N protein synthesis and folding capacity in the ER (ER stress), these sensors recognize the

May 28, 2018

N protein synthesis and folding capacity in the ER (ER stress), these sensors recognize the misfolded proteins in the lumen of the ER and trigger the unfolded protein response (UPR). Recent studies have revealed that ER stress is involved in the induction of inflammation by triggering signaling pathways to elicit an inflammatory response [12?4]. Furthermore, TLR signaling pathways have been shown to cross-talk with ER-stress [15?7]. Microglial cells are the main immune effector cells residing in the CNS and keep the brain environment under constant surveillance. Microglial activation is one of the hallmark features of HAND. In addition, the number of activated microglia is significantly increased among cocaine users [18]. This suggests that microglial activation could be a critical player in cocaine-induced neuroinflammation leading to CNS pathology. The underlying mechanism(s) of microglial activation in the presence of cocaine, however, remain unclear. The present study was aimed to elucidate the molecular mechanism(s) involved in cocaine-mediated microglial activation with a focus on the role of TLR2 and ER stress mediators. This study not only provides a novel mechanism of cocaine-induced microglial activation, but also sheds light on the implications for development of potential therapeutic targets aimed at mitigating neuroinflammation in cocaine abusers.eukaryotic initiation factor 2 (eIF2) (Cat# 5324S), peIF2 (Ser51) (Cat# 3398S), and histone H3 (Cat# 9715S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies including ATF4 (Cat# ab23760) and NF-B p65 (Cat# ab16502) were purchased from Abcam (Cambridge, MA, USA). Short interfering RNA (siRNA) of TLR2, ATF4, and myeloid differentiation protein 88 (MyD88) were purchased from Thermo Scientific (Hudson, NH, USA).AnimalsC57BL/6N male mice were purchased from Charles River Laboratories (Wilmington, MA, USA). All of animals were housed under conditions of constant temperature and humidity on a 12-h light, 12-h dark cycle, with lights on at 7:00 am. Food and water were available ad libitum. All animal procedures were performed according to the protocols approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center and the National Institute of Health. Animals were divided into two groups (n = 6): (1) saline and (2) cocaine. Cocaine was PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/28859980 injected at a dose of 20 mg/kg intraperitoneally for 7 days. On the seventh day, 1 h after the last cocaine injection, the mice were sacrificed, brains removed, and striatal homogenates assessed for levels of TLR2. Mice injected similarly with saline served as controls.Primary mouse microglial cell isolationMethodsReagentsCocaine hydrochloride (Cat# C5776), apocynin (Cat# A10809), and phenyl-N-t-butyl nitrone (PBN) (Cat# B7263) were purchased from Sigma-Aldrich (St Louis, MO, USA). Antibodies purchased from Santa Cruz Biotechnology (Dallas, TX, USA) include TLR2 (Cat# sc-10739), pPERK (Thr 981) (Cat# sc-32577), PERK (Cat# CEP-37440 web sc-13073), goat anti-rabbit (Cat# sc-2004), and goat anti-mouse (Cat# sc-2005). Antibodies includingPrimary mouse microglial cell isolation was performed as described previously [19, 20]. Primary microglia cells were obtained from 1- to 3-day-old C57BL/6 newborn pups. After digestion and dissociation of the dissected brain cortices in Hanks buffered salt solution (HBSS, Invitrogen, 14025076) supplemented with 0.25 trypsin (Invitrogen, 25300-054), mixed glial cultures we.