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

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.

Artemisinin inhibits TLR4 signaling by targeting co‐receptor MD2 in microglial BV‐2 cells and prevents lipopolysaccharide‐induced blood–brain barrier leakage in mice

“…After overnight incubation, cells were treated with LPS (200 ng/ml), and/or 100 μM artemisinin that was added 10 min earlier before LPS. The LPS treating time (1 hr) was based on the on literatures (Ali et al., 2019; Zusso et al., 2019) and the prelimary time course studies of LPS‐induced TLR4 dimerization and endocytosis (Figure S1). After 60 min of incubation with LPS, cells were washed twice with cold PBS and subsequently stained with PE‐Cy7‐IgG2a κ isotype antibody, APC‐IgG2a κ isotype antibody or TLR4 antibodies that assess dimerization (TLR4‐PE‐Cy7, clone MTS510) or endocytosis (TLR4‐APC, Clone SA15‐21) for 30 min on ice.…”

TLR4 and CD14 trafficking and its influence on LPS-induced pro-inflammatory signaling

“…Accordingly, in such conditions, the TRIF-dependent signaling was inhibited [40,74,95,155,[159][160][161]. In addition, dynasore attenuated the LPS-induced cleavage of caspase-3 in murine microglia and almost completely abolished expression of IL-1β in these cells and in rat astrocytes overexpressing TLR4 [156,159]. In HEK293 cells expressing TLR4/CD14 and a dominant negative dynamin II mutant (Dyn K44A), the NF-κB activation was upregulated in comparison to cells overexpressing wild-type dynamin, suggesting an upregulation of the MyD88-dependent signaling [41].…”

“…2, I) [180]. In murine microglia (BV2 cells), the TLR4 translocation to the Rab5-positive vesicles seems to be slower than in murine macrophages since a colocalization of TLR4 and Rab5 was visible only after 2 h of stimulation with the same LPS concentration [156], suggesting that the dynamics of TLR4 endocytosis is cell type specific. In the Rab5-positive endosomes TLR4 interacts with TRAM and activates the TRIF-dependent signaling cascade leading to the production of type I IFN and expression of IFN-induced genes.…”

“…The LPS-induced internalization of TLR4 occurs via clathrin-independent and/or clathrin-dependent pathways, while E. coli bacteria undergo phagocytosis in which TLR4 is involved [3,33,41,155,156]. The TLR4 endocytosis depends on dynamin, a GTPase responsible for pinching off of clathrin-coated buds, some macro-and micropinosomes, and phagosomes [157,158] inhibition of dynamin with dynasore prevented LPS-induced internalization of TLR4 in several cell types, including macrophages [40,141,155,159].…”

Transcriptome analysis of the hippocampus in environmental noise-exposed SAMP8 mice reveals regulatory pathways associated with Alzheimer’s disease neuropathology

“…TLRs, a family of receptor proteins, play a wide role in innate and adaptive immune responses upon the stimulations by exogenous and endogenous TLR ligands. There is an increased endocytosis of TLR4 in the AD model mice brain, which might be a key event for the neurodegeneration signaling in the brain [24]. TNF-α has been assessed in the pathophysiology of AD both in human [25] and animal studies [26], and in our previous study, chronic noise exposure increased levels of TNF-α in the rat hippocampus [20].…”