Activation of the α2A adrenoceptor in microglia promotes LPS-induced TNF-α production and cognitive impairment in mice

Abstract Background: a 2A adrenoceptor receptor (a 2A -AR) plays an important role in inflammatory response in Kupffer cells in sepsis. Blockage of a 2A -AR inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor a (TNF-a) production and protects the target organ functions in sepsis animal models; however, its expression and function in microglia have remained obscure. This study aimed to determine whether a 2A -AR was expressed in microglia and whether its activation would exacerbate microglial inflammation and sepsis-related neurological dysfunction. Methods: Western blotting and immunofluorescence were used to detect a 2A -AR expression in BV-2 microglia. Enzyme-linked immunosorbent assay (ELISA) was used to assess the TNF-a production in the supernatant after LPS induced BV-2 cells were pretreated with a 2A -AR agonist BHT933, and/or a 2A -AR antagonist BRL44408, and also in the supernatants derived from BV-2 cells treated with BHT933 and/or PKC inhibitor. Signaling pathways including JNK，P38，ERK，IκBa, CREB and PCK were detected by western blotting. a 2A -AR gene knock-out (KO) and wild type (WT) mice were prepared by intraperitoneal injection of LPS. Lectin /TNF-a labeled microglia and synaptophysin/NeuN expression in the hippocampus were localized by immunofluorescence. Morris water maze test, Rotating-stick test, Elevated plus maze test and Open-field test were conducted over 4 weeks. Results: a 2A -AR was constitutively expressed in BV-2 microglia, which was enhanced by LPS. Pretreatment with BHT933 promoted LPS-induced IκB and JNK phosphorylation, and TNF-a secretion in BV-2 microglia which were abrogated by BRL44408. Activation of a 2A -AR by BHT933 also increased PKC phosphorylation in LPS-treated BV-2 microglia. PKC inhibitor significantly reversed the promoting effects of BHT933 on IκB and JNK phosphorylation as well as TNF-a secretion in LPS-treated BV-2 microglia. Furthermore, LPS treatment significantly increased hippocampal microglia activation and TNF-a expression, decreased hippocampal synaptophysin expression, and impaired cognitive and motor functions in WT mice, all of which were markedly reversed by a 2A -AR gene knockout. Conclusion: a 2A -AR activation promotes LPS-induced IκB and JNK phosphorylation as well as TNF-a production in microglia through the PKC signaling pathway. Knockout of a 2A -AR gene significantly improves LPS-induced cognitive and motor impairments in mice, indicating that a 2A -AR is a potential therapeutic target for sepsis-associated encephalopathy.