The Role of Autophagy in Pyocyanin-Induced Neurotoxicity

Andrew K Davey1,2, Amelia J McFarland1,2, Gary D Grant1,2, Anthony V Perkins1,3, Cameron Flegg1,3, Katie L Powell1,3, Catherine M McDermott4, Tristan J Allsopp5, Gillian Renshaw6, Justin Kavanagh6, Shailendra Anoopkumar-Dukie1,2. 1Griffith Health Institute, Griffith University, Gold Coast, Queensland, Australia, 2School of Pharmacy, Griffith University, Gold Coast, Queensland, Australia, 3School of Medical Science, Griffith University, Gold Coast, Queensland, Australia, 4Faculty of Health Sciences and Medicine, Bond University, Gold Coast, Queensland, Australia, 5School of Medicine, University of Queensland, Brisbane, Queensland, Australia, 6School of Physiotherapy and Exercise Science, Griffith University, Gold Coast, Queensland, Australia

The role of autophagy in pyocyanin-induced toxicity in the CNS remains unclear with only evidence from our group identifying it as a mechanism underlying toxicity in 1321N1 astrocytoma cells.1 Therefore the aim of this study was to further examine the role of autophagy in PCN-induced toxicity in the CNS. To achieve this we exposed 1321N1 astrocytoma and SH-SY5Y neuroblastoma cells to PCN (0-100 μM) and tested the contribution of autophagy by measuring the impact of the autophagy inhibitor 3-methyladenine (3-MA) using a series of biochemical and molecular markers. Pre-treatment with 3-MA (5mM) in 1321N1 astrocytoma cells decreased the PCN-induced acidic vesicular organelle and autophagosome formation as measured using acridine orange and GFP-LC3 fluorescence respectively.2 Furthermore, 3-MA (5mM) significantly protected 1321N1 astrocytoma cells against PCN-induced (0.1-100µM) toxicity (100-104% survival versus 60-90% survival, respectively) measured using resazurin reduction to resorufin as index of cell viability (p<0.05; Tukey-Kramer multiple comparisons test). In contrast, pre-treatment with 3-MA (5mM) significantly increased PCN-induced toxicity (32-87% survival versus 43-101% survival, respectively) in SH-SY5Y neuroblastoma cells (p<0.05; Tukey-Kramer multiple comparisons test). Given the influence of autophagy in inflammatory responses we investigated if the observed effects in this study involved inflammatory mediators using ELISA.3 PCN (100 μM) significantly increased the production of IL-8 (40-160% increase) PGE2 (68-172% increase) and LTB4 (96-201% increase) in both cell lines (p<0.05; Tukey-Kramer multiple comparisons test). Consistent with its paradoxical role in modulating PCN-induced toxicity, 3-MA (5mM) significantly reduced the PCN-induced production of IL-8, PGE2 and LTB4 in 1321N1 astrocytoma cells back to baseline levels but augmented their production (230%, 211% and 247% respectively) in SH-SY5Y neuroblastoma cells (p<0.05; Tukey-Kramer multiple comparisons test ). In conclusion, we show here for the first time the paradoxical role of autophagy in mediating PCN-induced toxicity in 1321N1 astrocytoma and SH-SY5Y neuroblastoma cells and provide novel evidence that these actions may be mediated by effects on IL-8, PGE2 and LTB4 production.

1. McFarland AJ, Anoopkumar-Dukie S, Perkins AV, Davey AK, Grant GD. Inhibition of autophagy by 3-methyladenine protects 1321N1 astrocytoma cells against pyocyanin- and 1-hydroxyphenazine-induced toxicity. Arch Toxicol 2012;86:275-84.

2. Klionsky DJ, Abeliovich H, Agostinis P, et al. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 2008;4:151-75.

3. Plantinga TS, Joosten LA, van der Meer JW, Netea MG. Modulation of inflammation by autophagy: consequences for Crohn\\\'s disease. Curr Opin Pharmacol 2012;12:497-502.