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Research article
Hussein Yassine
University of Southern California
Corresponding Author
ORCiD: https://orcid.org/0000-0002-2483-649X
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Keywords
cPLA2, ApoE4, Alzheimer's disease, p38 MAPK, neuroinflammation, Oxidative stress
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This is a list of supplementary files associated with this preprint. Click to download.
- FigureS12.tif
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 .
- FigureS2.tif
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.
- FigureS3.tif
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.
- FigureS4.tif
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.
- FigureS5.tif
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.
- TableS1.docx
Supplementary Table 1
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On 12 Jan, 2021
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Reviewer #2 agreed
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Reviewers invited
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This preprint is under consideration at Molecular Neurodegeneration. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
Hussein Yassine
University of Southern California
Corresponding Author
ORCiD: https://orcid.org/0000-0002-2483-649X
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 3
Posted 11 Feb, 2021
No community comments so far
Editorial decision: Minor revision
On 10 Feb, 2021
Editor assigned
On 01 Feb, 2021
Submission checks complete
On 01 Feb, 2021
Editor invited
On 01 Feb, 2021
No comments provided
Editorial decision: Minor revision
On 21 Jan, 2021
Review #1 received
Received 19 Jan, 2021
Review #2 received
Received 17 Jan, 2021
Reviewer #2 agreed
On 13 Jan, 2021
Editor assigned
On 12 Jan, 2021
Reviewers invited
Invitations sent on 12 Jan, 2021
Reviewer #1 agreed
On 12 Jan, 2021
Submission checks complete
On 12 Jan, 2021
Editor invited
On 12 Jan, 2021
No comments provided
Editorial decision: Major revision
On 14 Oct, 2020
Review #2 received
Received 07 Oct, 2020
Review #1 received
Received 04 Oct, 2020
Reviewer #2 agreed
On 27 Sep, 2020
Reviewers invited
Invitations sent on 21 Sep, 2020
Reviewer #1 agreed
On 21 Sep, 2020
Editor assigned
On 08 Sep, 2020
First submitted
On 07 Sep, 2020
Submission checks complete
On 07 Sep, 2020
Editor invited
On 07 Sep, 2020
Subject Areas
Neurobiology of Disease
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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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