Figure 7

Delayed neurodegeneration induced by prion protein or PrP106-126 in the presence of iNOS expression is microglia-dependent and mediated by peroxynitrite. The addition of prion protein (5 μg/ml) or PrP106-126 (225 μg/ml) to LPS/IFN-γ treated neuronal-glial cultures induced delayed death of co-cultured neurons, over 6 days. Neuronal death, assessed by Hoechst 33342 to visualise chromatin condensation (CC; b) or PI for necrosis (a) was prevented by inhibitors of iNOS (25 μM 1400W) and NADPH oxidase (1 mM apocynin), a peroxynitrite scavenger (100 μM FeTPPS) or a blocker of the NMDA receptor (10 μM MK-801). Neuronal death was accompanied by proliferation of microglia (c). Microglial proliferation was inhibited by LPS/IFN-γ treatment alone but in the presence of prion protein or PrP106-126 it was stimulated and returned to basal levels. This stimulation of proliferation by prion protein or PrP106-126 (in the presence of LPS/IFN-γ) was completely prevented by apocynin. Additionally, nitrite/nitrate (NO_X) levels correlated with the number of microglia present (d). Statistical differences were established using ANOVA at *p < 0.05, **p < 0.01 and ***p < 0.001 are in comparison to untreated cultures (symbol *) or LPS/IFN-γ treatment (symbol ¶) or LPS/IFN-γ plus prion protein or PrP106-126 (symbol #); data expressed is mean ± SEM, n = 3 or more. In c & d, the differences are in comparison to prion protein or PrP106-126 alone (*), LPS/IFN-γ (¶) or LPS/IFN-γ plus prion protein or PrP106-126 (#). Data expressed is mean ± SEM, n = 3 or more.