Fig. 4.

Activation of neuronal toll-like receptor 2 induces an inflammatory response. a Retinoic acid-differentiated SHSY5Y cells were treated with agonists of MYD88-depedent TLR signaling for 6 h before RNA was extracted for measurement of TNFα mRNA expression by qRT-PCR (TLR2 agonist = 1 μg/ml PAM3CSK4, TLR4 agonist = 1 μg/ml lipopolysaccharide, TLR7 agonist = 1 μg/ml CLO97 and TLR9 agonist = 2 μM ODN 2336). TNFα mRNA was not detected in untreated samples, or samples treated with agonists other than TLR2. TNFα crossed the threshold at ~cycle 30, with GAPDH at ~cycle 20. b TNFα mRNA expression was measured by qRT-PCR following treatment of differentiated SHSY5Y cells with a panel of TLR2 agonists for 6 h. PAM2CSK4 and PAM3CSK4 are di- and tri-acylated bacterial lipopeptides, respectively, FSL1 is a synthetic lipoprotein derived from Mycoplasma salivarium, HKLM is heat killed Listeria monocytogenes, LPS-PG is lipopolysaccharide from Porphyomonas gingivalis, LTA is lipoteichoic acid, PGN is peptidoglycan and Zymosan is a yeast cell wall component. c ELISA assay of TNFα protein secreted into tissue culture by differentiated SHSY5Y cells treated with 1 μg/ml PAM3CSK4 over a 24 h time course. d PAM3CSK4-induced expression of TNFα mRNA at 6 h is blunted by co-treatment with a TLR2 neutralizing antibody. e Differentiated SHSY5Y cells were treated with PAM3CSK4 over a 2 h time course and lysates generated for immunoblot of phosphorylated NFκB P105 subunit and phosphorylated P38 MAPK. f Reactive oxygen species measured by DCFDA assay following 24 h treatment of differentiated SHSY5Y cells with 1 μg/ml PAM3CSK4. g Flow cytometry scatter plots of annexin V and propidium iodide median fluorescence intensity in SHSY5Y cells treated with or without 1 μg/ml PAM3CSK4 or 50 μM tert-butyl H₂O₂ (TBHP) for 24 h. h mRNA expression of TLR2 in differentiated SHSY5Y cells treated over 24 h with 1 μg/ml PAM3CSK4. i Increased expression of TLR2 protein following 12 h of 1 μg/ml PAM3CSK4 is blocked by 10 μg/ml of cyclohexamide. j mRNA expression of SNCA in differentiated SHSY5Y cells treated over 24 h with 1 μg/ml PAM3CSK4. For all graphs, data are presented as mean ± SEM with n = at least 6. All figures are representative of at least three independent experiments. *P < 0.05