amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, and some tauopathies [2,6,31,32,33,34,35,36,37]. Hirano bodies are highly ordered filamentous actin (F-actin) arranged in a paracrystalline structure [38,39]. Electron microscopy has shown that the F-actin appears arranged as either a herringbone or crosshatch pattern depending on the plane of section. Due to the lack of a model system, previous research has focused on studying their ultra- structure and identifying their components [3,33,40,41]. Recently, the creation of model Hirano bodies was discovered through the expression of a truncated version of the 34 kDa actin bundling protein. This truncation consists of the carboxyl-terminal region of 34 kDa protein (CT) [42]. Model Hirano bodies induced by CT expression have been successfully created in Dictyostelium, a variety of mammalian cell lines, and in transgenic mice [42,43,44]. Model Hirano bodies are indistinguishable from authentic Hirano bodies on an ultrastructural level and contain some of the same protein components including tau and c- terminal fragments of APP [6,35,43,45,46]. Previous work from our laboratory has shown that model Hirano bodies protect against AICDc58-dependent cell death in H4 cells [47]. This work also showed that model Hirano bodies lowered the transcriptional activation from exogenous AICDc58 [47]. We have extended these studies by investigating the effect of model Hirano bodies on the presence of exogenous APP, AICDc58, and c31. Since tau co-localized with Hirano bodies and tau exhibits pathology in neurodegenerative diseases, we have studied the effect of tau in the presence of APP, AICDc58, and c31 and model Hirano bodies. We provide direct evidence for enhanced cell death between APP/AICDc58, APP/c31 and 352PHPtau, and AICDc58/352PHPtau. We show that these enhanced cell death conditions are partially attributed to activation of apoptotic pathways. Furthermore, we show that model Hirano bodies protect against APP/AICDc58, AICDc58/ 352PHPtau, APP/c31, and APP/c31/352PHPtau-induced cell death. Understanding the physiological role of Hirano bodies and their interactions with tau and fragments of APP may provide fundamental new insights to understanding Alzheimer’s disease and the presence of Hirano bodies in other neurodegenerative diseases. Results Model Hirano bodies protect against APP and c31- induced cell death It was shown previously that APP enhances the level of cell death induced by c31 [48]. We investigated the effect of Hirano bodies on c31-induced cell death. We transiently expressed APPc31-myc and/or APP to induce cell death in N2A cells as described previously [48], and measured cell death in the presence or absence of model Hirano bodies as shown in Figure 1. Expression of either APP or APPc31-myc alone induced low levels of cell death in the presence or absence of model Hirano bodies. Cells lacking model Hirano bodies and expressing APP/c31 showed a significant increase in cell death as previously reported [48]. In contrast, cells expressing model Hirano bodies and both APP/c31 showed a significant reduction in cell death levels (**p,0.01 compared to cells without model Hirano bodies). Since N2A cells contain moderate levels of endogenous tau [49], we investigated whether APP/c31-induced cell death requires the presence of tau. To address this question, we utilized astroglioma derived H4 cells that have very low levels of endogenous tau [49,50]. Hirano bodies have been proposed to have a glial origin [51] and astrocytes have been shown to be important mediators of cell death induced by beta amyloid and tau [52]. APP and/or c31 Hirano Bodies Protect against Cell Death Figure 1. Model Hirano bodies protect against APP and c31- induced cell death in N2A cells. N2A cells were transfected with APP/APPc31 in the presence (CT-GFP, check bars) or absence (GFP, black bars) of model Hirano bodies. Either APP or APPc31-myc causes low amounts of cell death while the presence of both APP/c31 shows an enhanced increase in cell death. Furthermore, model Hirano bodies are able to protect against the enhanced effect of APP/c31-induced cell death (** p,0.01 between cells with and without Hirano bodies). Error bars represent the standard deviation. doi:10.1371/journal.pone.0044996.g001 were transiently expressed in H4 cells and cell death was measured as shown in Figure 2. Low levels of cell death were observed in the presence of exogenously expressed APPc31-myc as previously observed in N2A cells. Expression of exogenous APP increased the level of cell death in H4 cells similar to the results obtained in N2A cells. In the presence of very low amounts of endogenous tau, model Hirano bodies still protected H4 cells against APP-induced cell death (*p,0.05). In the presence of both exogenous APP and APPc31-myc, enhanced cell death occurred in H4 cells in the presence or absence of model Hirano bodies. The presence of model Hirano bodies significantly lowered the amount of cell death induced by APP/c31 expression (**p,0.01). Cell death pathways of APP and c31 can operate independently of tau. In addition, the presence of model Hirano bodies protect cells from APP/c31-induced cell death. Previously, we observed AICDc58-dependent cell death in H4 cells without expression of either exogenous APP or tau [47,53] and that model Hirano bodies protect against cell death. We investigated whether model Hirano bodies would protect against the enhanced cell death induced by APP/AICDc58 by cotransfec- tion of APPc58-myc plasmid and APP. Expression of either exogenous APPc58-myc alone or APP/AICDc58 caused a significant amount of cell death as shown in Figure 3. The presence of model Hirano bodies decreased the amount of cell death by approximately 50% (+p,0.05). This level of protection is in agreement with previous data [47]. AICDc58-induced cell death was increased approximately 60% in the presence of exogenous APP. The presence of model Hirano bodies signifi- cantly reduced APP/AICDc58 cell death (++p,0.01). Model Hirano bodies protect against tau-dependent cell death. The effect of tau on AICDc58-induced cell death was investigated. Wild type fetal human tau (352 residues, 3R0N) tau (352WTtau) or 352PHPtau fetal human tau (352 residues) with PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e44996