Figure 12. Model Hirano Bodies do not protect against etoposide-induced cell death. H4 cells were transfected with CT- GFP to induce Hirano body formation (check bars) or GFP (black bars) as a control. Expression of CT-GFP or GFP alone caused very low levels of cell death in the absence of etoposide. In the presence of 100 mM etoposide, cell death was significantly increased (***p,0.005). The presence of model Hirano bodies had no impact on etoposide-induced cell death. Error bars represent the standard deviation. doi:10.1371/journal.pone.0044996.g012 Model Hirano bodies protect against cell death Hirano bodies are found in increased frequency in autopsied brains with neurodegenerative diseases compared to control brains [4,37,70,71]. The physiological role of Hirano bodies is unknown. Development of cell culture and transgenic mouse models of Hirano bodies provide avenues of study that are not available using autopsy and necropsy samples [43,44] to determine the physiological role of Hirano bodies. Our results obtained using the cell culture model system show that cell death induced by either APP/c31 in the presence or absence of 352PHPtau or APP/ AICDc58 is significantly reduced in the presence of model Hirano bodies. 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]. In addition, the APP binding adaptor protein Fe65 has also been shown to co-localize with Hirano bodies in brain and cell cultures [6,47]. Both c31 and 352PHPtau co-localize with model Hirano bodies, but unlike 352PHPtau, the distribution of c31 changes from diffuse in control samples to concentrated and co-localized with Hirano bodies. The interaction of tau with Hirano bodies could occur through tau and its phosphorylated forms binding directly to F-actin [96]. Other findings in different experimental models suggest that tau promotes the formation of Hirano bodies and that actin promotes tau-dependent toxicity [96,97]. It is not known if specific isoforms of tau or whether phosphorylation of tau is required for interaction with model Hirano bodies. We suggest that model Hirano bodies could confer protection against cell death by sequestering c-terminal fragments of APP and possibly tau, preventing them from either participating in signaling pathways which contribute to cell death or gene transcription. Model Hirano bodies can be degraded by either autophagy or through the proteasome pathway [98]. Via these pathways, deleterious proteins bound to the model Hirano bodies are cleared from the cell, possibly conferring protection from cell death. We showed that model Hirano bodies are unable to protect cells against etoposide-induced apoptosis. This lack of protection Hirano Bodies Protect against Cell Death suggests that Hirano bodies may have a specific role in protection against cell death. It will be important in the future to determine the effects of model Hirano bodies on the progression of neurodegeneration in animal model systems to determine whether they represent a neuroprotective mechanism. Materials and Methods Plasmids pEGFPN-1 was obtained from Clontech. CT-EGFP was previously described [43]. APPc58-myc was generously provided by Dr. Bradley T. Hyman, Harvard Medical School [53]. APP was generously provided by Dr. Thomas C. Su¨ dhof, Stanford University School of Medicine [56,76]. 352tau is fetal human tau (352 residues) and 352PHP-tau is fetal human tau (352 residues) with S198, S199, S202, T231, S235, S396, S404, S409, S413, and S422 mutated to glutamate to mimic phosphorylated residues was generously provided by Dr. Roland Brandt, University of Heidelberg [54]. APPc31-myc was constructed by deleting the coding region of c58-myc from APPc58-myc, inserting the coding sequence of c31 (664–695 of APP) made by PCR amplification using APPc58-myc as a template, and ligating into plasmid that remained from APPc58-myc after deletion of the APPc58-myc coding sequence. Mutagenesis of Tip60 and Fe65 [56,76] (generously provided by Dr. Thomas C. Su¨ dhof, Stanford University School of Medicine) were performed using Stratagene Quickchange mutagenesis kit. DNA sequencing (Genewiz) was performed to confirm sequence fidelity of the Q377E G380E Tip60 and C655F Fe65 mutations and APPc31-myc. Cell Culture and Transfection. All cell lines were grown in DMEM supplemented with 10% fetal bovine serum (FBS) at 37uC and 5% CO2. Cells were plated in a 96-well plate (Nunc) or 35 mm dishes approximately 24 hours prior to transfection. Transient transfections were performed using Fugene 6 (Roche Diagnostics) or Lipofectamine LTX with Plus reagent (Invitrogen) according to the manufacturer’s recommendations. Approximate- ly 4 hours after transfection, cells were washed with PBS, resuspended in DMEM plus 10% FBS and grown for approxi- mately 48 hours before cell death assays were performed. N2A mouse neuroblastoma cells (ATCC) were transfected using Fugene 6 with 2.5 mg APPc58-myc, 1 mg APPc31-myc, and 2 mg pEGFPN-1 or CT-EGFP. H4 human astroglioma cells (ATCC) were transfected with Fugene 6 with 3 mg or 1.5 mg APP (tau- dependent and tau-independent conditions, respectively), 1 mg APPc31-myc and either 2 mg pEGFPN-1 or CT-EGFP for the tau-independent assays. For tau-dependent assays, 1 mg 352PHP- tau or 352WTtau was used. For AICDc58 experiments, 1 mg APPc58-myc, 1.5 mg APP, and 1 mg 352PHPtau or 352WTtau were used. Other experiments used 3 mg APP, 3.5 mg APPc58- myc, and 1 mg APPc31myc with either 2 mg of pEGFPN-1 or CT- EGFP. For experiments with Fe65 and Tip60, 1 mg Q377E G380E Tip60 or C655F Fe65 was used. Lipofectamine LTX transfection was used in Figures 4 and 11 with 250 ng of APP, APPc58-myc, 352PHPtau, 352WT tau, pEGFPN-1 or CT-EGFP. Flow cytometry was used to verify that the presence of multiple plasmids did not affect the level of protein expression (see Figure S1 and Method S1). Inhibitors used during the cell death assays were added immediately after removal of the Fugene 6 transfection reagent. A final concentration of 20 mM a-pifithrin (Sigma) and 100 mM caspase inhibitors Z-IGTD-FMK (caspase 8), Z-DEVD-FMK (caspase3), and Ac-VAD-CHO (broad caspases) (MP Biomedicals) were used and incubated for 48 hours before cell death assays were PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e44996