ApoE4 than in ApoE3-TR mice.
Both human and mouse models Gale et al [34] ApoE4-TR mice displayed enhanced plasma cytokines after systemic LPS compared with ApoE3 counterparts. After intravenous LPS, APOE3/4 patients had higher plasma TNF-α levels than APOE3/3 patients.
Human brain studies of in ammation and oxidative stress studies by APOE genotype Montine at al [35] Pyramidal neuron cytoplasm was immunoreactive for 4-hydroxy-2-nonenal (HNE) in 4 of 4 APOE4 homozygotes, 2 of 3 APOE3/4 heterozygotes, and none of 3 APOE3 homozygotes Ramassamy et al al. [20] In hippocampal homogenates from AD brains, APOE4 carriers had greater levels of thiobarbituric acid-reactive substances (TBARS), lower activities of catalase, glutathione peroxidase and glutathione than tissues from patients homozygous for the APOE3 allele (n = 10 per group).
Egensperger et al [36] The number of activated microglia and the tissue area occupied by these cells increased signi cantly with APOE4 gene dose (n = 20).

Author Key ndings
Friedberg et al [38] Cellular density of microglial marker-Iba1 was positively associated with tau pathology in APOE4 carrier participants only (n = 154).

Systemic in ammation and dementia risk by genotype
Tao et al [19] Participants with APOE4 and elevated plasma C reactive protein (CRP) levels had a shortened latency for onset of AD (n = 2562).
There is evidence from clinical studies implicating greater cPLA2 activation around AD brain plaques [12]. cPLA2 activity is also increased in the CSF of patients with AD [39]. cPLA2 activation can be indirectly assessed by the release of AA from membrane phospholipids [2]. 11 C AA brain uptake by PET and AA/DHA measurement in CSF are surrogate markers of brain cPLA2 activity. Indeed, greater incorporation coe cients of 11 C AA by PET scans was observed in the grey-matter region of the brain of AD patients compared to control subjects [40]. Moreover, a greater AA/DHA ratio in both CSF and plasma was present in APOE4 carriers with mild AD compared to APOE3 carriers after DHA supplementation [21]. A greater AA/DHA ratio in plasma phospholipids in cognitively healthy APOE4 carriers was associated with greater conversion to MCI/AD [41]. The greater plasma AA/DHA in APOE4 suggests a systemic (for example in the liver, adipose tissues) activation of cPLA2 that is not just con ned to the brain.
Greater cPLA2 activation is mechanistically involved in AD pathology and may represent one pathophysiological link between Aβ oligomers and neuroin ammatory responses [42]. An increase of phosphorylated cPLA2 but not of total cPLA2 was observed in the brains of AD mouse models compared with WT mice [14]. In vitro studies suggested that Aβ oligomers can trigger cPLA2 activation and PGE2 production in neurons eventually leading to neurodegeneration [43,44]. Inhibition of cPLA2 prevented synaptic loss and memory de cits induced by Aβ oligomers in mice [45]. Similar to Aβ, there is evidence that human prion peptide can also induce neurotoxicity by activating cPLA2, which can be prevented by cPLA2 inhibition [46]. In support of greater cPLA2 activity, hippocampal levels of AA and AA-derived metabolites were much greater in hAPP mice than in non-transgenic control mice [47].
The pattern of enhanced neuroin ammation of the APOE4 AD brains observed in this study does not support the induction of the NF-ĸB in ammasome (such as TNFα, IL1β, IL6 and Ccl2), as past ndings supporting this activation pattern were mostly a result of high doses LPS injections in cell culture and in vivo animal models (summarized in Table 2). Instead, we found greater level of leukotrienes (LTB4) in the cerebral cortex of AD with E3/E4 carriers compared to E3/E3 carriers, and in apoE4 astrocytes that was associated with the greater phosphorylation of cPLA2. These observations provide a mechanism for the greater levels of oxidative stress in the APOE4 brain [20,35]. It is plausible that not just astrocytes, but microglia as well contribute to the greater LTB4, ROS and iNOS production with APOE4. A large recent proteomic and lipidomic investigation in animal brains of ApoE-TR mice corroborate the enhanced eicosanoid signaling with APOE4 [48]. LTB4 signaling may have a prominent role in inducing oxidative stress. Chuang et al reported that ROS and NO production during microglia activation is reduced by inhibition of lipoxygenase but not cyclooxygenase [8], suggesting induced LOX signaling as the primary driver of oxidative stress.
Activation of cPLA2 may differ by cell type and within cellular compartments. Recently, astrocytic activation of cPLA2 bound directly with MAVS enhanced NF-ĸB pathways to produce pro-in ammatory factors such as Ccl2 and Nos2 in an animal model of multiple sclerosis (MS) [7]. Here, we did not observe greater CcL2 or Nos2 expression in APOE4 astrocytes, mouse or human brains suggesting that a different localization of cPLA2 activation within the astrocyte can lead to a distinct neuroin ammatory phenotype.
In addition to MS, the increase in AA release and its metabolisms to prostaglandins and leukotrienes, have been observed in cancers and other neurodegeneration diseases [49][50][51]. For example, PIK3CA mutant breast cancer tumors cells displayed dramatically elevated AA and eicosanoid levels, promoting tumor cells proliferation [50].
Activation of cPLA2 activity is associated with its phosphorylation [10]. cPLA2 phosphorylation is regulated by ERKs and p38 MAPK pathways, which phosphorylates cPLA2 at Ser-505 and increases its enzymatic activity [9]. cPLA2 phosphorylation and AA release in response to PMA and ATP stimulation in mouse astrocytes are mediated by ERKs and p38 MAPK pathways [10]. In the platelets, cPLA2 phosphorylation was induced by p38 MAPK activation [24]. Here, we found that ApoE4 selectively activated p38 but not ERKs. This is consistent with a previous report of greater p38 activation but not ERKs pathway in ApoE4-TR mice [52], suggesting the increase of phosphorylation of cPLA2 in ApoE4 is regulated by p38 MAPK but not ERKs. Interestingly, p38 inhibitors are in drug development pipelines for AD [53].
Our study has strengths and some limitations. We validated our ndings of greater cPLA2 activation in cells, in ApoE-TR animal models and in human brains matched by disease stage and differing by genotype. We identi ed the signaling pathway involved in cPLA2 activation-(MAPK-p38) and validated this in both animal and human brains. Some of the limitations include not examining the effects of ApoE4 on cPLA2 activation on microglia. It is plausible that ApoE4 enhances microglial activation through a cPLA2 dependent mechanism, with unique downstream activation patterns. In the clinical cohort, we did not study cPLA2 expression in APOE4 homozygote patients without cognitive impairment, as this condition is extremely rare. We also acknowledge that the small sample sizes for both humans and mice studies preclude the full examination of the effect of sex on the association between APOE4 and neuroin ammation. Future studies will include larger samples sizes and more speci c approaches (such as single cell sequencing) to capture cPLA2's activation ngerprint on distinct brain cells.

Conclusions
Overall, using multiple approaches, our study has identi ed that the activation of cPLA2 is implicated in neuroin ammation and oxidative stress associated with APOE4 (Fig. 9). Our ndings support that induction of the MAPK-p38 pathway as the driving factor for the activation of cPLA2-LTB4 signaling cascade, and our cellular studies prioritize astrocytes as the target cell type. Small molecular inhibitors of cPLA2 can be tested in vivo for their capacity to reduce the risk of AD dementia associated with carrying the APOE4 allele.

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 (

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 iso urane 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]. Brie y, 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 Scienti c, FB-12) and 1% antibioticantimycotic (Anti-anti) (Thermo Fisher, 15240062). The cells were dispersed into a single-cell level by repeated pipetting and ltered through 100µ m cell strainers (VWR, 10199-658). After ltering, 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 cm 2 ask 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 con uence 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.
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. To investigate arachidonic acid (AA) and docosahexaenoic acid (DHA) release by cPLA2 and iPLA2 activation respectively, we performed an AA and DHA e ux 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 lled 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 lled with 3 mL of scintillation cocktail. After mixing rigorously, the vials were counted in a Beckman LS6500 liquid scintillation counter (Beckman Coulter). The e ux 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 e ux 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 3 H-AA (1 µCi/mL) or 14 C-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 e ux 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.  ApoE4 increases cPLA2 but not iPLA2 expression in mouse primary astrocytes. A, cPLA2 mRNA levels in primary astrocytes from APOE3 and APOE4-TR mice. B, cPLA2 and phosphorylated cPLA2 (p-cPLA2) protein levels in primary astrocytes from ApoE3 and ApoE4-TR mice (left) were detected by densitometry (western blot -WB). Quanti cation of WB from three independent experiments (right). C, iPLA2 mRNA levels in primary astrocytes from ApoE3 and ApoE4-TR mice. D, iPLA2 protein levels in primary astrocyte cultures from ApoE3 and ApoE4-TR mice (left) was detected by WB. Quanti cation of WB from three independent experiments (right). E, F, Primary astrocytes from ApoE3 and ApoE4-TR mice were incubated with 3H-labelled AA (E) or 14C-labeled DHA (F) for 24h, followed by induction by 100nM ATP for 15min.
The e ux of 3H-AA (E) and 14C-DHA (F) from cells to media was measured by scintillation counting. G, H, Primary astrocytes from C57BL/6 wild type mice were labelled with 3H-AA (G) or 14C-DHA (H) for 24h and then treated with recombinant ApoE3 or ApoE4 for 24h, followed by induction with 100nM ATP for 15min. 3H-AA (G) and 14C-DHA (H) e ux was measured by scintillation counting.  Increased phosphorylated-cPLA2 in APOE4 is mediated by p38 MAPK. A, Phosphorylated and total p38 and ERK levels in primary astrocyte from ApoE3 and ApoE4-TR mice were detected by WB densitometry (left). Quanti cation of WB from three independent experiments (right). B, Phosphorylated and total p38 and ERK levels in cortical homogenates from APOE3 and APOE4-TR mice were detected by WB (left).
Quanti cation of WB from three independent experiments (right). WB: Western Blot  In ammatory responses in primary astrocytes, mouse and human cortex with different APOE genotypes.
A, mRNA levels of pro-in ammatory markers in the primary astrocyte from ApoE3-TR or ApoE4-TR mice.
B, mRNA levels of pro-in ammatory cytokines in the cortex of ApoE3-TR or ApoE4-TR mice. C, GFAP and Iba-1 expression in the cortex of ApoE3-TR or ApoE4-TR mice. (n=5, 3 males and 2 females for B and C).