Abstract
Acetylcholine activation of α7 nicotinic acetylcholine receptors (α7 nAChRs) in microglia attenuates neuroinflammation and regulates TNF‐α release. We used lipopolysaccharide to model inflammation in the microglial cell line EOC20 and examined signaling by the α7 nAChR. Co‐immunoprecipitation experiments confirm that α7 nAChRs bind heterotrimeric G proteins in EOC20 cells. Interaction with Gαi mediates α7 nAChR signaling via enhanced intracellular calcium release and a decrease in cAMP, p38 phosphorylation, and TNF‐α release. These α7 nAChR effects were blocked by the inhibition of Gαi signaling via pertussis toxin, PLC activity with U73122, and α7 nAChR channel activity with the selective antagonist α‐bungarotoxin. Moreover, α7 nAChR signaling in EOC20 cells was significantly diminished by the expression of a dominant‐negative α7 nAChR, α7345‐8A, shown to be impaired in G protein binding. These findings indicate an essential role for G protein coupling in α7 nAChR function in microglia leading to the regulation of inflammation in the nervous system.
Highlights
Acetylcholine activation of a7 nicotinic acetylcholine receptors (a7 α7 Nicotinic Acetylcholine Receptor (nAChR)) in microglia attenuates neuroinflammation and regulates tumor necrosis factor a (TNF-a) release
Studies show that ACh, as well as nicotine, Abbreviations ACh, acetylcholine; BGTX, a-bungarotoxin; cAMP, cyclic adenosine monophosphate; Chol, choline; CNS, central nervous system; Co-IP, co-immunoprecipitation; fluorescent-conjugated BGTX (fBGTX), fluorescent a-bungarotoxin; G Protein, heterotrimeric G protein; G proteinbinding cluster (GPBC), G protein-binding cluster; GPCR, G protein-coupled receptor; Gα, heterotrimeric G protein Subunit α; IP3, inositol triphosphate; IP3R, inositol triphosphate receptor; IP, immunoprecipitation; LPS, lipopolysaccharide; MEC, mecamylamine; NO, nitric oxide; p38, p38 mitogen-activated protein kinase; pheochromocytoma 12 (PC12), pheochromocytoma Cell line 12; phospho-p38, phosphorylated p38 mitogen-activated protein kinase; phosphor-CDC42, Cell Division Control protein 42; PLC, phospholipase C; PTX, pertussis toxin; ROI, region of interest; ROS, reactive oxygen species; TNF-α, tumor necrosis factor α; Xest
A better understanding of how a7 nAChRs operate in immune cells is essential for drug development, and our results indicate that a7
Summary
EOC20 cells (ATCCÒ CRL-2469, Manassas, VA, USA) were grown on plastic petri dishes or glass coverslips (Genesee Scientific, San Diego, CA, USA) coated with a poly-D-lysine (100 lgÁmLÀ1) matrix and maintained in DMEM (Thermo Fisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin (Thermo Fisher). A co-IP of the a7 nAChR protein complex was obtained from 500 lg cell membrane protein fraction using 5 lg of the C-20 antibody (Santa Cruz, Dallas, TX, USA) [19]. Proteins were transferred onto a nitrocellulose membrane (Thermo Fisher) for immunoblot detection using the following antibodies: anti-Gas (Rabbit) (New East Bioscience), anti-Gaq (Rabbit) (New East Bioscience), antiGai (Rabbit) (New East Bioscience), anti-Gb (T-20) (Santa Cruz), anti-phospho-p38MAPK (Thr180/Tyr182), antip38MAPK, anti-phospho-CDC42/Rac (Ser 71), and antiGAPDH (Cell Signaling, Danvers, MA, USA). EOC20 cells were transfected with the calcium sensor protein GCaMP5G [22] 3 days prior to the calcium imaging experiment. Imaging was performed using an inverted Zeiss LSM800 confocal microscope, and fluorescence signal was analyzed using ImageJ. A minimum statistical value P < 0.05 was considered significant
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
