Wnt-3a exacerbates production of TNF-α in LPS stimulated microglia independent of the β-catenin canonical pathway

Abstract Background Neuroinflammatory pathways are emerging therapeutic targets for neurological conditions such as Parkinson’s disease (PD). Studies have indicated Wnt-3a, a member of the wingless type MMTV integration (Wnt) signalling cascade, may exert anti-inflammatory effects via canonical pathway activation and β-catenin stabilisation. Furthermore, dysregulation of the Wnt/β-catenin pathway has been implicated in the degeneration of dopamine neurons in PD, however, stimulation of the canonical pathway via application of Wnt-3a to protect against inflammation and dopaminergic degeneration has not been explored. Methods Primary microglial cultures were stimulated with lipopolysaccharide (LPS) for 24 hours with or without Wnt-3a. TNF-α levels were measured via ELISA while changes in NFkB inflammatory pathway proteins and phosphorylated and non-phosphorylated β-catenin were analysed via capillary western blot. To assess Wnt pathway involvement, cultures were treated with DKK1 (β-catenin canonical pathway inhibitor), SP600125 (Wnt/Pcp pathway inhibitor) or U73122 (Wnt/Ca 2+ pathway inhibitor). Finally, C57BL/6 mice received continuous intracerebroventricular infusion of Wnt-3a via osmotic pumps to investigate the effects of Wnt-3a on dopaminergic neuron survival and on microglial numbers in the MPTP model of PD. Results Wnt-3a alone had no effect on TNF-α release from microglia. However, when co-administered with LPS, there was a significant increase in cytokine release beyond that seen with LPS alone. Protein analysis revealed that this exacerbation in TNF-α levels was not due to alterations in the NFkB pathway or differences in activation of β-catenin. Furthermore, DKK1 treated cells showed no changes in TNF-α levels, however both SP600125 and U723122 were able to block Wnt-3a + LPS induced TNF-α release, implicating the non-canonical pathways. Meanwhile Wnt-3a in vivo did not alter dopaminergic or microglial populations in the substantia nigra in MPTP lesioned animals. Conclusion Together, these results suggest a pro-inflammatory response to Wnt-3a in an inflammatory context with little or no effect on resting microglia. Importantly, this outcome was independent of the β-catenin canonical pathway, revealing that Wnt-3a can increase pro-inflammatory TNF-α release via non-canonical signaling in an inflammatory environment. This demonstrates the importance of cellular context when identifying potential therapies for neurodegenerative diseases where neuroinflammation is a critical mediator of pathology.