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

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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.

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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

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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

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.

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Authors and Affiliations

  1. Department of Integrative Physiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, 812-8582, Japan

    Fatma Ali

  2. Department of Neuroinflammation and Brain Fatigue Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, 812-8582, Japan

    Md. Shamim Hossain

  3. Department of Medicine and Biosystemic Science, Kyushu University Faculty of Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, 812-8582, Japan

    Sanyu Sejimo & Koichi Akashi

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  1. Fatma Ali
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Corresponding author

Correspondence to Md. Shamim Hossain.

Electronic supplementary material

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)

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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Ali, F., Hossain, M.S., Sejimo, S. et al. Plasmalogens Inhibit Endocytosis of Toll-like Receptor 4 to Attenuate the Inflammatory Signal in Microglial Cells. Mol Neurobiol 56, 3404–3419 (2019). https://doi.org/10.1007/s12035-018-1307-2

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