Fig. 6
LPS-activated microglia increase neuronal α-SNC secretion and JNK phosphorylation. a PC12 cells expressing α-SNC were incubated in monoculture (neurons) or together with primary microglia isolated from neonatal rats (neurons + microglia) with or without 100 ng/ml LPS overnight. Conditioned medium was then analyzed for secreted α-SNC. The blot shown is representative of four independent experiments. b Quantification of a secreted α-SNC mean fold increase + SEM relative to control (**p < 0.01 compared to control, N = 4). c PC12 cells expressing α-SNCA30P or α-SNCA30P/p25α were cultured together with Ra2 microglia or Ra2 overexpressing gp91phox (Ra2-gp91phox) for increased oxidant production. Conditioned media was analyzed for secreted α-SNC. Images are representative of two independent experiments. d Media collected from Ra2 or Ra2-gp91phox microglia with or without overnight exposure to 100 ng/ml LPS were transferred to differentiated PC12 cells expressing α-SNCA30P in monoculture. After overnight incubation, the conditioned medium was collected and analyzed by western blotting. The blot shown is representative of two independent experiments. e Ra2 microglia were exposed to 500 ng/ml LPS in HBSS (Ra2 + LPS) with or without NGF for 6 h before transferring Ra2-conditioned HBSS to differentiated PC12 cells expressing α-SNC in monoculture. After 6 h of incubation, whole cell lysates and conditioned media were collected for WB analysis of p-JNK and secreted α-SNC, respectively. The blot shown is representative of three independent experiments. f Quantification of p-JNK (lysate) and α-SNC (TCA) in e. Mean fold increase + SEM relative to LPS alone (*p < 0.05 compared to “Ra2”; # p < 0.05 compared to “NGF Ra2”). g PC12 cells expressing α-SNCwt in monoculture (PC12) or co-culture with Ra2 microglia (PC12 + Ra2) were incubated with 100 ng/ml LPS for 6 h prior to fixation and analysis for p-JNK and neurofilament (NF) by immunofluorescence. Slides were examined in a LSM510 confocal laser microscope. Representative images from two to three independent experiments are shown. Bars, 5 μm