Seven month-old homozygous female KO-Tau mice (Tucker et al., 2001) and littermate WT mice were used to assess the role of Tau in DNA protection in aged mice. All animals were maintained in standard animal cages under conventional laboratory conditions (12 h/12 h light/dark cycle, 22°C), with ad libitum access to food and water. The animals were maintained in compliance with institutional protocols and all animal experiments were performed in compliance with, and following the approval of the local Animal Resources Committee (CEEA 342012 on December 12, 2012), standards for the care and use of laboratory animals, and the French and European Community guidelines. Three different mice have been used in each group for all experiments.

We designed an in vivo mouse model of transient hyperthermic stress based on the rat model described previously by Papasozomenos (1996). The mice were anesthetized using xylazine (20 mg/kg) and ketamine (100 mg/kg) and maintained in a 37°C environment for 30 min to avoid anesthesia related hypothermia and Tau hyperphosphorylation as previously described (Planel et al., 2007). The mice were then maintained at 37°C (control (C) group) or heat stressed (HS group) by being placed in an incubator containing ambient air heated to 44°C for 20 min. The rectal temperature of the mice was monitored every 10 min and did not exceed 41°C. In the (HS+24H) group, mice were subjected to HS during 20 min and then returned to room temperature during 24 h.

Protein oxidation was analyzed using an OxyIHC oxidative stress-detection kit (Millipore) according to the manufacturer’s directions. Protein carbonyl groups generated by oxidative stress were visualized using immunolabeling after reaction with 2,4-dinitrophenylhydrazine (DNPH).

The mice were euthanized through cervical dislocation, and their brains were rapidly removed. One hemisphere of each brain was post-fixed for 24 h in 4% paraformaldehyde and embedded in paraffin. The hippocampus and cortex were dissected from the other hemisphere and used for biochemical analyses.

Mouse tissues were harvested in ice-cold buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.15% NP-40) supplemented with protease inhibitors (Complete Mini-Roche) and phosphatase inhibitors (125 nM okadaic acid and 1 mM orthovanadate). The tissues were mechanically homogenized using a 50-ml all-glass homogenizer on ice and centrifuged at 100 g for 1 min. The supernatant was collected, and a second homogenization was conducted. The supernatant was collected as the cytoplasmic fraction after centrifugation at 1000 g for 10 min. The pelleted nuclei were washed three times and lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (Complete Mini-Roche) and phosphatase inhibitors (125 nM okadaic acid and 1 mM orthovanadate). The samples were sonicated and centrifuged at 12000 g at 4°C for 20 min to yield the supernatant as the nuclear fraction. The protein concentrations were determined using a bicinchoninic acid assay (BCA) kit. Lamin B and synaptophysin (SYP) were used as specific nuclear and cytoplasmic markers, respectively.

Electrophoresis and immunoblotting were performed as described previously (Sultan et al., 2011) using a Tau C-terminal antibody as described previously (Galas et al., 2006). The results are expressed as the mean ± S.E.M. of three different mice. ImageJ software was used for quantification.

The alkaline in vivo comet assay was specifically developed in the cortex for this project. The mouse cortices were dissected and mechanical disaggregation of each tissue was performed by using the Medimachine® system (Becton Dickinson). A small piece of cortex was inserted into a Medicon (i.e., a disposable chamber containing an immobile stainless steel screen allowing for efficient cutting) with approximately 1.0 mL of PBS buffer. The Medicon was thus inserted into the Medimachine® which was then run for 5 s. Once the tissue was processed, the cell suspension was recovered and viability was assessed using the trypan blue exclusion method. Cell viability was assessed using the trypan blue exclusion method. The comet assay was performed as described previously (Sultan et al., 2011). The Olive tail moment (OTM; Olive et al., 1990) was used to evaluate DNA damage. The OTM, expressed in arbitrary units, is calculated by multiplying the percent of DNA fluorescence in the tail by the length of the tail in micrometers. The tail length is measured between the edge of comet head and the end of the comet tail. A major advantage of using the OTM as an index of DNA damage is that both the amount of damaged DNA and the distance of migration of the genetic material in the tail are represented by a single number.

Terminal deoxyribonucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining was conducted on tissue slices using the TUNEL Apoptosis Detection Kit (Millipore) according to the manufacturer’s instructions. Tissue slices were pre-treated with low concentration of DNAse (1 µg/mL during 1 h) to perform positive controls. The TUNEL assay is often used to detect late apoptosis-induced DNA breaks; however, it also detects accessible 3′-hydroxyl (3′-OH) groups that are generated from DNA single- or double-strand breaks (DSB) under non-apoptotic conditions (Liu et al., 2005).

Brain slices from heat-stressed KO-Tau mice were incubated with DNAse-free RNAse (0.5 mg/mL, 3 h, Roche), RNAse-free DNAse (0.2 mg/mL, 3 h, Millipore #17-141 h) or a mixture of DNAse/RNAse prior to the TUNEL assay.

Sagittal (5 µM) brain slices were deparaffinized and unmasked using citrate buffer (3.75 mM acid citrate, 2.5 mM disodium phosphate, pH 6) for 10 min in a domestic microwave. The slices were submerged for 1 h in 1% horse serum (Vector Laboratories), and the primary antibodies were incubated overnight at 4°C in the presence of PBS-0.2% Triton using the following primary antibodies: total Tau (Tau CTer) and Tau1 antibodies (Galas et al., 2006) and the phospho-histone H2A.X (Ser 139) antibody from Millipore.

These antibodies were revealed via secondary antibodies coupled to Alexa 488 or 568 (Life Technologies). The sections were counterstained and mounted with Vectashield/DAPI (Vector Laboratories). 4′,6-diamidino-2-phenylindole (DAPI) was used as a chromatin counterstain.

Mouse hippocampal sections were acquired using an LSM 710 confocal laser-scanning microscope (Carl Zeiss). The confocal microscope was equipped with a 488-nm Argon laser, 561-nm diode-pumped solid-state laser and a 405-nm ultraviolet laser. The images were acquired using an oil 40x Plan-NEOFLUAR objective (1.3 NA) and an oil 63X Plan-APOCHROMAT objective (1.4 NA). All recordings were performed using the appropriate sampling frequency (8 bits, 1024-1024 images and a line average of 4). Serial sections from the three-dimensional reconstruction were acquired using Z-steps of 0.2 µm.

Images is an array of pixel and each pixel contains information about the different light intensity or color. This information is encoded in grid as a gray level. The gray values or gray scale describe the fluorescence intensity of every pixel. Acquisitions in confocal microscope are executed in 8 bits, therefore the different values of gray level of a pixel that can take are from 0 to 255 levels (0 no signal and 255 maximum signal).

The elliptical selection tool of ImageJ marked 20–30 representative nuclei based on DAPI staining (Figure 1). All immunofluorescence quantifications from the nuclear mean intensity fluorescence are expressed in gray values, and image analyses of the raw data were obtained using ImageJ (http://rsb.info.nih.gov.gate2.inist.fr/ij/, NIH, USA) and ZEN (Carl Zeiss) software programs. The results are expressed as the mean ± S.D. of the gray value from 20–30 different nuclei or at least three different areas of the CA1 cell layer.

Student’s t-test (BiostaTGV software, Jussieu, France) was used to determine the significance (p-value) between groups for immunoblotting and immunofluorescence analysis. A p-value < 0.05 was considered to indicate a significant difference.

We have shown previously that nuclear Tau protects DNA integrity in primary neuronal cultures of embryonic origin (Sultan et al., 2011). We designed an in vivo mouse model of transient hyperthermia/HS that induced ROS production in the cortex and the hippocampus, a region in the mouse brain particularly sensitive to oxidative stress, to investigate the physiological relevance of the DNA-protective function of Tau.

The ability of HS to induce oxidative stress (Flanagan et al., 1998) in the cells of 7-month (7 m) WT and KO-Tau mice was qualitatively analyzed through the immunohistochemical detection of carbonyl groups added to proteins (Figure 2). HS induced an increase of the carbonyl immunolabeling both in WT and KO-Tau cells in sagittal hippocampal sections.

The data obtained after Western blot analysis indicated that Tau was present in the nuclei of neurons from the cortex and hippocampus of WT mice under physiological conditions (Figures 3A, B), which is consistent with previous results in neuronal cultures (Sultan et al., 2011). HS increased Tau nuclear localization, also as observed previously in neuronal cultures (Sultan et al., 2011). The HS-induced nuclear accumulation of Tau in both regions was reversible. Tau in cortical and hippocampal nuclear extracts from WT mice was positively labeled with Tau1 antibody in physiological and HS conditions showing that nuclear Tau was predominantly dephosphorylated at epitope Ser195-202 (Figures 3A, B), as previously described in cultured neurons (Sultan et al., 2011).

The degree of DNA damage was monitored using a single-cell gel electrophoresis (Comet) assay in the cortices of 7 m WT and KO-Tau mice under control and HS conditions to analyze the capacity of Tau to protect neuronal DNA integrity in vivo. A highly significant enhancement of the median OTM, which reflects DNA fragmentation, was observed in KO-Tau mice compared with WT mice under physiological conditions (2.5-fold in KO-Tau C vs. WT C, p < 0.001), which shows that the Tau protein plays a major role in the protection of DNA integrity in vivo in the adult mouse brain (Figure 4A). HS treatment also selectively increased the OTM in KO-Tau mouse cells (1.4-fold in KO-Tau HS vs. KO-Tau C, p < 0.05); however, no significant induction of DNA damage was observed after HS treatment in WT mice (Figure 4A).

The TUNEL assay was performed to specifically visualize and quantify DNA breaks in vivo. Sagittal hippocampal sections from non-treated or HS-treated WT and KO-Tau mice were subjected to TUNEL assays, and the results were imaged using laser-scanning confocal microscopy (Figure 4B). Nuclear TUNEL fluorescence was specifically quantified in the neurons (Figure 4C) of two distinct hippocampal areas, the DG and the CA1 subfield. A strong and highly significant difference was observed between WT and KO-Tau mice under control and HS conditions, consistent with the Comet assays. TUNEL nuclear intensity was dramatically higher in the DG and CA1 of KO-Tau mice compared with WT mice under control condition (≈20-fold increase in DG and CA1 KO-Tau C vs. WT C, p < 0.001). Altogether, these observations support an essential physiological role for Tau in the protection of neuronal DNA integrity. HS significantly increased TUNEL-positive cells in KO-Tau mice compared with control (C) non-treated cells (CA1, 1.6-fold in KO-Tau HS vs. KO-Tau C DG, p < 0.001; DG, 2.5-fold in KO-Tau HS vs. KO-Tau C, p < 0.001). Only a fraction of the hippocampal neurons were TUNEL-positive after HS, which reflects the heterogeneity of the stress response between neurons (Figure 4B). As a positive control, DG and CA1 sagittal sections from 7-month-old WT mice in control condition were pretreated with a low concentration of DNAse to create substrate for the end-labeling réaction (Figure 4B). The level of gray was quantified within the nuclei in cells from DG and CA1 subfields in the positive control and compared to the averaged gray levels in the DG and CA1 regions in HS condition (DG: 70% in KO-Tau HS vs. positive control; CA1: 100% in KO-Tau HS vs. positive control), showing the potent effect of HS to induce nucleic acid breaks in KO-Tau neurons.

KO-Tau mice were allowed to recover for 24 h at room temperature after HS to investigate the later effects of HS-induced DNA damage. The nuclear TUNEL fluorescence in DG neurons returned to basal levels (p < 0.001); however, the nuclear TUNEL fluorescence only partially decreased in CA1 cells (1.2-fold in KO-Tau HS+24 h compared with KO-Tau HS, p < 0.05; Figure 4C). These results demonstrate the persistence of DNA damage selectively in CA1 neurons.

No change in cell density was detected (Figure 4D), which demonstrates the absence of HS-induced cell toxicity in KO-Tau mice in our in vivo HS model.

The production of DNA DSB leads to H2AX phosphorylation (γ-H2AX) under normal conditions (Kuo and Yang, 2008), which is necessary to initiate DSB repair. We performed fluorescent immunohistochemical labeling using an anti-γ-H2AX antibody in hippocampal sections from WT and KO-Tau mice before and after HS treatment to investigate DSB formation (Figure 5A). No significant increase in γ-H2AX labeling was observed between 7 m WT and KO-Tau mice under control conditions. However, a strong increase in nuclear γ-H2AX foci was observed in KO-Tau hippocampal (DG and CA1) cells after HS treatment (Figure 5A).

A strong increase in γ-H2AX levels (3.2 fold in HS KO-Tau vs. HS WT, p < 0.05) was also specifically observed in lysates from KO-Tau hippocampi after HS treatment (Figure 5B) using immunoblotting. These results indicate that only HS-induced neuronal DNA damage in KO-Tau mice led to γ-H2AX foci production.

γ-H2AX fluorescent labeling and TUNEL assays were performed concomitantly in hippocampal slices of 7 m KO-Tau mice (Figure 5C). HS specifically evoked γ-H2AX foci formation on chromatin in certain TUNEL-positive neurons, as observed in the TUNEL/γ-H2AX, γ-H2AX-DAPI and TUNEL-DAPI overlays.

γ-H2AX labeling in DG cells from KO-Tau mice decreased to basal levels 24 h after HS (Figure 5D), which is similar to the TUNEL staining. This result further confirms the capacity of the DG cells in KO-Tau mice to repair HS-induced DNA damage. In contrast, discrete γ-H2AX foci persisted in the nuclei of CA1 neurons, which indicated the reduced capacity of CA1 neurons to fully restore DNA integrity after HS.

The TUNEL-positive staining described in Figure 3B was also present in the cytoplasm of KO-Tau neurons (Figure 6). Cytoplasmic TUNEL staining was diffuse and, therefore, did not correspond to mitochondrial DNA fragmentation. We hypothesized that the cytoplasmic TUNEL labeling reflected RNA fragmentation because the TUNEL assay is based on fluorochrome labeling of 3′-OH termini after nucleic acid breaks.

Hippocampal sagittal sections from heat-stressed 7 m KO-Tau mice were incubated with high concentrations of DNAse-free RNAse, RNAse-free DNAse or a mixture of DNAse and RNAse to investigate the possibility that the cytoplasmic TUNEL staining corresponded to RNA strand breaks (Figure 6).

Pre-treatment with RNAse fully abolished cytoplasmic TUNEL staining and partially abolished nuclear TUNEL staining, which suggested that the cytoplasmic staining and a portion of the nuclear staining was HS-induced RNA damage in KO-Tau neurons. Pre-treatment with DNAse only partially removed nuclear TUNEL staining despite the complete disappearance of DAPI staining, which indicates complete DNA degradation. This result suggests that nuclear RNA is damaged after HS in KO-Tau neurons. Concomitant pre-treatment with DNAse and RNAse fully abolished cytoplasmic and nuclear TUNEL staining, which confirms the specific alterations of these nucleic acids in stressed KO-Tau neurons. These results suggest that DNA and nuclear and cytoplasmic RNA are damaged after HS in KO-Tau neurons.

The present study demonstrates a novel, major physiological role for nuclear Tau in the protection of neuronal DNA integrity in vivo in the adult mouse brain under physiological conditions; the absence of Tau rendered neuronal cells abnormally susceptible to HS-induced DNA damage.

Neurons in the brain encounter recurrent oxidative stress throughout their lifespan. The high basal levels of DNA damage in KO-Tau compared with WT neurons in aged mice under physiological conditions likely reflected a loss of the intrinsic protective function of Tau against chronic endogenous oxidative stress in the brain. It may contribute to the different deficits observed with age in Tau KO mice (Ke et al., 2012).

We designed a novel in vivo mouse model of transient hyperthermia/HS as a valuable and useful tool to easily investigate the effects of a transient and acute ROS increase in the whole brain of WT or transgenic mice. This model can be used to delineate the role of Tau in DNA protection under oxidative stress conditions. However, we cannot exclude the possibility that effects other than oxidative stress are involved in hyperthermia (Morano et al., 2012). This model demonstrated that hyperthermia potentiated DNA alterations in the absence of Tau in vivo, which reproduces the DNA protective role of Tau under HS conditions in primary neuronal cultures (Sultan et al., 2011).

Altogether these results show that Tau plays an essential role to preserve DNA integrity in adult neurons in vivo under physiological and HS conditions.

DNA damage in neurons triggers a cascade of highly potent DNA repair mechanisms to maintain genome integrity (Canugovi et al., 2013). DNA damage responses can induce highly dynamic post-translational modifications of histones that are critical for the DNA repair process (Lukas et al., 2011). DBS, one of the most toxic forms of DNA damage, quickly induce phosphorylation at serine 139 in the C-terminal sequence of histone H2AX (γ-H2AX), which promotes the recruitment of multiple DNA repair factors.

The absence of strong γ-H2AX accumulation in KO-Tau neurons under control conditions notwithstanding the strong increase in neuronal DNA damage in KO-Tau compared with WT mice observed using Comet and TUNEL assays, indicated that the DNA damage accumulation in Tau-deficient mice under physiological conditions differs from the HS-induced damage. This result suggests that chronic oxidative stress primarily induces single breaks rather than DSB or that the DSB repair process is impaired in KO-Tau neurons under physiological conditions.

Conversely, hyperthermia induced a strong and transient increase in γ-H2AX foci selectively in the nuclei of KO-Tau neurons, indicating that Tau deficiency induced the accumulation of non-repaired DNA DSB through altered DNA damage-induced chromatin post-translational modifications. This result suggests that Tau modulates double-strand break DNA repair responses under hyperthermia.

Hyperthermia-induced nucleic acid damage is reversible, which suggests that a Tau deficiency delays, but is not critical for, the DNA repair process. Tau likely plays a modulating rather than an essential role in double-strand break DNA repair mechanisms, and other proteins could compensate for its loss of function.

These results show an essential role for Tau in the control of DNA breaks under physiological conditions in adult neurons in vivo and a modulating role in the DNA double-strand break repair process under hyperthermia. Overall, this study suggests an important role of Tau in DNA repair mechanisms although we cannot exclude that Tau may only be involved in DNA protection.

DNA from hippocampal neurons in the CA1 subfield showed a higher susceptibility to damage than DG neurons 24 h after hyperthermia. This result highlights the relative weakness of KO-Tau CA1 neurons to restore DNA integrity compared with DG neurons.

Selective deficiency in oxidized DNA repair has been reported in CA1 neurons compared to others hippocampal neurons under oxidative stress conditions, hypoxia, ischemia and neurodegeneration in Alzheimer’s disease (AD; Wang and Michaelis, 2010).

Our data suggest that impaired Tau-dependent DNA repair plays a role in the selective vulnerability of CA1 neurons.

The present in vivo study suggests that Tau deficiency triggers alterations in RNA integrity in hippocampal neurons under HS.

Indeed, RNA oxidation predominantly leads to strand breaks (Poulsen et al., 2012). Cytoplasmic TUNEL staining has been described, but RNA fragmentation is rarely suggested as a possible cause. Nevertheless, TUNEL protocols often advise the use of RNAse treatment to clear the so-called cytoplasmic background (Zhang et al., 2006).

However, as RNA integrity has been analyzed in an indirect way, further experiments like mass spectrometry would be necessary to confirm the alteration of RNA in KO-Tau mice.

It is generally acknowledged that altered RNA is degraded rather than repaired because very few RNA repair mechanisms have been described in mammalian cells (Aas et al., 2003; Nunomura et al., 2009). Cleaved RNA is particularly harmful for neurons because it can lead to the translation of dysfunctional truncated or mutated proteins. RNA damage accumulation in KO-Tau neurons suggests that Tau plays a role in the RNA quality control process in addition to its previously described genomic DNA protective function. Tau is an RNA-binding protein (Kampers et al., 1996), and Tau may protect RNA partially through direct or indirect interactions. Further experiments are necessary to elucidate the potential role of Tau in RNA metabolism.