Hirano Bodies Protect against Cell Death Figure 7. Tip60 contributes to both tau-independent and tau- dependent cell death. Cells were transfected with either APP/c31or APP/c31/352PHPtau in the absence (black bars) or presence (dotted bars) of mutant Q377E G380E Tip60 (mTip60) [53]. The expression of mutant Tip60 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.g007 Our results show that Hirano bodies protect against cell death induced by APP/c31 and AICDc58 in the presence or absence of 352PHPtau. Both tau and the c-terminal region of APP have been shown to co-localize with Hirano bodies in post mortem samples as well as in model Hirano bodies in cell culture [6,35,43,47]. This suggests that the protective role of Hirano bodies may be due to sequestering these proteins. To determine if c31 and 352PHPtau co-localize with model Hirano bodies, H4 cells were transfected with c31/352PHPtau and either CT-GFP (CT fused to EGFP) to induce model Hirano bodies or pEGFPN-1 as a control. In GFP control cells, 352PHPtau is predominantly found in the cytoplasm while APPc31-myc is uniformly distributed throughout the cell as shown in Figure 11. Surprisingly, in the presence of model Hirano bodies, APPc31- myc and 352PHPtau are predominantly co-localized with model Hirano bodies. Although APPc31-myc and 352PHPtau co- localize with model Hirano bodies (Figure 11) and cell death induced by these proteins is reduced in the presence of model Hirano bodies, it does not prove that model Hirano bodies reduce cell death by sequestration of these proteins. To address whether the presence of model Hirano bodies protect cells from death specifically, H4 cells expressing GFP or CT-GFP were incubated with and without 100 mM etoposide, a well charac- terized inducer of apoptosis. Expression of either GFP of CT- GFP in the absence of etoposide results in very low levels of cell death as shown in Figure 12. Cells expressing both GFP and CT- GFP incubated with etoposide had a significant increase in cell death (***p,0.005). The presence of model Hirano bodies had no impact on cell death induced by etoposide. Thus, model Hirano bodies have some specificity protecting against cell death. Figure 8. p53 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 (dotted bars) of a-pifithrin. a-pifithrin significantly reduced tau-independent (*** p,0.005) and tau-dependent cell death (** p,0.01). Error bars represent the standard deviation. doi:10.1371/journal.pone.0044996.g008 Figure 9. Caspases contribute 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 broad caspase inhibitor (Ac-VAD-CHO; striped bar) or caspase 8 inhibitor (Z-IGTD-FMK; white bars) or caspase 3 inhibitor (Z-DEVD- FMK; grey bars). The broad caspase inhibitor and caspase 8 inhibitor significantly reduced tau-independent (APP/c31) cell death (** p,0.01) and tau-dependent cell death (APP/c31/352PHPtau) (* p,0.05). Caspase 3 inhibitor had no significant effect on either kind of cell death. Error bars represent the standard deviation. doi:10.1371/journal.pone.0044996.g009 PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e44996