Hirano Bodies Protect against Cell Death Figure 2. Model Hirano bodies protect against APP and c31- induced cell death (tau-independent) in H4 cells. Cells were transfected with APP/APPc31 in the presence (CT-GFP, check bars) or absence (GFP, black bars) of model Hirano bodies. Expression of either APP or APPc31-myc cause low amounts of cell death while the presence of both APP/c31 caused an enhanced increase in cell death. Furthermore, model Hirano bodies are able to protect against cell death induced by APP (*p,0.05 between cells with and without Hirano bodies) and APP/c31 (** p,0.01 between cells with and without Hirano bodies). Error bars represent the standard deviation. doi:10.1371/journal.pone.0044996.g002 S198, S199, S202, T231, S235, S396, S404, S409, S413, and S422 mutated to glutamate to mimic phosphorylated residues [54] was utilized. Tau phosphorylation has been implicated in neurodegen- eration and expression of 352PHPtau has been shown to induce death in a cell culture system at an earlier time point than wild type tau [54,55]. Exogenous APPc58-myc was transfected alone and with either 352WTtau or 352PHPtau in the presence or absence of Hirano bodies. 352WTtau, like 352PHPtau, causes low levels of cell death when co-expresssed with GFP shown in Figure 4. Expression of exogenous AICDc58 alone causes a significant amount of cell death. AICDc58/352WTtau slightly increases cell death compared to AICDc58 alone. In contrast, 352PHPtau/AICDc58 increases cell death and is significantly higher than both AICDc58-induced cell death (***p,0.005) and AICDc58/352WTtau (**p,0.01). The presence of model Hirano bodies significantly reduced AICDc58, AICDc58/352WTtau, and AICDc58/352PHP-induced cell death (++p,0.01). The effect of tau on APP/c31-induced cell death was investigated. H4 cells were transfected with APP or APP/c31 and 352WTtau or 352PHPtau in the presence or absence of model Hirano bodies. Surprisingly, the 3.0 mg of APP plasmid used to initiate APP and APPc31-myc-induced cell death in H4 cells killed virtually every cell in the presence of APP, 352PHPtau and APPc31-myc (data not shown). Therefore, the amount of APP plasmid was reduced to 1.5 mg in order to achieve a maximum of approximately 50% cell death in the presence of exogenous APPc31-myc and 352PHPtau as shown in Figure 5. A small amount of cell death occurred with expression of 352PHPtau or c31/352PHPtau. At this new concentration of APP plasmid, APP/ c31 had significantly lower cell death than with APP/c31/ 352PHPtau, indicating that the addition of 352PHPtau is responsible for most of the death (++p,0.01 compared to APP/ Figure 3. Model Hirano bodies protect against AICDc58 and APP/AICDc58-induced cell death. Cells were transfected with APP and/or AICDc58 in the presence (CT-GFP, check bars) or absence (GFP, black bars) of model Hirano bodies. AICDc58 causes significant cell death in H4 cells in the absence of exogenously expressed APP. Transfection of H4 cells with APP/AICDc58 causes a significant increase in cell death (* p,0.05 in comparison to GFP/AICDc58). The presence of model Hirano bodies significantly protected against AICDc58 and APP- induced cell death (+ p,.05 between with and without model Hirano bodies) and APP/AICDc58-induced cell death (++ p,0.01 between with and without model Hirano bodies). Error bars represent the standard deviation. doi:10.1371/journal.pone.0044996.g003 c31). When model Hirano bodies were present, the amount of APP/c31/352PHPtau-induced cell death was significantly re- duced (**p,0.01 compared to cells without model Hirano bodies). Both tau-independent and -dependent cell death involve apoptosis. To discover how model Hirano bodies protect against tau-independent and tau-dependent cell death, we investigated possible mechanisms of cell death. We previously showed that AICDc58 and Fe65 co-localize with model Hirano bodies and that model Hirano bodies significantly reduced AICDc58-induced cell death and the transcriptional activity mediated by AICDc58, Fe65, and Tip60 [47]. In addition, it has been previously shown that AICDc58-induced cell death involves Tip60 [53]. The contribution of Fe65 and Tip60 in cell death was investigated by utilizing mutations in these proteins. We utilized a previously characterized dominant negative mutation in Fe65, C655F, which abolished its ability to interact with the YENPTY domain of APP or its c-terminal fragments [56]. Exogenous expression of C655F Fe65 significantly reduced both tau-independent (APP/c31) (***p,0.005) and tau–dependent (APP/c31/352PHPtau) cell death (***p,0.005) as shown in Figure 6, implicating Fe65 in both cell death pathways. We utilized a previously characterized dominant negative mutant form of Tip60, Q377E G380E, which abolished the catalytic activity of Tip60 [53]. Expression of Q377E G380E Tip60 (mTip60) significantly reduced both tau-independent (APP/c31) and tau– dependent (APP/c31/352PHPtau) cell death by at least 50% as shown in Figure 7 (***p,0.005). This result indicates a role of Tip60 in both cell death pathways. It has been reported that AICD and presenilin mediate the upregulation of p53 in cells [57]. Furthermore, Tip60 has been shown to acetylate p53 directly, causing it to initiate transcription PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e44996