α-Synuclein potentiates interleukin-1β-induced CXCL10 expression in human A172 astrocytoma cells

Abstract

Neuroinflammation and neuronal degeneration observed in Parkinson's disease (PD) has been attributed in part to glial-mediated events. Increased expression of proinflammatory cytokines and abnormal accumulation of the neuronal protein, α-synuclein in the brain are also characteristic of PD. While increasing evidence suggests that astrocytes contribute to neuroinflammation and dopaminergic neuronal degeneration associated with PD, there remains much to learn about these astroglial-mediated events. Therefore, we investigated the in vitro effects of interleukin-1β (IL-1β) and α-synuclein on astroglial expression of interferon-γ inducible protein-10 (CXCL10), a proinflammatory and neurotoxic chemokine. IL-1β-induced CXCL10 protein expression was potentiated by co-exposure to α-synuclein. α-Synuclein did not significantly affect IL-1β-induced CXCL10 mRNA expression, but did mediate increased CXCL10 mRNA stability, which may explain, in part, the increased levels of secreted CXCL10 protein. Future investigations are warranted to more fully define the mechanism by which α-synuclein enhances IL-1β-induced astroglial CXCL10 expression. These findings highlight the importance of α-synuclein in modulating inflammatory events in astroglia. These events may be particularly relevant to the pathology of CNS disorders involving α-synuclein accumulation, including PD and HIV-1 associated dementia.

Figure 1. α-Synuclein potentiated IL-1β-induced CXCL10 protein expression in human A172 astroglial cells

Cells were either unstimulated or exposed to IL-1β (250 pg/ml) in the presence or absence of α-synuclein (480 pg/ml) in serum-free medium for 24 h. CXCL10 protein in the media was quantitated by ELISA. Data represent the mean ± S.E.M. (n = 3–8). Significant differences were determined by one-way ANOVA with SNK pair-wise comparisons. ***p < 0.001 vs IL-1β, **p < 0.01 vs IL-1β.

Figure 2. α-Synuclein did not alter IL-1β-induced NF-κB activation in human A172 astroglial cells

Cells were exposed to IL-1β (250 pg/ml) in the presence or absence of α-synuclein (480 pg/ml) in serum-free medium for 2–120 minutes. The levels of active (A) NF-κB p65 and (B) p50 in the nucleus were determined using the Transcription Factor kits (Thermo Scientific, Rockford, IL). Luminescence of the labeled NF-κB-DNA product was then measured. Data represent the mean ± S.E.M. (n = 2–4). Two-way ANOVA (treatment × time) with SNK pair-wise comparisons indicated a significant effect of time (p65: **p < 0.009 vs. 2 min, #p < 0.002 vs. 60 min; p50: **p < 0.002 vs. 2 min, *p < 0.05 vs. 2 min, #p < 0.03 vs. 60 min), whereas, α-synuclein did not significantly (p > 0.89) affect IL-1β-induced increases in nuclear levels of NF-κB.

Figure 3. α-Synuclein did not affect IL-1β-stimulated CXCL10 mRNA expression in human A172 astroglial cells

Cells were exposed to IL-1β (250 pg/ml) in the presence or absence of α-synuclein (480 pg/ml) in serum-free medium for 2–48 h. CXCL10 mRNA levels were assessed using real time RT-PCR. Data were normalized to GAPDH mRNA within each independent experiment and expressed as fold change (compared to IL-1β-stimulated group at 2 h). Data represent the mean ± S.E.M. (n = 7–15). Two-way ANOVA (treatment × time) with SNK pair-wise comparisons indicated a significant effect of time (*p < 0.04, vs. 2 min), whereas, α-synuclein did not significantly (p = 0.72) affect IL-1β-induced increases in CXCL10 mRNA expression.

Figure 4. Co-exposure to α-synuclein results in enhanced CXCL10 mRNA stability in IL-1β-stimulated human A172 astroglial cells

Cells were exposed to IL-1β (250 pg/ml) in the presence or absence of α-synuclein (480 pg/ml) in serum-free medium for 2.5 h. Cultures were replenished with media alone or media containing 10 µg/ml actinomycin D (ActD). The time-course (3–270 min) of CXCL10 and GAPDH mRNA expression was assessed using quantitative real time RT-PCR. Data were normalized to GAPDH within each independent experiment and expressed as fold change (relative to IL-1β treated cells at time 0). Data represent the mean ± S.E.M. (n = 2–6). Two-way ANOVA (treatment × time) indicated that treatment, time and the interaction were each highly significant (p < 0.001). **p < 0.01 vs. IL-1β + α-Syn + ActD as determined by SNK pair-wise comparisons.