Figure 10. Model Hirano bodies lower tau-dependent and tau- independent apoptosis. H4 cells were transfected with either APP/ c31 or APP/c31/352PHP/tau in the absence (GFP-black bars) or presence of model Hirano bodies (CT-GFP-check bars). H4 cells were stained with the early apoptotic marker annexin V. Sytox orange was used as a control for annexin V false positive cells with compromised membranes. Cells transfected with either GFP/APP/c31 (tau-independent) or GFP/ APP/c31/352PHPtau (tau-dependent) showed significant levels of annexin V positive staining. The presence of model Hirano bodies lowered the percent of cells that were annexin V positive in both tau- independent and -dependent cell death (* p,0.05 in comparison to cells without model Hirano bodies). Error bars represent the standard deviation. doi:10.1371/journal.pone.0044996.g010 Discussion The cause of Alzheimer’s disease is unknown [11]. The amyloid cascade hypothesis and subsequent refinement that oligomeric species of abeta contribute to neuronal cell death provide a framework for investigation and explains a wide range of data [14,16,62,63]. AICD is an intracellular product of the processing of APP that produces amyloid-beta. The role of AICD in neurodegeneration is controversial. The lifetime of AICD is short [64,65] (but tagged versions have a longer lifetime [66,67,68]) impeding efforts to identify which cellular pathways it participates in. In spite of this, it has been shown that AICD does activate gene transcription but there are many targets that vary depending on the cell line and strategy used in the investigation (reviewed in [12,22,69]). All of these factors lead to controversy and caution in ascribing signaling activity to AICD. In this study, we have investigated the role of Hirano bodies, intracellular F-actin-rich inclusions, found in a variety of post- mortem neurodegenerative diseases including Alzheimer’s disease [4,37,70,71] and cell death induced by APP, AICDc58 and c31 and tau. Previously, we had shown that the presence of model Hirano bodies reduced transcription of AICDc58 and Fe65 and reduced AICDc58-induced cell death [47]. We have shown that the intracellular c-terminal fragments of APP, AICDc58 and c31 both induce cell death but that c31 requires the presence of APP to achieve significant levels of cell death. In addition, AICDc58- induced death increases in the presence of APP. These results are in agreement with previous results [47,48,53,72,73,74]. The Hirano Bodies Protect against Cell Death presence of model Hirano bodies significantly reduce the level of cell death. Transcription and Cell Death Since the transactivating activity of the APP/Fe65/Tip60 complex has been consistently observed (reviewed in [22,23,74,75]), we utilized mutants in either Fe65 or Tip60 to elucidate whether they play a role in cell death induced by exogenous AICDc58 or APP/c31. Mutations within the active site of Tip60 reduce AICD transcriptional activity and prevent AICD- induced cell death [53,56,76]. Our data is consistent with the gene activation activity of this complex since expression of dominant negative mutant Fe65 and Tip60 significantly reduce both APP/ c31 and APP/c31/352PHPtau-induced cell death. Our results disagree with a prior study which showed a mutant form of AICD (Y682A/Y687A) that was suggested not to bind Fe65, was still active in promoting cell death [53]. A possible explanation for this discrepancy is that the AICD mutation (Y682A/Y687A) was not proven to prevent interaction with Fe65 [53]. Furthermore, AICD mutants with either Y682A or Y687A reduce, but do not abolish the interaction of AICD with Fe65. In contrast, the C655F Fe65 mutant used in our study has been shown to prevent this interaction [56]. Thus, all work is consistent with the finding that Fe65 and Tip60 contribute to AICD-induced cell death. While the mechanism of AICD and tau-dependent cell death is unknown, AICD-induced cell death in the absence of tau involves induction of apoptosis [53]. We have shown that APP/c31 also induces apoptosis. Tip60 has been shown to regulate the specificity of p53, activating transcriptional preference for pro-apoptotic genes [58,59,60]. Furthermore, presenilin-dependent release of AICD is reported to enhance activity and expression of p53 to promote neurodegeneration [57]. An additional report shows that Fe65 can either activate or repress target genes, and promote cell death by controlling expression of caspase 4 [77]. Our data is consistent with these findings since our results show that cell death requires both p53 and caspase activation and cells were stained with Annexin V. While the cause of neurodegenerative diseases is unknown, there are data that suggests that AICD and/or c31 may play a role. AICD levels have been shown to be significantly higher in AD patient brains compared to age matched controls and correlates to levels of phosphorylated tau in these brains [78,79]. Caspase cleaved APP is also higher in AD brains suggesting that c31 is produced at a much higher rate in AD patients [80]. Additionally, there is evidence that c-terminal fragments of APP could associate with amyloid beta-induced cell death by a mechanism involving amyloid beta binding directly to APP, inducing its cleavage [48,81]. In addition, AICD expression in mice has been shown to increase levels of insoluble tau and phosphorylated tau, impaired neurogenesis, and promoted neu- rodegeneration compared to wild type mice [78,82]. These transgenic mice also show susceptibility to excitotoxicity stressors, which do not affect wild type mice [83,84]. In contrast, a small subset of the familial mutations found in presenilin 1 have been shown to decrease AICD production, suggesting that AICD might not play a role in Alzheimer’s disease [85]. The mutant presenilins also lower notch intracellular domain levels [85]. However, the gamma secretase complex has over 50 substrates in cells and other signaling pathways may also be affected by these presenilin mutations [86]. In addition, the role of AICD and Fe65 has been investigated in model mice overexpressing these proteins. Transgenic mice have elevated levels of active GSK-3ß, which is also observed with AD PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e44996