In LPS treated BV cells we found that the upregulated
In LPS-treated BV-2 cells, we found that the upregulated CysLT2R was translocated into the nucleus. The nuclear localization of CysLT2Rs in microglia has not been reported previously. It is known that CysLT1Rs in endothelial DOI hydrochloride translocated to the nucleus in a ligand-independent manner after ischemic insult in vitro, which mediated the ischemic injury (Fang et al., 2012). Recently, we reported that the PD neurotoxin rotenone induced CysLT1R expression and nuclear translocation which is relevant to microglial activation (Zhang et al., 2014a). In intestinal epithelial cells, the nuclear CysLT1R has been linked to inflammation-related carcinogenesis, and increased nuclear localization of CysLT1R modulated leukotriene D4-induced ERK1/2 proliferative signaling and cell proliferation (Nielsen et al., 2005, Parhamifar et al., 2010). However, the functional role of nuclear CysLT2R in microglial cells needs further investigation. Furthermore, we demonstrated the CysLT2R mediated neuronal toxicity of LPS-activated microglia for the first time. CysLT2R antagonist HAMI 3379 and RNA interference of CysLT2R protected PC12 cells from neurotoxicity via reducing cytokine release from LPS-stimulated BV-2 cells. Overproduction of the proinflammatory cytokines TNF-α, IL-1β and IL-6 by activated microglia has been implicated as key mechanisms of neuronal death in experimental neurodegenerative disorders (Glass et al., 2010, Kwon et al., 2008, More et al., 2013, Nolan et al., 2013, Ramsey and Tansey, 2014, Tansey and Goldberg, 2010). It has been reported that LPS increased the production of IL-1β and TNF-α in BV-2 cells, which caused neurotoxicity in the murine mesencephalic cell line MN9D cells. The synthetic triterpenoid CDDO-methyl ester reduced levels of specific subsets of cytokines induced by LPS and thereby attenuated death of MN9D cells induced by CM from LPS-stimulated BV-2 cells (Tran et al., 2008). CM derived from amyloid β-treated BV-2 cells increased the production of the inflammatory molecules nitric oxide, TNF-α and IL-6, and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive primary hippocampal cells; and sinomenine protected against the indirect neurotoxicity of hippocampal neurons by reducing generation of these active substances in BV-2 cells (Shukla and Sharma, 2011). We recently reported that the proinflammatory cytokines were required for microglia-mediated neurotoxicity in an in vitro rotenone- or OGD-induced model (Zhang et al., 2013, Zhang et al., 2014a, Zhang et al., 2014b). It is noteworthy that in this study LPS enhanced release of TNF-α, IL-6 and IL-1β from BV-2 cells, and these neurotoxic cytokines in LPS-treated BV-2 CM might be responsible for the consequent PC12 cell death. Pretreatment of BV-2 cells with HAMI 3379 and CysLT2R shRNA decreased production of microglial cytokines induced by LPS and indirectly attenuated PC12 cell death. Our results indicate that microglia, not neurons, may serve as the target of HAMI 3379 effects on LPS-induced neurotoxicity. The CysLT signaling molecules including 5-lipoxygenase and CysLT1Rs has been implicated in ischemic neuronal injury and related inflammatory responses (Härtig et al., 2013, Huang et al., 2012, Jatana et al., 2006, Schaal et al., 2012, Tintinger et al., 2010, Yu et al., 2005, Yu et al., 2014, Zhang et al., 2014a, Zhao et al., 2011b). However, it is well known that 5-lipoxygenase inhibitors possess a wide range of effects. Similarly, the CysLT1R antagonist montelukast was also reported to have the broad pharmacological activities in addition to its CysLT1R antagonism action. For example, montelukast protected against ischemic brain damage by inhibiting cyclic nucleotide phosphodiesterases (Schaal et al., 2012, Tintinger et al., 2010) and P2Y-like receptor G protein-coupled receptor 17 (Zhao et al., 2012). We found that montelukast attenuated OGD-induced ischemic neuronal injury through CysLT1R-independent and -dependent mechanisms (Zhang et al., 2013). Moreover, montelukast was reported to exhibit its antioxidative effects in rat primary cultured neurons (Zhang et al., 2013) and in peripheral tissues as well (Mohamadin et al., 2011). Therefore, it is necessary to elucidate the roles of the CysLT2R and its selective antagonist(s) in microglial inflammation and microglia-dependent neuronal death in the present study.