Interferon-γ Potentiates α-Synuclein-induced Neurotoxicity Linked to Toll-like Receptors 2 and 3 and Tumor Necrosis Factor-α in Murine Astrocytes

alpha-Synuclein (alpha-syn), a metabolite of neurons, induces glial activation and neuroinflammation and participates in pathogenesis of neurodegenerative diseases. This inflammatory response involves activation of toll-like receptors (TLRs) and its neurotoxic outcomes such as cytokine expression and release. However, regulatory role of cytokines on alpha-syn-induced neurotoxicity is still unclear. In this study, we used interferon (IFN)-gamma to costimulate primary astrocytes with wild-type or A53T mutant alpha-syn, and evaluated inflammatory pathway activation. Four alpha-syn concentrations (0.5, 2, 8 and 20 mu g/mL, 24 h) and four alpha-syn time-points (3, 12, 24 and 48 h, 2 mu g/mL) were chosen to coincubate with one IFN-gamma concentration (2 ng/mL). IFN-gamma alone upregulated expressions of TLR3 and tumor necrosis factor (TNF)-alpha (mRNA level), and A53T mutant or wild-type alpha-syn alone activated the pathway components including TLR2, TLR3, nuclear factor-kappa B, TNF-alpha and interleukin (IL)-1 beta. Additive application of IFN-gamma amplified this activation effect except for IL-1 beta at mRNA and protein levels or TNF-alpha release, displaying a synergistic effect of alpha-syn and IFN-gamma. Blocking TLR2 other than TLR4 suppressed TLR3, TLR2 and TNF-alpha expressions induced by alpha-syn or plus IFN-gamma, reflecting an interaction of TLR2 and TLR3 in TNF-alpha expression. These data collectively showed that IFN-gamma potentiated alpha-syn stimulation and inflammatory outcomes via TLR2, TLR3 and TNF-alpha other than IL-1 beta in astrocytes, suggesting that involvement of IFN-gamma in alpha-syn-induced innate immunity may be required for initiation and maintenance of glial activation, a novel neurotoxic mechanism underlying pathogenesis of neurodegenerative diseases. Graphical IFN-gamma potentiates alpha-synuclein (A53T or wild-type)-induced innate immunity, involving expressions of TLR2, TLR3, NF-kappa B, and TNF-alpha, other than IL-1 beta. This effect is suppressed by blockage of TLR2 other than TLR4, reflecting an interaction of TLR2 and TLR3 in TNF-alpha expression. Thus, involvement of IFN-gamma in alpha-syn-induced neurotoxicity may be required for initiation and maintenance of glial activation, a novel neurotoxic mechanism underlying pathogenesis of neurodegenerative diseases.