Fig 2: 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 (n = 3 for each genotype). B Phosphorylated and total p38 and ERK levels in cortical homogenates from ApoE3 and ApoE4-TR mice were detected by WB. (n = 5 for each genotype). C ApoE4 primary astrocytes from mouse were pre-treated with p38 inhibitors SB 202190 (10 μM) or SB 203580 (10 μM) for 20 minutes and then treated with medium or TNFα plus IFNγ together for 30 minutes. The total and phosphorylated cPLA2 and p38 were detected in the cell lysate by WB. Quantification was obtained from 3 independent repeats. D cPLA2 bound with p38. Immunoprecipitation was performed in the cell lysate of immortalized ApoE4 astrocytes using anti-cPLA2 antibody or species-matched IgG. cPLA2 and p38 were co-detected after immunoprecipitation by WB. WB: Western Blot. Data are represented as mean ± SEM and analyzed by Student’s t-test (two-tailed). *p < 0.05

Fig 3: cPLA2 and p-cPLA2 levels in 8-month old ApoE3-TR and ApoE4-TR mouse brains. The cortex of 8-month old APOE3-TR and ApoE4-TR mice were collected. a, cPLA2 mRNA level in the cortex was detected by qPCR. b, p-cPLA2 and total cPLA2 protein levels in the cortex were detected by western blot. c. Densitometric quantification from b. d, cPLA2 activity in mouse cortex homogenates were measured by cPLA2 activity assay kit. e, LTB4 levels in mouse cortical homogenates were measured by LTB4 assay kit. (n = 5 for each genotype, 3 males and 2 females)

Fig 4: rE4 and Aβ42 induce greater cPLA2 translocation to synaptic vesicles (cytosolic fraction) in human synaptosomes than rE3. A Frozen postmortem human cortex samples were prepared to collect the supernatant (P1 fraction) and pellets (P2 fraction, synaptosomes) and the different cell type markers were measured by WB. B cPLA2 expression was detected in different fractions. C The LDH assay was performed to test the membrane integrity of the isolated synaptosomes after incubation for 1 hour at different temperatures. D One group of synaptosomes was treated with KR alone (control), and the remaining groups were treated with rE3, rE4, Aβ42 and C-1-P for 30 minutes, followed with C-1-P treatment for 15 minutes. After treatment, the cytosolic and membrane fractions of synaptosomes were isolated by centrifugation. cPLA2 was enriched by immunoprecipitation with anti-cPLA2 antibody and then phosphorylated and total cPLA2 were measured by WB. A representative blot for total cPLA2 is shown. Phosphorylated cPLA2 levels were low and not shown. E Quantification of cPLA2 protein expression in the cytosol fraction. cPLA2 was normalized to β-actin for each group and then adjusted to KR alone for all groups. F Quantification of cPLA2 protein expression in membrane fraction. cPLA2 protein expression was normalized to Na,K ATPase for each group and then adjusted to KR alone for all groups. For E and F, data was obtained from 7 independent repeats. WB. Western blot; C, cytosol; M, Membrane; rE3, recombinant ApoE3; rE4, recombinant ApoE4; C-1-P, Ceramide-1-phosphate; KR, Krebs Ringer buffer. Data presented as mean ± SEM and analyzed by Student’s t-test (two-tailed). *p < 0.05

Fig 5: p38-MAPK levels are greater in the frontal cortex of persons with AD dementia with APOE3/E4 compared with APOE3/E3. The frozen inferior frontal lobe tissue from persons with NCI or AD dementia with different APOE genotypes was homogenized to test phosphorylated p38 (p-p38) and total p38-MAPK protein levels using WB. A p-p38 and p38 protein levels in the cortex from persons with NCI (n = 6 for E3/E3; n = 6 for E3/4). B p-p38 and p38 protein levels in the cortex from persons with AD dementia (n = 6 for E3/E3; n = 8 for E3/E4). NCI, no cognitive impairment; AD, Alzheimer’s disease. WB: Western Blot. Data are represented as mean ± SEM and analyzed by Student’s t-test (two-tailed). *p < 0.05