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LPS or ethanol triggers clathrin‐ and rafts/caveolae‐dependent endocytosis of TLR 4 in cortical astrocytes
Author(s) -
PascualLucas Maya,
FernandezLizarbe Sara,
Montesinos Jorge,
Guerri Consuelo
Publication year - 2014
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/jnc.12639
Subject(s) - endocytic cycle , clathrin , microbiology and biotechnology , endocytosis , caveolae , internalization , lipid raft , dynamin , biology , tlr4 , signal transduction , caveolin , receptor mediated endocytosis , neuroinflammation , chemistry , receptor , biochemistry , immunology , inflammation
Abstract Toll‐like receptor 4 (TLR4) activation and signalling in glial cells play critical roles in neurological disorders and in alcohol‐induced brain damage. TLR4 endocytosis upon lipopolysaccharide (LPS) stimulation regulates which signalling pathway is activated, the MyD88‐dependent or the TIR‐domain‐containing adapter‐inducing interferon‐β ( TRIF )‐dependent pathway. However, it remains elusive whether ethanol‐induced TLR 4 signalling is associated with receptor internalization and trafficking, and which endocytic pathway(s) are used in cortical astrocytes. Using the adenoviral over‐expression of TLR 4 GFP , confocal microscopy and the imagestream technique, we show that upon ethanol or LPS stimulation, TLR4 co‐localizes with markers of the clathrin and caveolin endocytic pathways, and that this endocytosis is dependent on dynamin. Using chlorpromazin and filipin as inhibitors of the clathrin and rafts/caveolae endocytic pathways, respectively, we demostrate that TRIF‐dependent signalling relies on an intact clathrin pathway, whereas disruption of rafts/caveolae inhibits the MyD88‐ and TRIF‐dependent signalling pathways. Immunofluorescence studies also suggest that lipid rafts and clathrin cooperate for appropriate TLR 4 internalization. We also show that ethanol can trigger similar endocytic pathways as LPS does, although ethanol delays clathrin internalization and alters TLR 4 vesicular trafficking. Our results provide new insights into the effects of ethanol or LPS on TLR 4 signalling in cortical astrocytes, events that may underlie neuroinflammation and brain damage.The results demonstrate that ethanol or lipopolysaccharide (LPS) triggers toll‐like receptor 4 (TLR4) endocytosis by caveolae and clathrin‐dependent pathways in astrocytes. We proposed that while clathrin is the protein responsible for TLR4 internalization, caveolin‐1/lipid rafts membrane microdomains are required for TLR4 signaling. The results provide new insights into the effects of ethanol on TLR4 signalling in astrocytes, events that may underlie neuroinflammation.