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Plasmalogens Inhibit Endocytosis of Toll-like Receptor 4 to Attenuate the Inflammatory Signal in Microglial Cells

  • Fatma Ali
  • Md. Shamim HossainEmail author
  • Sanyu Sejimo
  • Koichi Akashi
Article
  • 213 Downloads

Abstract

Microglial activation is a pathological feature of many neurodegenerative diseases and the role of cellular lipids in these diseases is mostly unknown. It was known that the special ether lipid plasmalogens (Pls) were reduced in the brain and blood samples of Alzheimer’s disease (AD) patients. It has recently been reported that the oral ingestion of scallop-derived Pls (sPls) improved cognition among mild AD patients, which led us to investigate the role of sPls in the microglial activation. We used the lipopolysaccharides (LPS)-induced microglial activation model and found that sPls inhibit the LPS-mediated TLR4 endocytosis and the downstream caspases activation. By using the specific inhibitors, we also confirmed that the TLR4 endocytosis and the caspases activation strictly controlled the pro-inflammatory cytokine expression. In addition, the reduction of cellular Pls by sh-RNA-mediated knockdown of GNPAT (glyceronephosphate O-acyltransferase), a Pls synthesizing enzyme, enhanced the endocytosis of TLR4 and activation of caspase-3 which resulted in the enhanced pro-inflammatory cytokine expression. We also report for the first time that the TLR4 endocytosis was significantly higher in the cortex of aged mice and AD model mice brains, proposing a significant link between the age-related reduction of Pls and microglial activation. Interestingly, the sPls drinking in AD model mice significantly reduced the TLR4 endocytosis. Our cumulative data indicates that the cellular Pls attenuate the microglial activation by maintaining the endocytosis of TLR4, suggesting a possible mechanism of the cognition improvement effect of sPls among mild AD patients.

Keywords

Endocytosis Caspase-3 TLR4 Plasmalogens 

Abbreviations

AD

Alzheimer’s disease

ANOVA

one-way analysis of variance

BCA

bicinchoninic acid

BSA

bovine serum albumin

CDE

clathrin-dependent endocytic

CHO

Chinese hamster ovary

CIE

clathrin-independent endocytic

C/LR

caveolin/lipid raft-mediated endocytic

DAPI

4′,6-diamidino-2-phenylindole

DEVD

Z-DEVD-fmk

DMEM

Dulbecco’s modified Eagle medium

FBS

fetal bovine serum

GAPDH

glyceraldehyde-3-phosphae dehydrogenase

GNPAT

glycerone phosphate O-acyltransferase

HEK

human embryonic kidney

IL-1β

interleukin-1β

IL-1R

interleukin-I receptor

IRF

interferon regulatory factor-3

LPS

lipopolysaccharide

MβCD

methyl-β-cyclodextrin

NF-κB

nuclear factor-κB

PARP1

polyadenosine diphosphate ribosepolymerase1

PBS

phosphate-buffered saline

PCR

polymerase chain reaction

Pls

plasmalogens

Pls-Etn

ethanolamine plasmalogens

RCDP

rhizomelic chondrodysplasia punctate

RIPA

radio-immunoprecipitation assay

SDS-PAGE

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

sh-RNA

short hairpin-RNA

TBS

Tris-buffered saline

TDU

transduction unites

TIRAP

Toll/IL-1R domain-containing adaptor protein

TLR4

toll-like receptor 4

TNF-α

tumor necrosis factor-α

TRAM

Toll/interleukin-1 receptor domain-containing adaptor inducing type I interferons-related adaptor molecule

TRIF

Toll/interleukin-1 receptor domain-containing adaptor inducing type I interferons

VAD

Z-VAD-fmk

Notes

Acknowledgements

We appreciate the technical assistance of the Research Support Center, Graduate School of Medical Sciences, Kyushu University. We thank Dr. A. Ibrahim for insightful discussion and suggestions to continue this study. We appreciate the technical assistance from Ayako Tajima to perform experiments.

Funding Information

This work was supported by JSPS KAKENHI grant number 26460320 to Toshihiko Katafuchi, JSPS Wakate B (16K19007) to MSH and Egyptian ministry of higher education scholarship for young scientist fellowship to F. Ali.

Supplementary material

12035_2018_1307_MOESM1_ESM.pptx (154 kb)
Figure S1. Pls pretreatments attenuate the LPS-mediated cleavages of caspase-8 and caspase-3 proteins in microglial cells. Western blotting data showed that LPS (1 μg/ml) treatments for 6 hours induced cleavage of caspase-8 and caspase-3 (as shown in Fig. 1) in BV2 microglial cells, which were attenuated by pretreatments with 5 μg/ml of sPls for 12 hours. Here, we show the representative data of two independent experiments. (PPTX 153 kb)
12035_2018_1307_MOESM2_ESM.pptx (58 kb)
Figure S2. MβCD, an inhibitor of lipid raft function, abolishes the attenuating effect of Pls on LPS-induced IL-1β expression. BV2 cells were pretreated with Pls (5 μg/ml) for 12 hours then MβCD (5 μM) was applied 1 hour before LPS (1 μg/ml) treatment. Real time PCR analysis performed 6 hours after LPS treatment demonstrated that the LPS-induced increase in IL-1β mRNA expression was attenuated by the pretreatment with Pls. However, the attenuation was completely blocked by application of MβCD. MβCD treatment itself did not affect the LPS-induced IL-1β expression (**P<0.01 vs. control group, ##P<0.01 vs. LPS group, n=5) (PPTX 57 kb)

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Authors and Affiliations

  1. 1.Department of Integrative Physiology, Graduate School of Medical SciencesKyushu UniversityFukuokaJapan
  2. 2.Department of Neuroinflammation and Brain Fatigue Science, Graduate School of Medical SciencesKyushu UniversityFukuokaJapan
  3. 3.Department of Medicine and Biosystemic Science, Kyushu University Faculty of Medicine, Graduate School of Medical SciencesKyushu UniversityFukuokaJapan

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