Fig. 7

Inhibition of protein kinase C and IP3 reduced neurotoxicity. a Pre-treatment with the phospholipase C inhibitor U73122 for 30 min significantly reduced neurotoxicity caused by 3 days of exposure to granzyme B (10 nM) and IL-1β (20 ng/ml). All doses of U73122 were effective at reducing toxicity relative to maximum IL-1β augmented granzyme B-mediated toxicity. The 1 and 10 μM doses reduced toxicity to levels identical to that of control media. ***p < 0.001, ****p < 0.0001 relative to control media with 10% ethanol (vehicle), ####p < 0.0001 relative to granzyme B + IL-1β condition, one-way ANOVA with post hoc Tukey’s test, n = 5 per condition. Data are shown as the mean ± standard error of the mean from five separate experiments. b Several doses of the selective IP3 inhibitors 2-APB and xestospongin significantly reduced the cumulative toxicity typically seen after 3 days. *p < 0.05, ****p < 0.0001 relative to control media and ##p < 0.01, ####p < 0.0001 relative to granzyme B + IL-1β, one-way ANOVA with post hoc Tukey’s test, n = 5 per condition. Data are shown as the mean ± standard error of the mean from four separate experiments. c Neuronal viability was preserved in an acute, single day exposure to 10 nM granzyme B following pre-treatment with only 100 μM 2-APB, ****p < 0.0001 relative to control media, ####p < 0.0001 relative to granzyme B + IL-1β, one-way ANOVA with post hoc Tukey’s test, n = 5 per condition. Data are shown as the mean ± standard error of the mean from duplicate experiments. d Proposed mechanism of action for granzyme B-mediated neurotoxicity with IL-1β assistance. IL-1β promotes increased numbers of PAR1 receptors on neuronal membrane surface. Granzyme B cleaves the PAR1 receptors’ extracellular domains. Activated PAR1 directly couples with phospholipase Cβ which in turn yields increased IP3 production and leads to the well-described cellular processes that compromise mitochondrial function and eventually culminate in neuronal demise