Background: Apolipoprotein E4 (APOE4) is associated with a greater response to neuroinflammation and the risk of developing late-onset Alzheimer's disease (AD), but the mechanisms for this association are not clear. The activation of calcium-dependent cytosolic phospholipase A2 (cPLA2) is involved in inflammatory signaling and is elevated within the plaques of AD brains. The relation between APOE4 genotype and cPLA2 activity is not known.
Methods: Mouse primary astrocytes, mouse and human brain samples differing by APOE genotypes were collected for measuring cPLA2 expression, phosphorylation, and activity in relation to measures of inflammation and oxidative stress.
Results: Greater cPLA2 phosphorylation, cPLA2 activity and leukotriene B4 (LTB4) levels were identified in ApoE4 compared to ApoE3 in primary astrocytes, brains of ApoE-targeted replacement (ApoE-TR) mice, and in human brain homogenates from the inferior frontal cortex of patients with AD carrying APOE3/4 compared to APOE3/3. Greater cPLA2 phosphorylation was also observed in human postmortem frontal cortical synaptosomes and primary astrocytes after treatment with recombinant ApoE4 ex vivo. In ApoE4 astrocytes, the greater levels of LTB4, reactive oxygen species (ROS), and inducible nitric oxide synthase (iNOS) were reduced after cPLA2 inhibition.
Conclusions: Our findings implicate greater activation of cPLA2 signaling system with APOE4, which could represent a potential drug target for mitigating the increased neuroinflammation with APOE4 and AD.
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Supplementary Figure 1. ApoE4 increases cPLA2 expression in immortalized ApoE astrocytic cultures. A, cPLA2 mRNA levels in immortalized ApoE3 or ApoE4 astrocytes. B, cPLA2 and phosphorylated cPLA2 (p-cPLA2) protein levels in immortalized ApoE3 or ApoE4 astrocytes were detected by WB. C, cPLA2 and phosphorylated cPLA2 (p-cPLA2) protein levels in primary microglial cells from ApoE3 or ApoE4-TR mice were detected by WB. WB: Western blot .
Supplementary Figure 2. cPLA2 distribution in cytosol and membrane of primary astrocytes. A, ApoE3, and ApoE4 primary astrocytes were labeled with biotin, and the membrane proteins were purified with Avidin agarose beads. Phosphorylated and total cPLA2 levels were detected by western blot. Beta-actin was used as the loading control for cytosolic fraction, and Na,K ATPase, was the loading control for the membranous fraction. B, C, Densitometric quantification of blotting shown in A.
Supplementary Figure 3. Aβ and APP levels in the cortex of AD patients with different APOE genotypes. Aβ and APP protein levels in the inferior frontal cortex from AD patients were detected by WB (left panel, n=12, AD E3/E3; n=10, AD E3/E4). WB of the lysate of astrocytes treated with Aβ42 addition as the positive control. WB: Western blot.
Supplementary Figure 4. Total and activated cPLA2 and p38 levels in the hippocampus of persons with different APOE genotypes and disease conditions. (A) p-cPLA2 and cPLA2 protein levels and (B) p-p38 and p38 protein levels in the hippocampus from persons with no cognitive impairment (NCI) carrying E3/E3 and AD patients carrying E4/E4 were detected by western blot.
Supplementary Figure 5. Correlation of p-cPLA2/cPLA2 with GFAP or Iba1 levels in the inferior frontal cortex from AD patients. The Kendall rank correlation coefficient was used to estimate a rank-based measure of association.
Supplementary Table 1
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Posted 11 Feb, 2021
On 10 Feb, 2021
On 01 Feb, 2021
On 01 Feb, 2021
On 01 Feb, 2021
On 21 Jan, 2021
Received 19 Jan, 2021
Received 17 Jan, 2021
On 13 Jan, 2021
On 12 Jan, 2021
Invitations sent on 12 Jan, 2021
On 12 Jan, 2021
On 12 Jan, 2021
On 12 Jan, 2021
On 14 Oct, 2020
Received 07 Oct, 2020
Received 04 Oct, 2020
On 27 Sep, 2020
Invitations sent on 21 Sep, 2020
On 21 Sep, 2020
On 08 Sep, 2020
On 07 Sep, 2020
On 07 Sep, 2020
On 07 Sep, 2020
Posted 11 Feb, 2021
On 10 Feb, 2021
On 01 Feb, 2021
On 01 Feb, 2021
On 01 Feb, 2021
On 21 Jan, 2021
Received 19 Jan, 2021
Received 17 Jan, 2021
On 13 Jan, 2021
On 12 Jan, 2021
Invitations sent on 12 Jan, 2021
On 12 Jan, 2021
On 12 Jan, 2021
On 12 Jan, 2021
On 14 Oct, 2020
Received 07 Oct, 2020
Received 04 Oct, 2020
On 27 Sep, 2020
Invitations sent on 21 Sep, 2020
On 21 Sep, 2020
On 08 Sep, 2020
On 07 Sep, 2020
On 07 Sep, 2020
On 07 Sep, 2020
Background: Apolipoprotein E4 (APOE4) is associated with a greater response to neuroinflammation and the risk of developing late-onset Alzheimer's disease (AD), but the mechanisms for this association are not clear. The activation of calcium-dependent cytosolic phospholipase A2 (cPLA2) is involved in inflammatory signaling and is elevated within the plaques of AD brains. The relation between APOE4 genotype and cPLA2 activity is not known.
Methods: Mouse primary astrocytes, mouse and human brain samples differing by APOE genotypes were collected for measuring cPLA2 expression, phosphorylation, and activity in relation to measures of inflammation and oxidative stress.
Results: Greater cPLA2 phosphorylation, cPLA2 activity and leukotriene B4 (LTB4) levels were identified in ApoE4 compared to ApoE3 in primary astrocytes, brains of ApoE-targeted replacement (ApoE-TR) mice, and in human brain homogenates from the inferior frontal cortex of patients with AD carrying APOE3/4 compared to APOE3/3. Greater cPLA2 phosphorylation was also observed in human postmortem frontal cortical synaptosomes and primary astrocytes after treatment with recombinant ApoE4 ex vivo. In ApoE4 astrocytes, the greater levels of LTB4, reactive oxygen species (ROS), and inducible nitric oxide synthase (iNOS) were reduced after cPLA2 inhibition.
Conclusions: Our findings implicate greater activation of cPLA2 signaling system with APOE4, which could represent a potential drug target for mitigating the increased neuroinflammation with APOE4 and AD.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 9
Figure 10
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