Hirano Bodies Protect against Cell Death Figure 4. AICDc58 potentiates 352PHPtau, but not 352WTtau- induced cell death. Cells were transfected with AICDc58/352WTtau or AICDc58/352PHPtau in the presence (CT-GFP, check bars) or absence (GFP, black bars) of model Hirano bodies. Co-transfection of H4 cells with GFP and AICDc58/352WTtau seems to result in cell death that approximates GFP/352WTtau plus GFP/AICDc58 cells. Co-transfection of GFP and AICDc58/352PHPtau significantly enhances cell death com- pared to GFP/AICDc58 only (***p,0.005) or GFP/AICDc58/352WTtau (**p,0.01). The presence of model Hirano bodies significantly protected against AICDc58-, AICDc58/352WTtau-, and AICDc58/ 352PHPtau-induced cell death (++p,0.01 compared to cells without model Hirano bodies). Error bars represent the standard deviation. doi:10.1371/journal.pone.0044996.g004 of apoptotic genes [58,59,60]. We tested the role of p53 in APP/ c31-induced cell death in the presence or absence of 352PHPtau using a cell permeable inhibitor of p53, a-pifithrin [61]. Cell death in H4 cells was induced by exogenous expression of APP/ c31 or APP/c31/352PHPtau. Cell viability was measured in the presence or absence of a-pifithrin as shown in Figure 8. a- pifithrin significantly reduced both tau-independent (APP/c31) (***p,0.005) and tau-dependent (APP/c31/352PHPtau) cell death (**p,0.01). APP and c31-induced cell death in N2A cells and AICD- induced cell death in H4 cells have been shown to involve caspases [48,53]. Caspase inhibitors were utilized to investigate whether caspases are involved in APP/c31 and APP/c31/352PHPtau death pathways. Broad-spectrum caspase inhibitor (Ac-VAD- CHO) as well as inhibitors of caspase 8 (Z-IGTD-FMK) and caspase 3 (Z-DEVD-FMK) was utilized in cell death assays in H4 cells as shown in Figure 9. Both the broad and the caspase 8 inhibitors, but not caspase 3 inhibitor, significantly reduced both tau-independent (APP/c31) (**p,0.01) and tau-dependent (APP/ c31/352PHPtau) cell death (*p,0.05). Since both p53 and caspases were involved, we hypothesized that apoptosis is likely the mechanism of cell death. To verify that p53 and caspase activation is leading to apoptosis, H4 cells undergoing both tau- independent (APP/c31) and tau-dependent (APP/c31/ 352PHPtau) cell death were stained with Annexin V. A large population of H4 cells undergoing both tau-independent and tau- dependent cell death were stained by Annexin V, indicating apoptosis is initiated in cell death as shown in Figure 10. H4 cells with model Hirano bodies showed a significant reduction in the amount of Annexin V positive cells compared to H4 cells without model Hirano bodies (*p,0.05) as shown in Figure 10. This Figure 5. Model Hirano bodies protect against tau-dependent cell death. H4 cells were transfected with APP/c31, and or 353PHPtau in the presence (CT-GFP, check bars) or absence (GFP, black bars) of model Hirano bodies. Transfection of 352PHP tau decreases the amount of exogenous APP (1.5 mg versus 3.0 mg of APP in Figure 2) required to achieve significant enhancement of cell death. Cell death induced by APP/c31/352PHPtau expression is significantly higher than APP/c31 alone (++ p,0.01). Model Hirano bodies protect against tau-dependent cell death (** p,0.01 between with and without model Hirano bodies). Error bars represent the standard deviation. doi:10.1371/journal.pone.0044996.g005 observation is consistent with the results above that Fe65, Tip60, caspases, and p53 are involved in both tau-independent and tau- dependent cell death and that model Hirano bodies protect against cell death. Figure 6. Fe65 contributes to both tau-independent and tau- dependent cell death. Cells were transfected with either APP/c31 or APP/c31/352PHP/tau in the absence (black bars) or presence of C655F Fe65 [56] (dotted bars). The expression of mutant C655F Fe65 significantly reduced both tau-independent and tau-dependent cell death (*** p,0.005). Error bars represent the standard deviation. doi:10.1371/journal.pone.0044996.g006 PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e44996