Pneumolysin and H₂O₂ are each sufficient to induce apoptosis in microglia and neurons. (a) Microglia cells were incubated for 8 hours with purified pneumolysin (0.1 μg/ml) or H₂O₂ (0.2 mM) and stained with AO and EB. Either treatment resulted in shrinkage and condensation typical of apoptosis induced by wild-type pneumococci. The apoptosis-inducing activity of pneumolysin correlates with its cytolytic (Lys) but not its complement-activating (Compl) function. Microglial cells were incubated with wild-type or mutant pneumolysin (0.1 μg/ml) for 12 hours and stained with AO and EB. A point mutant in the domain required for cytolytic activity of pneumolysin (W433F) failed to induce apoptosis, whereas a point mutant in the domain required for complement activation (D385N) retained full apoptosis-inducing capacity. Bars = 10 μm. (b) Quantification of the effects of wild-type or mutant pneumolysin on primary rat hippocampal neurons. Results shown (means + SD) are representative for three independent experiments performed in triplicate. (c) Neurotoxicity (primary rat hippocampal neurons) of various concentrations of H₂O₂.