Mustapha Aoubala

Fiona Murray-Zmijewski

Marie P Khoury

Kenneth Fernandes

Stephane Perrier

Hugo Bernard

Anne-Catherine Prats

David P Lane

Jean-Christophe C Bourdon
email: j.bourdon@dundee.ac.uk

p53 directly transactivates ∆133p53α, regulating cell fate outcome in response to DNA damage

p53RE: p53 responsive element; siRNA: small interfering RNA; siNS: non-specific siRNA; si133a: siRNA specific for ∆133p53 mRNAs; si133b: siRNA specific

Introduction

The p53 tumor suppressor protein is a transcription factor that, in response to various forms of cellular stresses, regulates the expression of numerous genes involved in several biological activities such as cell cycle, apoptosis, differentiation and metabolism [START_REF] Royds | p53 and disease: when the guardian angel fails[END_REF][START_REF] Braithwaite | p53: more research and more questions[END_REF][START_REF] Vousden | p53 in health and disease[END_REF].

The activation of p53 allows the preservation of the genome integrity and prevents genomic abnormalities being passed on to daughter cells, which could ultimately lead to cancer formation [START_REF] Vousden | p53 in health and disease[END_REF]. The p53 protein itself is rapidly stabilized and activated in response to a wide variety of cellular stresses such as DNA damage, hypoxia, nutrient deprivation and viral infection. However, the ways by which the cell fate outcome is determined by p53 in response to different cellular stresses remain, to an extent, elusive [START_REF] Lavin | The complexity of p53 stabilization and activation[END_REF].

Furthermore, the molecular mechanisms by which p53 accomplishes its different activities are still not completely understood. So far, the best described mechanism is its ability to modulate gene expression by binding directly and specifically to p53 responsive elements (p53REs) on DNA [START_REF] Laptenko | Transcriptional regulation by p53: one protein, many possibilities[END_REF].

It is now well established that the p53 gene belongs to the same gene family as p63 and p73, two other transcription factors that regulate transcription using similar responsive elements (p53REs) [START_REF] Mondal | The tumor suppressor protein p53 functions similarly to p63 and p73 in activating transcription in vitro[END_REF]. Like most of the human genes, it has been shown that the p63 and p73 genes express multiple mRNA variants due to alternative splicing and usage of different promoters. Hence, genes encoding these two family members can express various isoforms of the p63 and p73 proteins which contain different domains of the protein [START_REF] Moll | p63 and p73: roles in development and tumor formation[END_REF]. The p53 gene was first thought to have a much simpler organization than the p63 and p73 genes, with transcription being initiated from two distinct sites upstream of exon-1 (P1 and P1') [START_REF] Moll | p63 and p73: roles in development and tumor formation[END_REF]. We have assessed the structure of the p53 gene, using a PCR-based technique that specifically amplifies capped mRNAs [START_REF] Bourdon | p53 isoforms can regulate p53 transcriptional activity[END_REF]. This showed that, similarly to p63 and p73, nine distinct human p53 protein isoforms (p53, p53β, p53γ, ∆40p53α, ∆40p53β, ∆40p53γ, ∆133p53α, ∆133p53β, ∆133p53γ) can be produced through alternative initiation of translation, usage of an internal promoter and alternative splicing [START_REF] Murray-Zmijewski | p53/p63/p73 isoforms: an orchestra of isoforms to harmonise cell differentiation and response to stress[END_REF]. In this study, we refer to p53 as full-length p53 (FLp53). Expression of these different p53 isoforms, at the mRNA level, has been confirmed both in normal human tissues and in tumors [START_REF] Boldrup | Expression of p53 isoforms in squamous cell carcinoma of the head and neck[END_REF][START_REF] Ebrahimi | Expression of novel p53 isoforms in oral lichen planus[END_REF][START_REF] Bourdon | p53 and its isoforms in cancer[END_REF]. Moreover, the p53 isoforms have been shown to be differentially expressed in breast tumors, head and neck tumors, ovarian cancer, acute myeloid leukaemia, colorectal carcinoma and melanoma [START_REF] Boldrup | Expression of p53 isoforms in squamous cell carcinoma of the head and neck[END_REF][START_REF] Bourdon | p53 and its isoforms in cancer[END_REF][START_REF] Bourdon | p53 Family isoforms[END_REF][START_REF] Anensen | A distinct p53 protein isoform signature reflects the onset of induction chemotherapy for acute myeloid leukemia[END_REF][START_REF] Avery-Kiejda | Small molecular weight variants of p53 are expressed in human melanoma cells and are induced by the DNA-damaging agent cisplatin[END_REF][START_REF] Marabese | Expression levels of p53 and p73 isoforms in stage I and stage III ovarian cancer[END_REF][START_REF] Fujita | p53 isoforms Delta133p53 and p53beta are endogenous regulators of replicative cellular senescence[END_REF]. All this may suggest an important physiological function for the p53 isoforms.

We have previously shown that ectopic overexpression of ∆133p53α (an N-terminally truncated p53 isoform), negatively regulates p53 tumor suppressor activity by inhibiting p53-mediated apoptosis [START_REF] Bourdon | p53 isoforms can regulate p53 transcriptional activity[END_REF]. Using the zebrafish animal model, we have recently reported that, in response to DNA-damaging agents and embryo development defects, the zebrafish p53 internal promoter is induced by p53, leading to ∆113p53 protein expression (zebrafish homologue of human ∆133p53α), which prevents p53-mediated apoptosis [START_REF] Chen | p53 isoform delta113p53 is a p53 target gene that antagonizes p53 apoptotic activity via BclxL activation in zebrafish[END_REF]. However, little is known about the molecular mechanisms by which human ∆133p53α modulates p53 activities at the physiological level.

In this study, we aimed to investigate the regulation of ∆133p53α expression and its physiological role in modulating the cellular response to DNA damage. Our results clearly show that the human p53 internal promoter is directly transactivated by p53 in response to genotoxic stress, leading to ∆133p53α protein induction. The induced ∆133p53α then inhibits p53-dependent apoptosis and G1 arrest, without inhibiting p53dependent G2 arrest in U2OS cells. Our results further demonstrate that physiologically, ∆133p53α does not exclusively act in a dominant-negative manner toward p53 to differentially regulate cell cycle arrest and apoptosis.

Results

Mapping of the p53 internal promoter within intron-4 of the human p53 gene.

In the p63 and p73 genes, ∆N isoforms are transcribed from an internal promoter (P2), located within intron-3 of both genes [START_REF] Moll | p63 and p73: roles in development and tumor formation[END_REF]. Although p63 and p73 are related to p53, no such internal promoter had been described for the p53 gene. In our previous study, Gene-Racer PCR results showed that a transcription initiation site lies in the 3' end of intron-4 of the p53 gene [START_REF] Bourdon | p53 isoforms can regulate p53 transcriptional activity[END_REF]. Therefore, we sought to map the initiation site using deletion constructs within this region.

We first amplified by PCR a region of 1555 bp upstream of exon-5 of the human p53 gene. This PCR fragment corresponding to nucleotides 11523-13076 of the human p53 gene (accession no. X54156, NCBI), was cloned into the promoterless pGL3-basic plasmid, upstream of the firefly luciferase (luc) reporter gene, to generate the pi3i4-luc construct (Figure 1A,a). Subsequently, a number of deletion constructs (Figure 1A, b-f) of the recombinant pi3i4-luc construct were made by enzymatic digestion, followed by re-ligation or sub-cloning of fragments, as shown in Figure 1A.

To define the minimum promoter region, the different luciferase constructs were then transfected into p53-null H1299 cells (non-small cell lung carcinoma) and cell extracts were tested for luciferase activity. As shown in Figure 1B, the 1555 bp fragment (a) contains a promoter activity in H1299 cells. Its activity is similar to the basal promoter activity of Bax and is approximately 4 times greater than the basal activity of the promoterless pGL3-basic plasmid. Deletion of residues 1 to 1304 (Figure 1A, constructs c, d and f) did not abolish the promoter activity (Figure 1B, c, d andf), suggesting that the minimum p53 internal promoter region is located within the last 251 bp of the intron-4 (Figure 1A,f).

Moreover, the minimum p53 internal promoter (Figure 1B, f) showed a significant increase in the basal promoter activity (4-fold), as compared to the basal activity of the pi3i4-luc construct (Figure 1B Human ∆133p53 is a direct p53 target gene.

Using our bioinformatic software and our previously refined consensus p53RE (20), we investigated whether the p53 internal promoter contains p53REs. The consensus p53RE was defined as two consensus decamers (RRRCWWGYYY) flanked by at least two additional ones. A maximum of 3 differences to the consensus was accepted in total for the two consensus decamers, as well as for each additional decamer. Each additional decamer is allowed to bear one base insertion or deletion between the median C and G, if the two consensus decamers are contiguous. This set of criteria was validated by identifying within the promoter region of the IGF-BP3 gene, the existence of a cluster of 11 decamers (p53REs) whose functional significance was verified [START_REF] Bourdon | Further characterisation of the p53 responsive element--identification of new candidate genes for trans-activation by p53[END_REF]. We identified five putative p53REs at the junction of exon-4 and intron-4 of the human p53 gene corresponding to the residues 757-804 in the pi3i4-luc construct (Figure 2A, dashed box), suggesting that p53 may directly transactivate its own internal promoter.

To test this hypothesis, deletion constructs c and d (Figure 1A) were assessed for their responsiveness to p53 (Figure 2A, c and d respectively), in H1299 cells co-transfected with p53 expression vector and each of the generated luciferase constructs. A luciferase construct driven by the p21 promoter was used as a positive control. As shown in Figure 2B, p53 induced the luciferase activity in all constructs containing the putative p53REs (Figure 2A, a andc). Interestingly, the greatest induction (6-fold) was obtained with the construct containing the 723-1555 bp fragment (Figure 2A, construct c), which contains all five p53REs but lacks the upstream 722 bp region of the p53 internal promoter, suggesting that this region may contain negative regulatory elements. Interestingly, deletion of the five putative p53REs (Figure 2A, construct d) resulted in a significant decrease in p53 induction (Figure 2B,d compared to c). However, the induction by p53 of the promoter deleted of the region encompassing the p53REs (723-1042), (Figure 2B,d) is not totally abolished, suggesting that this region retains a weak responsiveness to p53, either through direct DNA binding and/or interaction with other transcription factors or co-activators.

To further confirm that the putative identified sites correspond to p53REs, four single base mutations were generated by site-directed mutagenesis within the first two p53REs of the internal promoter. As shown in Figure 2C, the four mutations are sufficient to significantly decrease p53 transactivation of the entire p53 internal promoter (construct a). This result clearly demonstrates that intron-4 of the p53 gene contains functional p53REs. However, these mutations do not completely abolish p53 activation probably because of the presence of the remaining p53REs. Further analysis will be required to establish whether the remaining p53REs can confer responsiveness to p53.

Endogenous p53 can directly bind to and transactivate its internal promoter.

We next investigated whether endogenous p53 could directly bind to and transactivate its own internal promoter. MCF7 cells, (a breast cancer cell line expressing WTp53) were left untreated or treated with 60 ng/ml of Actinomycin D (Act D) for 2h and a chromatin immunoprecipitation (ChIP) assay was performed, using the mouse anti-p53 monoclonal antibody DO-1. The immunoprecipitated DNA was quantified by RT-qPCR (Figure 3A).

A probe and primers specific for a region within the GAPDH gene that is not bound by p53, was used as a negative control. All GAPDH amplifications gave a cycle number greater than 40, indicating that no DNA was present in the ChIP samples and confirming the specificity of the assay. The binding of p53 to the p21 promoter was analyzed as a positive control (Supplementary Figure B1). We further quantified the genomic DNA corresponding to either the p53REs (intron-4) or exon-8 of the p53 gene, which is co-immunoprecipitated with the p53 protein. As illustrated in Figure 3A, in unstressed cells, no significant binding of p53 to either intron-4 or exon-8 was observed. However, following Act D treatment, we observed an enhancement of the specific binding of endogenous p53 to intron-4 but not to exon-8. This clearly indicates that, in response to stress, endogenous p53 directly binds to its own internal promoter located within intron-4.

RT-qPCR experiments were carried out in MCF7 and MCF7-DDp53 (a stable cell line overexpressing a dominant-negative form of p53) cells, to determine whether there is a p53-dependent induction of ∆133p53 mRNAs (Figure 3B). Since the PCR fragments corresponding to the ∆133p53 isoforms (∆133p53α, ∆133p53β and ∆133p53γ) are all around 800 bp long and mRNA quantification by RT-qPCR is not reliable for large DNA fragments, it is not technically possible to specifically quantify each of the human ∆133p53 isoforms mRNAs. Therefore, we designed a probe and primers from the unique 5'UTR region of ∆133p53 mRNAs, encoding all three ∆133p53 isoforms (∆133p53α, ∆133p53β and ∆133p53γ). MCF7 and MCF7-DDp53 cells were then treated with Act D. p21 mRNA induction was determined as a positive control (Supplementary Figure B2).

As illustrated in Figure 3B, upon cellular stress, the expression of ∆133p53 mRNAs is significantly induced 2h following Act D treatment. No induction of p21 or ∆133p53 mRNAs was seen in MCF7-DDp53 cells, confirming that the induction of ∆133p53 mRNAs is p53-dependent.

To assess whether ∆133p53 mRNAs induction could occur in other cell lines and in response to other DNA-damaging agents, we compared the expression of ∆133p53 mRNAs in response to doxorubicin treatment in colorectal carcinoma cell lines, expressing WTp53 (HCT116+/+) or devoid of FLp53 expression (HCT116-/-). In HCT116-/-cells, the neomycin gene has been integrated within exon-2 of the p53 gene, resulting in the loss of FLp53 expression [START_REF] Bunz | Requirement for p53 and p21 to sustain G2 arrest after DNA damage[END_REF]. However, HCT116-/-cells still express p53 isoforms at the mRNA and protein levels, due to initiation of translation from codon 40 located in exon-4 of the p53 gene [START_REF] Murray-Zmijewski | p53/p63/p73 isoforms: an orchestra of isoforms to harmonise cell differentiation and response to stress[END_REF][START_REF] Courtois | DeltaNp53, a natural isoform of p53 lacking the first transactivation domain, counteracts growth suppression by wild-type p53[END_REF]. We show here that ∆133p53 mRNAs are inducible in response to doxorubicin only in HCT116+/+ but not in HCT116 -/-cells (Figure 3C). Similar results were obtained in response to etoposide treatment (data not shown). We further investigated ∆133p53 mRNAs induction after treating U2OS osteosarcoma cell line (expressing WTp53) and primary normal human dermal fibroblasts (NHDF) with doxorubicin (Figure 3D and3E, respectively). Similarly to MCF7 and HCT116+/+ cells, ∆133p53 mRNAs expression is significantly induced upon doxorubicin treatment in both cell lines. The data indicate that induction of ∆133p53 mRNAs expression is p53-dependent in multiple cell lines. Altogether, these results demonstrate that in vivo, the p53 internal promoter located within intron-4, is directly transactivated by p53 in response to DNA damage ∆133p53α protein expression is induced by p53 in response to DNA damage.

Western blot analyses were performed to determine whether the increase of ∆133p53α protein is associated with the expression of ∆133p53 mRNAs. The rabbit polyclonal antibody CM1 was raised against full-length recombinant human p53 protein and can detect all p53 protein isoforms, including ∆133p53α. We thus analyzed ∆133p53α protein expression in HCT116+/+ and HCT116-/-cells, treated or not with doxorubicin.

To prevent any in vitro protein degradation, cells were directly lyzed in SDS-Laemmli buffer. The results in HCT116+/+ cells show that doxorubicin treatment clearly induces p53 and some p53 isoforms migrating at 48 kD, 45 kD and 35 kD (Figure 4A). In HCT116-/-cells, a strong expression of ∆40p53 was detected but neither ∆40p53 nor other p53 isoforms were induced in response to doxorubicin treatment. However, it is not technically possible with the CM1 antibody to specifically identify the ∆133p53α isoform by western blot analysis. Therefore, we designed two distinct siRNAs (si133a and si133b) specific for the ∆133p53 isoforms (∆133p53α, ∆133p53β and ∆133p53γ), which target the 5'UTR of ∆133p53 mRNA variants. The specificity and efficiency of the designed siRNAs were first assessed by RT-qPCR analysis in U2OS cells (Supplementary Figure C1).

Having confirmed the specificity and efficiency of the ∆133p53 siRNAs (si133a or si133b), we then attempted to identify by western blot the bands corresponding to endogenous ∆133p53α protein in HCT116 cells, transfected with either si133a, si133b or siNS as a negative control (Figure 4B). Doxorubicin treatment induced the expression of p53 and also of some p53 isoforms migrating at 48 kD, 45 kD and 35 kD (Figure 4B The induction of ∆133p53α protein was further confirmed by western blot analysis in primary normal human dermal fibroblasts (NHDF) treated with doxorubicin (Figure 4C).

Altogether, these results demonstrate that, in the cancer derived cell lines (HCT116 WTp53) and in the normal primary fibroblasts, ∆133p53α isoform is induced by p53 at both mRNA and protein levels in response to DNA damage.

Generation and characterization of U2OS-ctrl and U2OS-∆133p53 cells.

To determine the role of ∆133p53α in the cellular response to DNA damage, we investigated by BrdU cell cycle analysis and apoptosis assay (annexinV assay) the experimental conditions which enable the U2OS cells to undergo cell cycle arrest rather than apoptosis, in response to doxorubicin. We established that U2OS cells grown for 24h at 50% confluence and treated with a low dose of doxorubicin (0.5µM for 1h), trigger G2 cell cycle arrest without promoting apoptosis 24h after treatment. Those experimental conditions were then used in all the following experiments.

Two cellular models were generated in order to define the biological relevance of ∆133p53α induction in response to DNA damage. U2OS cells were stably transfected with the empty pcDNA3 or pcDNA3-∆133p53α expression vectors, to generate U2OSctrl and U2OS-∆133p53 cells respectively. U2OS-∆133p53 cells were used to assess whether the biological effects of ∆133p53 mRNAs depletion by si133a or si133b could be rescued by restoration of ∆133p53α expression.

First, we analyzed ectopic ∆133p53α protein expression in U2OS-∆133p53 cells by western blot analysis, using the CM1 antibody (Figure 5A). U2OS-∆133p53 cells transfected with either si133a, si133b or siNS were left untreated or treated with doxorubicin (0.5µM, 1h). As shown in Figure 5A, lane 1, ectopic ∆133p53α protein was expressed at 35kD. A weaker band at 32kD could also be detected and may correspond to a post-translationally modified form of ∆133p53α. Transfection of U2OS-∆133p53 cells with si133a or si133b specifically knocked-down the bands at 35kD and 32kD corresponding to ectopic ∆133p53α protein, without affecting p53 protein level (Figure 5A, lanes 2, 3 compared to 1). In U2OS-∆133p53 cells, doxorubicin treatment induced p53 protein expression but had no detectable effect on ∆133p53α protein expression (Figure 5A, lane 4 compared to 1). This is because the ectopic expression of ∆133p53α, driven by the CMV promoter (pcDNA3 plasmid), is largely above the endogenous expression level and therefore masks the induction of endogenous ∆133p53α (Figure 5B lane 1 compared to 7). Depletion of ∆133p53α by si133a or si133b was equally efficient in doxorubicin-treated and untreated U2OS-∆133p53 cells (Figure 5A, lanes 2, 3 compared to 1 and lanes 5, 6 compared to 4). The induction of p53 in response to doxorubicin is comparable in U2OS-∆133p53 cells transfected with either si133a, si133b or siNS (Figure 5A, lanes 5, 6 compared to 4). This clearly shows that p53 can be induced in U2OS-∆133p53 cells in response to doxorubicin treatment.

To compare the expression level of ∆133p53α protein in U2OS-ctrl and U2OS-∆133p53 cells, the same amount of protein from U2OS-∆133p53 cells was loaded in Figure 5A, lane 1 and in Figure 5B, lane 1. Endogenous ∆133p53α protein was not detectable in untreated U2OS-ctrl cells (Figure 5B, lanes 2, 3). Transfection of si133a or si133b did not affect the expression of p53 in untreated U2OS-ctrl cells (Figure 5B, lanes 4,5). In response to doxorubicin (0.5µM, 1h), endogenous ∆133p53α was induced in U2OS-ctrl cells (Figure 5B, lane 3 compared to 7). Of note, in U2OS-ctrl cells treated with doxorubicin, endogenous ∆133p53α protein level is far below the expression levels of ectopic ∆133p53α protein in U2OS-∆133p53 cells (Figure 5B lane 7 compared to 1).

Transfection of U2OS-ctrl cells with si133a or si133b inhibited the induction of endogenous ∆133p53α protein in response to doxorubicin (Figure 5B, lanes 8, 9 compared to 7). To determine whether the induction of ∆133p53α in doxorubicin-treated U2OS-ctrl cells is p53-dependent, cells were transfected with siTA, an siRNA that specifically targets p53 mRNAs containing exon-2 of the p53 gene (p53, p53β, p53γ, ∆40p53α, ∆40p53β and ∆40p53γ), without targeting ∆133p53 mRNAs. The results show that ∆133p53α protein induction is inhibited in doxorubicin-treated U2OS-ctrl cells depleted of p53 expression, confirming that ∆133p53α is induced by p53 (Figure 5B, lane 6 compared to 7).

Human ∆133p53α antagonizes p53-mediated apoptosis and G1 cell cycle arrest without preventing G2 cell cycle arrest.

We next investigated whether the cellular response to DNA damage could be regulated by modulating ∆133p53α expression. U2OS-ctrl and U2OS-∆133p53 cells were transfected with either si133a, si133b or siNS and were left untreated or treated with doxorubicin (0.5µM, 1h) for 24h. In parallel to the western blots presented in Figures 5A and5B, cell cycle progression (Figures 5C, 5D and5E) and apoptosis (Figure 5F) were analyzed by BrdU assay and Annexin-V assay respectively. A representative BrdU experiment is shown in Figure 5C. Untreated U2OS-ctrl and U2OS-∆133p53 cells transfected with siNS showed similar cell cycle progression and a low percentage of spontaneous apoptosis (8%), indicating that overexpression of ∆133p53α does not alter cell cycle progression and apoptosis in untreated cells (Figures 5C-a, 5C-b, 5D, 5E and 5F). Similar results were obtained in untreated and untransfected cell lines.

U2OS-ctrl and U2OS-∆133p53 cells transfected with siNS and treated with a low dose of doxorubicin show statistically similar cell cycle progression with about 3% of cells arrested in G1 and 79% of cells arrested in G2 (Figures 5C-c, 5C-d and5E). In both cell lines, the low dose of doxorubicin did not significantly increase apoptosis compared to untreated cells (Figure 5F). Altogether, this indicates that overexpression of ∆133p53α in U2OS-∆133p53 cells does not alter the cellular response to a low dose of doxorubicin, compared to U2OS-ctrl cells (Figures 5C-c, 5C-d, 5D, 5E and 5F). To determine whether spontaneous apoptosis and the induction of G2 cell cycle arrest in response to a low dose of doxorubicin is p53-dependent, U2OS-ctrl cells were transfected with siTA. We detected a statistically significant decrease in the percentage of cells arrested in G2 (Figure 5E) and in the percentage of apoptotic cells, compared to U2OS-ctrl cells transfected with siNS and treated with a low dose of doxorubicin (Figure 5F). This is consistent with previous publications reporting that doxorubicin induces G2 cell cycle arrest and apoptosis in a p53-dependent manner in U2OS cells [START_REF] Allan | p53-dependent apoptosis or growth arrest induced by different forms of radiation in U2OS cells: p21WAF1/CIP1 repression in UV induced apoptosis[END_REF]. As we have previously reported that overexpression of ∆133p53α can act in a dominant negative manner toward p53 [START_REF] Bourdon | p53 isoforms can regulate p53 transcriptional activity[END_REF][START_REF] Murray-Zmijewski | p53/p63/p73 isoforms: an orchestra of isoforms to harmonise cell differentiation and response to stress[END_REF], it is surprising that the overexpression of ∆133p53α in U2OS-∆133p53 cells does not inhibit p53-mediated G2 arrest. It implies that ∆133p53α does not exclusively act in a dominant-negative manner toward p53.

Strikingly, in response to doxorubicin treatment, depletion of the ∆133p53 isoforms with si133a or si133b significantly enhanced the percentage of U2OS-ctrl cells in G1 arrest (16% and 26% respectively, Figures 5C-e and5D), suggesting that endogenous ∆133p53α inhibits G1 cell cycle arrest in U2OS-ctrl cells. To confirm that the G1 arrest is due to the specific depletion of ∆133p53α and not to off-target effects of the siRNAs, U2OS cells overexpressing ∆133p53α (U2OS-∆133p53) were transfected with si133a or si133b. Although both siRNAs inhibit ∆133p53α expression in U2OS-∆133p53 cells, ∆133p53α protein level was still overexpressed compared to endogenous ∆133p53α protein level in U2OS-ctrl cells (Figure 5A). The results showed that transfection of si133a or si133b in U2OS-∆133p53 does not significantly change the percentage of cells in G1 in response to a low dose of doxorubicin (Figure 5C-f and 5D), confirming that si133a and si133b are specific and that ∆133p53α inhibits G1 arrest in U2OS-ctrl cells.

Of note, no statistically significant alteration in the percentage of cells in S phase was observed (Figure 5C).

Regarding apoptosis (Figure 5F), in U2OS-ctrl cells transfected with si133a or si133b and compared to cells transfected with siNS, a statistically significant increase in the percentage of apoptotic cells was detected, suggesting that a low dose of doxorubicin can trigger apoptosis if cells are depleted of ∆133p53α. Consistent with this, in U2OS-∆133p53 cells transfected with si133a or si133b and compared to cells transfected with siNS, a statistically significant lower increase in the percentage of apoptotic cells was observed in response to a low dose of doxorubicin. This indicates that overexpression of ∆133p53α rescued the resistance to apoptosis in response to a low dose of doxorubicin confirming that endogenous ∆133p53α inhibits p53-mediated apoptosis in U2OS-ctrl cells.

Altogether, our results show that in response to a low dose of doxorubicin, endogenous ∆133p53α antagonizes p53-mediated apoptosis and G1 arrest, without inhibiting p53dependent G2 arrest. This indicates that ∆133p53α does not exclusively inactivate p53. Moreover, it demonstrates that the cellular response to DNA damage could be regulated by modulating ∆133p53α expression.

Human ∆133p53α can form a protein complex with p53 and can differentially regulate p21, HDM2 and Bcl-2 expression in response to stress.

We have previously reported that ectopic ∆133p53α can act in a dominant-negative manner toward p53 [START_REF] Murray-Zmijewski | p53/p63/p73 isoforms: an orchestra of isoforms to harmonise cell differentiation and response to stress[END_REF]. In order to explain the regulation of p53 activities by ∆133p53α, we investigated whether ∆133p53α could directly interact with p53. Coimmunoprecipitation assays were carried out on p53-null H1299 cells transfected with ∆133p53α and/or p53, using the mouse monoclonal antibody DO-1, recognising FLp53 but not ∆133p53α. Immunoprecipitated proteins were then analyzed by western blot with the rabbit polyclonal antibody (CM1), recognizing FLp53 and ∆133p53α. The results clearly show that ∆133p53α can form a protein complex with p53 (Figure 6A).

We then studied whether endogenous ∆133p53α could regulate p53 target gene expression in response to DNA damage. In this study, we focused our investigation on a few p53 target genes (p21, HDM2 and Bcl-2), implicated in cell cycle progression and apoptosis. p21 encodes a cyclin-dependent kinase inhibitor involved in G1 and G2 cell cycle arrest [START_REF] Medema | p21waf1 can block cells at two points in the cell cycle, but does not interfere with processive DNAreplication or stress-activated kinases[END_REF], while HDM2 is an E3-ubiquitin ligase that regulates p53 protein degradation [START_REF] Juven | Wild type p53 can mediate sequence-specific transactivation of an internal promoter within the mdm2 gene[END_REF][START_REF] Barak | Regulation of mdm2 expression by p53: alternative promoters produce transcripts with nonidentical translation potential[END_REF]. Both p21 and HDM2 genes are directly transactivated by p53 in response to stress, whereas Bcl-2 is an anti-apoptotic gene repressed by p53 (27) [START_REF] Bredow | Identification of a novel Bcl-2 promoter region that counteracts in a p53-dependent manner the inhibitory P2 region[END_REF]. and Bcl-2 at both protein and mRNA levels. This suggests that ∆133p53α can increase Bcl-2 expression and contributes to the induction of HDM2 expression, in response to a low dose of doxorubicin. Therefore, our results show that ∆133p53α can differentially regulate p21, HDM2 and Bcl-2 expression and that ∆133p53α can directly interact with p53.

Altogether, we show that p53-mediated expression of ∆133p53α in response to a low dose of doxorubicin, leads to the inhibition of p53-mediated apoptosis and G1 cell cycle arrest without impairing p53-mediated G2 cell cycle arrest. Moreover, we show that ∆133p53α can directly interact with p53 and can differentially regulate the expression of p21, HDM2 and Bcl-2 in response to a low dose of doxorubicin. This indicates that ∆133p53α does not exclusively inhibit p53 but rather modulates p53 activities.

Discussion

We have recently reported that the human p53 gene encodes nine different p53 isoforms, two of which can modulate p53 transcriptional activity and apoptosis. We have previously shown that p53β and ∆133p53α isoforms can modulate p53 target gene expression, in a promoter-dependent manner. In addition, we have reported that ectopic overexpression of the N-terminally truncated isoform ∆133p53α inhibits p53-mediated apoptosis [START_REF] Bourdon | p53 isoforms can regulate p53 transcriptional activity[END_REF]. In this report, we investigated the regulation of p53's internal promoter within intron-4 of the p53 gene, and the biological activities of endogenous ∆133p53α in response to a low dose of doxorubicin.

We determined that the initiation site of the p53 internal promoter is located within the last 250 bp of intron-4. Moreover, we identified five putative p53REs at the junction of exon-4 and intron-4 of the human p53 gene. By ChIP assay, luciferase reporter assay and site-directed mutagenesis, we showed that p53 binds directly to the p53REs and thus transactivates its own internal promoter in response to doxorubicin treatment. Further analysis will be required to establish whether all five p53REs can confer responsiveness to p53. It would be interesting to determine whether the p53 internal promoter can be regulated by p63 and/or p73. By bioinformatics analysis, we determined that the p53 internal promoter contains also numerous responsive elements to transcription factors that promote cell proliferation (AP1, Elk1, N-myc, E2F), are responsive to hypoxia (HIF-1α), to heat shock (HSF1) or to hormones (estrogen receptor). In addition, we identified a silencer region (position 1042-1304) within the p53 internal promoter suggesting that it can also be repressed by other transcription factors (ZNF219, CBF1). Therefore, the p53 internal promoter is responsive to stress and proliferation signals which would regulate p53 tumour suppressor activity by modulating ∆133p53α expression.

To date, it is still technically difficult to specifically quantify by RT-qPCR large DNA fragments. Therefore, it was not possible for us to design primers and probe sets to specifically quantify the expression of each separate ∆133p53 isoform (∆133p53α, ∆133p53β and ∆133p53γ), encoded from the p53 internal promoter. Hence, to quantify at the mRNA level the expression of the ∆133p53 isoforms (∆133p53α, ∆133p53β and ∆133p53γ), we designed primers and probe specific for the 5'UTR of ∆133p53 mRNAs, which is not present in FLp53 mRNA. We then showed that the human p53 internal promoter is induced in response to doxorubicin or Actinomycin D treatment, in different cancer derived cell lines expressing WTp53 (HCT116, U2OS and MCF7), as well as in primary normal human fibroblasts (NHDF). This indicates that the induction of the p53 internal promoter is not specific for cancer cells. By comparing ∆133p53 mRNAs expression in response to DNA damage in MCF7 (WTp53) and MCF7-DDp53 (overexpressing dominant negative p53 form) cells or in HCT116+/+ (WTp53) and HCT116-/-cells (devoid of FLp53 expression), we showed that the activation of the p53 internal promoter leading to ∆133p53 mRNAs expression is p53-dependent in response to DNA damage. This is consistent with our recent report on the induction of zebrafish p53 internal promoter by p53 in response to DNA-damaging agents, suggesting that p53mediated regulation of its own internal promoter is conserved through evolution [START_REF] Chen | p53 isoform delta113p53 is a p53 target gene that antagonizes p53 apoptotic activity via BclxL activation in zebrafish[END_REF].

Moreover, p63 and p73 internal promoters, which respectively lead to ∆Np63 and ∆Np73 expression, are also regulated by full-length p63 and p73 [START_REF] Moll | p63 and p73: roles in development and tumor formation[END_REF]. Therefore, the regulation of p53/p63/p73 internal promoters respectively by p53, p63 and p73 is a feedback-loop conserved among the p53 gene family, suggesting that the regulation of p53/p63/p73 internal promoters plays an important role in the biological activities of the p53 family members. It is worth noting that the p53 internal and the p53 proximal promoters are distinct, therefore the ratio of these isoforms could be independently modulated in a tissue-specific manner and in response to different stresses.

Western blot analyses were performed to determine whether the expression of ∆133p53 mRNAs is associated with an increase of ∆133p53α protein level. The rabbit polyclonal antibody CM1, which was raised against full-length recombinant human p53 protein, has a strong affinity for p53, recognizing several epitopes in the N-terminal and C-terminal domains of p53. CM1 can thus detect all forms of the p53 protein, including ∆133p53α (Figures 4 and5). However, we would like to point out that it is not possible to compare the expression level of ∆133p53α with p53 protein by western blot analysis using the CM1 antibody, since the N-terminal domain of p53, which contains most of the CM1 epitopes, is not present in the ∆133p53α protein. Therefore, the CM1 antibody does not detect ∆133p53α as efficiently as the p53 protein.

We showed by western blot that doxorubicin treatment induces accumulation of the p53 protein and its isoforms in HCT116+/+, U2OS and NHDF cells but not in HCT116-/cells, devoid of FLp53 expression. To identify the band corresponding to the ∆133p53α isoform, we inhibited its expression with two distinct siRNAs (si133a and si133b), specific for the 5'UTR of the ∆133p53 mRNA variants. We determined that endogenous ∆133p53α protein corresponds to a band at 35 kD induced in response to DNA damage in HCT116+/+ and U2OS cancer cells, as well as in primary NHDF, indicating that the induction of the ∆133p53α protein is not specific for cancer cells. Of note, ectopic and endogenous ∆133p53α give a band at 35kD and a weaker band at 32kD, in U2OS cells.

We assume that the band at 32 kD corresponds to a post-translationally modified form of ∆133p53α and we are currently characterizing its origin.

We then investigated the biological relevance to ∆133p53α induction in response to DNA damage. We have recently published that ∆113p53, the zebrafish homologue of human ∆133p53α, prevents p53-mediated apoptosis in response to embryonic defects or DNA damage [START_REF] Chen | p53 isoform delta113p53 is a p53 target gene that antagonizes p53 apoptotic activity via BclxL activation in zebrafish[END_REF] and that human ∆133p53α can inhibit replicative senescence [START_REF] Fujita | p53 isoforms Delta133p53 and p53beta are endogenous regulators of replicative cellular senescence[END_REF].

Here, we investigated whether the cellular response to DNA damage could be regulated by modulating ∆133p53α expression. In this aim, we explored the experimental conditions inducing p53-mediated cell cycle arrest without promoting p53-mediated apoptosis, in response to doxorubicin treatment. We determined that treatment of U2OSctrl cells with a low dose of doxorubicin (0.5µM, 1h) induces exclusively p53-dependent G2 cell cycle arrest without promoting G1 cell cycle arrest or p53-dependent apoptosis.

Then, we established that the cellular response to the same dose of doxorubicin can be changed by regulating ∆133p53α expression. Indeed, U2OS-ctrl cells depleted of ∆133p53 mRNAs by si133a or si133b and treated with doxorubicin (0.5µM, 1h) increases the proportion of cells undergoing apoptosis and G1 cell cycle arrest.

Overexpression of ∆133p53α restores resistance to apoptosis and abolition of G1 cell cycle arrest without preventing p53-mediated G2 cell cycle arrest. The inhibition of p53mediated apoptosis and G1 cell cycle arrest in response to ∆133p53α expression is consistent with our previous reports showing that ∆133p53α can act in a dominant-negative manner toward p53 [START_REF] Bourdon | p53 isoforms can regulate p53 transcriptional activity[END_REF][START_REF] Murray-Zmijewski | p53/p63/p73 isoforms: an orchestra of isoforms to harmonise cell differentiation and response to stress[END_REF][START_REF] Fujita | p53 isoforms Delta133p53 and p53beta are endogenous regulators of replicative cellular senescence[END_REF]. The direct interaction of ∆133p53α with p53 is in support of this mechanism. However, as the overexpression of ∆133p53α did not inhibit p53-dependent G2 cell cycle arrest in response to doxorubicin treatment, it indicates that ∆133p53α does not exclusively act by inactivating p53. This is confirmed by the fact that p21, Bcl-2 and HDM2 genes are differentially regulated by ∆133p53α in response to DNA damage. ∆133p53α represses p21, while it induces Bcl-2 and HDM2 expression.

The repression of p21 is consistent with a dominant-negative effect of ∆133p53α toward p53. However, as ∆133p53α is required for HDM2 induction, it confirms that ∆133p53α does not exclusively act by inactivating p53. It suggests that many genes are differentially regulated by ∆133p53α to modulate cell fate outcome in response to DNA damage. Regulation of gene expression is complex and involves transcriptional and posttranscriptional pathways. Further experiments will be required to identify the genes regulated by ∆133p53α and decipher the role of ∆133p53α in the regulation of each of those genes. Our findings may have profound significance to extend our understanding of the mechanisms by which p53 exerts its tumor suppressor activity. They demonstrate a new feedback loop in which p53-induced ∆133p53α acts to modulate the cellular response to DNA damage.

Materials and Methods

p53 internal promoter constructs

The p53 internal promoter, corresponding to the region 11523-13076 bp of the human p53 gene (accession no. X54156, NCBI), was generated by PCR using the primers p53RE-a fwd and p53RE-a rev (Table -1). The PCR fragment was first cloned into the pCR ® 4-TOPO ® vector using the TOPO TA Cloning ® kit (Invitrogen) and was further sub-cloned into the pGL3-basic plasmid, upstream of the luciferase reporter gene, to generate the pi3i4-luc construct (construct a in Figure 1A). Subsequently, a number of deletion constructs of pi3i4-luc (b, c, d and e in Figure 1A) were made by enzymatic digestion using various restriction enzymes, followed by re-ligation and sub-cloning of the obtained fragments. Two other pi3i4-luc constructs (constructs c and d in Figure 2A), with or without p53REs, were generated by PCR using the primers' combination p53REb fwd/p53RE-a rev and p53RE-c fwd/p53RE-a rev for constructs b and c, respectively (Figure 2A andTable-1). Site-directed mutagenesis of the identified p53REs was carried out by PCR on the pi3i4-luc, using the primers Mut-p53RE fwd and Mut-p53RE rev (Table -1). All generated constructs were verified by DNA sequencing.

Dual luciferase assay

H1299 cells (3x10 4 ) were seeded in 24-well plates, 24 hours prior to transfection. Two hours before transfection, media was replaced with 0.5 ml of fresh complete DMEM.

Each transfection was performed in duplicate, and all results shown are the average of at least three separate individual experiments. For each well, 0.5 µg of each p53 internal promoter construct (as indicated in the legends) and 50 ng of the Renilla luciferase reporter plasmid were co-transfected with 200 ng of the FLp53 expression vector or with the pSI (empty expression vector), used as negative control. The Renilla luciferase reporter plasmid was used as an internal control. Luciferase assays were performed as previously described [START_REF] Bourdon | Scotin, a novel p53-inducible proapoptotic protein located in the ER and the nuclear membrane[END_REF].

Cell culture and generation of stable cell lines

The different human cell lines used in our study were: the non small lung carcinoma cell line H1229 (p53-null), the osteosarcoma cell line U2OS (WTp53), The colon cell lines HCT116 (WTp53), the breast cancer cell line MCF7 (WTp53) and the primary normal human dermal fibroblasts NHDF. The MCF7 stable line overexpressing a dominant negative form of p53 (MCF7-DDp53) [START_REF] Shaulian | Transcriptional repression by the Cterminal domain of p53[END_REF] was also used. The HCT116 -/-, devoid of FLp53 expression, were kindly provided by Pr. Vogelstein's lab [START_REF] Bunz | Requirement for p53 and p21 to sustain G2 arrest after DNA damage[END_REF]. Two U2OS stable cell lines were generated after transfection with either the empty pcDNA-3 expression vector (U2OS-ctrl) or with the pcDNA3-∆133p53 expression vector (U2OS-∆133p53).

The heterogeneous populations of both cells lines were used in the experiments, no clones were isolated. NHDF cells were kindly provided by Dr C. Pourreyron. H1299 cells were cultured in RPMI medium supplemented with 10% fetal calf serum and 100 µg/ml penicillin/streptomycin. All the other cell lines were cultured in DMEM medium supplemented with 10% fetal calf serum and 100 µg/ml penicillin/streptomycin.

RNA extraction, cDNA synthesis, RT-qPCR and Chromatin Immunoprecipitation (ChIP) Assay

Total RNA was extracted with the RNeasy Mini Kit (Qiagen) and treated with DNase (Qiagen) prior to reverse transcription, which was carried out using random hexamers (Promega) and Superscript II reverse transcriptase (Invitrogen). qPCR was performed on Stratagene MX3005P. p53 specific primers and probes were designed in our laboratory (Table -1) and purchased from MWG. RT-qPCR analyses for the p21, Bax, HDM2 and Bcl2 genes were performed as previously described (31,[START_REF] Stevenson | The deubiquitinating enzyme USP2a regulates the p53 pathway by targeting Mdm2[END_REF]. All measurements were normalized to the expression of the TATA box-binding protein (TBP) gene. p53 specific ChIP assay was conducted on MCF7 cells, as previously described using anti-p53 monoclonal antibody, DO1 [START_REF] Bourdon | p53 isoforms can regulate p53 transcriptional activity[END_REF] .

Western blot analysis

Cells lysates were separated by 4-12% SDS PAGE (Novagen system), transferred to a nitrocellulose membrane and blotted with various antibodies. The primary antibodies used were the following: anti-p53 CM1 [START_REF] Midgley | Analysis of p53 expression in human tumours: an antibody raised against human p53 expressed in Escherichia coli[END_REF], anti-HDM2 4B2 (31), p21 (Calbiochem), PARP (Roche), Bax, Bcl2 and actin (Santa Cruz). All secondary Horseradish peroxidase (HRP) conjugated antibodies were purchased from Jackson ImmunoResearch Laboratories.

Immunoprecipitation assay H1299 (10 6 cells/10-cm dish) were co-transfected with 2µg pSV-p53 expression vector driven by the SV40 promoter and/or 2µg pSV-∆133p53 expression vector driven by the SV40 promoter. For each transfection mix, the concentration of the SV40 promoter was balanced with the SV40 empty expression vector. Fugene (Boehringer) was used as a transfection reagent. Immunoprecipitation was carried out as previously described [START_REF] Bourdon | p53 isoforms can regulate p53 transcriptional activity[END_REF] with anti-p53 mouse monoclonal antibody DO-1.

Protein content was then analyzed by western blot with anti-p53 rabbit polyclonal antibody (CM1), as previously described [START_REF] Bourdon | p53 isoforms can regulate p53 transcriptional activity[END_REF].

Small interfering RNA (siRNA) transfection, Apoptosis and Cell cycle analyses U2OS (10 5 cells/6-well plate) were transfected with the appropriate siRNA using oligofectamine (Invitrogen), treated with doxorubicin and further incubated for 24h before harvesting for apoptosis and cell cycle assays. The efficiency of si133a, si133b and siTA was assessed by RT-qPCR (Supplementary Figures C1 andC2). Apoptotic cells were quantified using an Annexin V-fluorescein isothiocyanate kit (Biosciences), according to the manufacturer's instructions and were analyzed by flow cytometry (Becton Dickinson). Cell cycle analysis was carried out using BrdU (bromodeoxyuridine, Sigma) pulse labeling and flow cytometry, as previously described [START_REF] Hoy | Bromodeoxyuridine/DNA analysis of replication in CHO cells after exposure to UV light[END_REF] and according to the manufacturer's instructions.

Titles and legends to figures

  , a), suggesting that the region 1-1304 bp is likely to contain negative regulatory elements such as silencers. In fact, the basal promoter activity of the internal p53 promoter (1304-1555, construct f) is increased (3-fold) compared to the basal promoter activity of the internal p53 promoter (1042-1555, construct d), suggesting that the region 1042-1304 contains a silencer element. Bioinformatics analysis revealed that the p53 internal promoter contains responsive elements to several transcription factors including p53, AP1, elk-1, ets, N-myc, E2F, HIF-1α, HSF1, ER, raising the possibility that it can be regulated by p53 and other transcription factors (Supplementary Figure A). Deletion of the last 251 bp from the 3' end of intron-4 (Figure 1A, constructs b and e) completely abolishes the promoter activity (Figure 1B, b and e), confirming that the initiation site of the p53 internal promoter is located within the last 250 bp of intron-4 of the human p53 gene.

  lane 5 compared to 1). The two ∆133p53 siRNAs, si133a and si133b, did not alter the level of endogenous p53 or of the other p53 isoforms (corresponding to the bands at 48 kD, 45 kD) but specifically reduced the expression of the 35 kD band (Figure4B, lanes 4, 6 compared to 5), indicating that the 35kD band corresponds to endogenous ∆133p53α protein.

  Figure D and Figure 6B lane 5 compared to 2). This confirms that in U2OS-ctrl cells,

Figure 1 .

 1 Figure 1. Mapping of the promoter activity within intron-4 of the p53 gene. (A) Diagram

Figure 2 .

 2 Figure 2. Identification and characterization of five p53REs within the p53 internal

Figure 3 .

 3 Figure 3. Human p53 internal promoter is directly transactivated by p53. (A) ChIP assay

Acknowledgements

Mustapha Aoubala is supported by Breast Cancer Campaign, Fiona Murray-Zmijewski was supported by Breast Cancer Campaign and MRC. Marie P. Khoury and Stephane Perrier are supported by Inserm (Institut National de la Santé Et de la Recherche Médicale), Hugo Bernard is supported by Ligue Nationale Contre le Cancer. Kenneth Fernandes and Jean-Christophe Bourdon are supported by Cancer-Research UK. The authors wish to thank Drs Mark Saville and Alice Marchado-Silva for their constructive discussions.

All results shown are the average of at least three separate individual experiments.

Table 1. PCR primers used for cloning, ChIP and RT-qPCR assays.

Primers for cloning and mutagenesis of the p53 internal promoter p53RE-a fwd: 5' ACAGGAGGTGGGAGCAGGGCACGGTA 3' p53RE-a rev: 5' GTTGGCAAAACATCTTGTTGAGGGC 3' p53RE-b fwd: 5'AAAACCTACCAGGGCAGCTACGGT 3' p53RE-c fwd: 5' GGAACTTTGGGATTCCTCTTCACCCTTA 3'

 (B-E) Quantification of endogenous p53 internal promoter activity by RT-qPCR in human cancer cells (MCF7, MCF7-DDp53, HCT116-/-, HCT116+/+ and U2OS) or normal human primary fibroblasts (NHDF). (B) MCF7 (WTp53) and MCF7-DDp53 (mutant p53) cells were left untreated or treated for 30min, 1h or 2h with 60 ng/ml of Act (A) HCT116+/+ (p53WT) and HCT116-/-(devoid of FLp53 expression) cells were treated with 0.5µM doxorubicin for 1h and proteins were extracted 24h after treatment in SDS-Laemmli buffer. Western blot analysis was performed using the CM1 rabbit polyclonal antibody which recognizes all p53 isoforms. (B) HCT116+/+ transfected for 24h with 50nM of either si133a, si133b (two distinct siRNAs specific for the 5'UTR of ∆133p53 mRNAs) or siNS (non-specific siRNA used as a negative control), were left untreated (-) or treated (+) for 1h with 0.5µM of doxorubicin, as indicated. Proteins were extracted 24h after treatment and analyzed by western blotting. p53 and its isoforms were revealed with the CM1 antibody. Actin was used as a protein loading control. (C) Western blot analysis of endogenous ∆133p53α expression in untreated (-) or doxorubicin-treated (+) NHDF cells. p53 and its isoforms were revealed with the CM1 antibody. Tubulin was used as a protein loading control. Extracted proteins were immunoprecipitated with the mouse monoclonal antibody DO-1 or with a non-relevant mouse monoclonal IgG antibody, as indicated. DO-1 is specific for FLp53 and does not bind ∆133p53α. Protein content was then analyzed by western blot analysis using the rabbit polyclonal antibody (CM1), recognizing FLp53 and ∆133p53α. 10% of the input was loaded as a control. (B) Western blot analysis of p53 target genes (p21, HDM2 and Bcl-2). In parallel to the cell cycle analysis and apoptosis assay, protein extracts from U2OS-ctrl cells treated as described in Figure 5B, were analyzed by western blotting using the corresponding antibodies. Actin was used as a protein loading control.

Titles and legends to supplementary figures

Supplementary

Supplementary Figure B. p53 directly binds and transactivates the p21 promoter. (A1)

ChIP assay of p21 promoter DNA was carried out by RT-qPCR in MCF7 cells that were left untreated or treated for 2h or 6h with 60 ng/ml of actinomycin D (Act D), using primers encompassing the p53REs. The amounts of the p21 promoter contained in the input or immunoprecipitated with the DO-1 antibody, were quantified by RT-qPCR, as previously described [START_REF] Kaeser | Chromatin immunoprecipitation analysis fails to support the latency model for regulation of p53 DNA binding activity in vivo[END_REF]. The results are expressed as a percentage of promoter specifically immunoprecipitated by the DO-1 antibody, compared to the total amount of promoter contained in 10% of the input. The results shown are the average of three In parallel to the cell cycle analysis and apoptosis assay, RNA samples from U2OS-ctrl cells treated as described in Figure 5B, were analyzed by RT-qPCR (Taqman).


Dr Carsten Culmsee

Svenja Tobaben

Julia Grohm

Alexander Seiler

Marcus Conrad

Nikolaus Plesnila

Bid-mediated mitochondrial damage is a key mechanism in glutamate-induced oxidative stress and AIF-dependent cell death in immortalized HT-22 hippocampal neurons

Keywords: glutamate, neuronal cell death, apoptosis, mitochondria, lipid peroxidation, reactive oxygen species, apoptosis inducing factor 2

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Glutamate toxicity is a well established cause for neuronal dysfunction and cell death in many acute and chronic neurological diseases. For example, increases in extracellular glutamate levels after acute brain damage by ischemic stroke, epilepsy or brain trauma may reach millimolar concentrations and induce massive Ca 2+ influx and excitotoxic damage through activation of glutamate receptors such as NMDA receptors or AMPA/Kainate receptors. [START_REF] Culmsee | Emerging pharmacotherapeutic strategies for the treatment of ischemic stroke[END_REF][START_REF] Lo | Mechanisms, challenges and opportunities in stroke[END_REF] The initial increase in intracellular Ca 2+ levels after stimulation of these glutamate receptors is rather short and the following molecular mechanisms of glutamate excitotoxicity in neurons are poorly defined.

While inhibition of the glutamate-induced Ca 2+ influx by NMDA-receptor antagonists or antagonists of AMPA/Kainate receptors protected neurons from glutamate excitotoxicity in experimental settings, the therapeutic time window of such neuroprotective effects is limited in vivo, and (post-)treatment strategies with glutamate receptor antagonists have failed in clinical studies to date. [START_REF] Culmsee | Combination therapy in ischemic stroke: synergistic neuroprotective effects of memantine and clenbuterol[END_REF][START_REF] Fisher | An overview of acute stroke therapy: past, present, and future[END_REF] Therefore, understanding the mechanisms of glutamate toxicity beyond the initial stimulation of Ca 2+ influx is of utter importance to provide efficient strategies of neuroprotection by targeting sustained mechanisms of glutamate-induced neuronal cell death. Such mechanisms include, for example, increased formation of reactive oxygen species (ROS), the activation of apoptosis-related death signaling, mitochondrial damage and DNA degradation.

In particular, oxidative stress has been considered to cause neuronal dysfunction and cell death triggered by glutamate after acute brain injury and in age-related chronic neurodegenerative diseases. Therefore, recent research has focused on better understanding ROS formation and dissecting ROS-triggered neuronal death signaling pathways in order to identify novel therapeutic strategies against glutamate neurotoxicity. Under physiological conditions, cellular ROS levels are tightly controlled by low molecular radical scavengers and by a complex intracellular network of enzymes, such as catalases, superoxide dismutases Cu/Zn-SOD and Mn-SOD (SOD-2), and enzymes of the glutathione (GSH)-and thioredoxin-dependent families. [START_REF] Matsuda | Alterations of oxidative stress markers and apoptosis markers in the striatum after transient focal cerebral ischemia in rats[END_REF][START_REF] Murphy | Immature cortical neurons are uniquely sensitive to glutamate toxicity by inhibition of cystine uptake[END_REF] Under conditions of lethal stress associated with glutamate toxicity, i.e. acute or chronic brain injury, these endogenous defense systems fail to detoxify increasing ROS levels and the cell is determined for oxidative death. Prominent sources of physiological intracellular ROS formation that may further be stimulated under conditions of cellular stress are, for example, lipoxygenases (LOX), cyclooxygenases (COX), NADPH-oxidases (NOX) and the uncoupled mitochondrial respiratory chain. [START_REF] Brennan | NADPH oxidase is the primary source of superoxide induced by NMDA receptor activation[END_REF][START_REF] Nagley | Multifaceted deaths orchestrated by mitochondria in neurones[END_REF] After exposure to glutamate, dysfunctional mitochondria may generate toxic amounts of intracellular ROS, which may further severely perturb cellular redox balance. [START_REF] Liot | Complex II inhibition by 3-NP causes mitochondrial fragmentation and neuronal cell death via an[END_REF] In addition, these organelles host pro-apoptotic proteins, e.g., AIF, cytochrome c or SMAC/DIABLO that may trigger caspase-dependent or caspase-independent death when released into the cytosol. Increasing evidence suggests a key role for mitochondrial AIF and related caspase-independent death in glutamate-induced neuronal death and in the injured adult brain. [START_REF] Culmsee | Apoptosisinducing factor triggered by poly(ADP-ribose) polymerase and Bid mediates neuronal cell death after oxygen-glucose deprivation and focal cerebral ischemia[END_REF][START_REF] Oppenheim | Developing postmitotic mammalian neurons in vivo lacking Apaf-1 undergo programmed cell death by a caspase-independent, nonapoptotic pathway involving autophagy[END_REF][START_REF] Plesnila | Nuclear translocation of apoptosis-inducing factor after focal cerebral ischemia[END_REF] In contrast to cytochrome cmediated caspase-dependent cell death, AIF translocates to the nucleus and induces nuclear condensation, DNA fragmentation and cell death immediately after release from mitochondria. [START_REF] Landshamer | Bidinduced release of AIF from mitochondria causes immediate neuronal cell death[END_REF] Neuronal HT-22 cells have been established as a suitable model system [START_REF] Li | A role for 12-lipoxygenase in nerve cell death caused by glutathione depletion[END_REF] to investigate glutamate-induced death signaling pathways that enhance ROS formation and lethal oxidative stress independent of NMDA receptor stimulation, as these neuronal cells lack glutamate receptor expression and are thus not susceptible to glutamate-induced rapid calcium influx. Glutamate-induced death signaling in these cells involves glutathione depletion and enhanced accumulation of ROS. [START_REF] Murphy | Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress[END_REF] Our recent study identified glutathione peroxidase 4 (GPx4) as an important mediator of oxidative stress-induced cell death pathway after glutathione depletion. [START_REF] Seiler | Glutathione peroxidase 4 senses and translates oxidative stress into 12/15-lipoxygenase dependent-and AIF-mediated cell death[END_REF] Experimental GSH depletion or GPx4 disruption caused 12/15-lipoxygenase (12/15-LOX)-dependent lipid peroxidation, nuclear AIF translocation, and cell death in fibroblasts. In the present study, we sought to address whether glutamate-induced cell death in HT-22 cells occurs through LOX-dependent lipid peroxidation and mitochondrial death signaling. Our results expose Bid as a key link between glutamate-induced activation of LOX and mitochondrial pathways of programmed cell death.

Results

Glutamate-induced oxidative stress in HT-22 cells involves lipid peroxidation by 12/15-lipoxygenase

Glutamate induces death in HT-22 immortalized hippocampal cells in a dose-and time dependent manner (Figure 1a,b). Real time recording of cellular impedance by the xCELLigence system and MTT assays revealed that cell death occurred 8-10 h after onset of exposure to 3 mM and 5 mM glutamate, and according to these measurements execution of cell death was concluded within 2 to 4 h, respectively.

As detected by the fluorescent dye BODIPY and FACS analyses glutamate-induced cell death was accompanied by an accumulation of lipid peroxides. Notably, lipid peroxidation appeared moderate, i.e. up to twofold, within 6-8 h of glutamate exposure, followed by a more pronounced, secondary increase within 8-18 h after glutamate treatment (Figure 1c). Measurements with DCF confirmed a nearly twofold increase in ROS formation 6 to 8 h after glutamate treatment followed by a secondary boost of ROS formation at 18 h after the glutamate challenge (Figure 1d).

To identify the source of lipid peroxidation, we tested the effect of LOX-inhibitors in our current model system. As illustrated in Figure 2a, the LOX-inhibitors PD146176 and AA861 prevented the first glutamate-induced increase in lipid peroxidation after 6 to 8 h. In addition, LOX inhibition significantly attenuated the boost of lipid peroxides detected at 17 h after onset of glutamate treatment (Figure 2b). For these experiments, two different glutamate concentrations were applied to demonstrate the effects of LOX inhibitors on glutamate-induced lipid peroxidation. For induction of substantial amounts of lipid peroxides at early time points, i.e. within 8 h after onset of glutamate treatment, we applied 3 mM glutamate (Figure 2a) , whereas 2 mM glutamate was used for the long term endpoint (17 h, Figure 2b), because at glutamate concentrations higher than 3 mM the cells were severely damaged at the later time point and could not be used for subsequent BODIPY loading and FACS analyses.

The LOX-inhibitor PD146176 significantly prevented glutamate-induced cell death in a concentration dependent manner. Notably, PD146176 fully protected HT-22 cells against glutamate toxicity at a concentration of 0.5 µM (Figure 3a,c) and significantly reduced the annexin-V/propidium iodide positive cells (Figure 3b). Comparable results were obtained by using the LOX inhibitor AA861 (Supplementary Figure 1), and the anti-oxidants N-acetyl cysteine and Trolox (Supplementary Figure 2 a,b). In contrast, the COX-inhibitor indomethacin (5 to 100 µM) failed to protect the cells against glutamate toxicity (Figure 3d). These findings demonstrate that LOX but not COX plays a major role in oxidative stress-induced cell death after glutamate exposure in HT-22 cells. In primary neurons, it has been suggested that glutamate-induced excitotoxicity involved both, disruption of the intracellular Ca 2+ homeostasis and increased oxidative stress. Here, we investigated the effect of PD146176 in glutamate-induced cell death in primary cortical neurons.

The 12/15-LOX inhibitor PD146176 significantly reduced cell death in primary neurons (Figure 3e). Further, primary neuronal cultures from Alox15-mice (15-LOX knock-out mice) were significantly protected from glutamate toxicity compared to wild type cultures (Figure 3f). These results strongly suggest that activation of 12/15-LOX plays an important role in both model systems of glutamate toxicity, i.e. in glutamate-induced oxytosis in HT-22 cells and in excitotoxic cell death in primary neurons.

We next determined the protective time window of LOX-dependent lethal oxidative stress in neuronal cell death and added the 12/15-LOX-inhibitor PD146176 at different time points between 2 h and 15 h after onset of the glutamate treatment. HT-22 cells were protected against glutamate toxicity even when PD146176 was added up to 8 h after the glutamate challenge (Figure 4a) indicating that beyond that time point glutamate-induced cell death proceeded too far for protection by LOX inhibition.

Similar results were obtained with Trolox, which also protected the cells against glutamate toxicity with a similar post-treatment time window of 8 h (Figure 4b).

12/15-LOX inhibitor PD146176 protects against glutamate-induced mitochondrial fission and prevents AIF translocation to the nucleus

Glutamate-induced cell death in neurons involves mitochondrial damage and release of pro-apoptotic mitochondrial proteins such as AIF. [START_REF] Culmsee | Apoptosisinducing factor triggered by poly(ADP-ribose) polymerase and Bid mediates neuronal cell death after oxygen-glucose deprivation and focal cerebral ischemia[END_REF][START_REF] Landshamer | Bidinduced release of AIF from mitochondria causes immediate neuronal cell death[END_REF] Further, the time pattern of the increase in lethal oxidative stress after glutamate exposure indicated LOXdependent induction of cell death signaling upstream of mitochondria. Therefore, we next investigated whether 12/15-LOX inhibition was also sufficient to prevent the hallmarks of mitochondrial damage such as mitochondrial fission and translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus. After glutamate treatment, most cells showed significant fragmentation of the mitochondria appearing as small round organelles in the damaged cells in contrast to long tubular mitochondria observed under control conditions (Figure 5a,b). The 12/15-LOX inhibitor PD146176 preserved the mitochondrial morphology despite glutamate exposure and in cells co-treated with the inhibitor the mitochondria appeared as a network of long tubular organelles similar to controls (Figure 5a). Since these data implied preserved mitochondrial integrity, we next explored the effect of PD146176 on AIF release from mitochondria after the glutamate challenge. AIF translocation has been identified as the final step of caspase-independent mitochondrial death signaling in neurons. [START_REF] Culmsee | Apoptosisinducing factor triggered by poly(ADP-ribose) polymerase and Bid mediates neuronal cell death after oxygen-glucose deprivation and focal cerebral ischemia[END_REF][START_REF] Landshamer | Bidinduced release of AIF from mitochondria causes immediate neuronal cell death[END_REF] In line with these earlier findings, HT-22 cells immunostained for AIF clearly showed AIF relocation and cell death at 18 h after the glutamate challenge. 12/15-LOX inhibition by PD146176 prevented AIF translocation to the nucleus and preserved cell morphology (Figure 5c). These findings were confirmed by Western Blot analysis as AIF was upregulated 33 % over control level following glutamate treatment, whereas PD146176 could avert glutamate induced AIF relocation (Figure 5d). These data further support a role for 12/15-LOX activity upstream of mitochondrial release of AIF to the nucleus.

The small molecule Bid-inhibitor BI-6C9 attenuates glutamate-induced oxidative stress

Recently, we reported a major role for the pro-apoptotic BH3-only protein Bid in mitochondrial demise upstream of AIF-mediated execution of caspase-independent death in neuronal cells exposed to glutamate. [START_REF] Culmsee | Apoptosisinducing factor triggered by poly(ADP-ribose) polymerase and Bid mediates neuronal cell death after oxygen-glucose deprivation and focal cerebral ischemia[END_REF][START_REF] Landshamer | Bidinduced release of AIF from mitochondria causes immediate neuronal cell death[END_REF] Here, we further investigated whether pro-apoptotic activation of Bid occurred upstream of LOX-dependent lipid peroxidation and related cell death mechanisms. Measuring lipid peroxidation by BODIPY staining and FACS analysis revealed that Bid inhibition did not affect early increases in lipid peroxidation within 6 to 8 h after glutamate treatment, thus excluding direct radical scavenger activities of the Bid inhibitor (Figure 6a). Notably, BI-6C9 significantly reduced the pronounced accumulation of lipid peroxides that was associated with cell death in the vehicle-treated controls at 17 h after glutamate exposure (Figure 6b), and the Bid inhibitor also significantly protected HT-22 cells against the glutamate challenge up to control levels confirming the important role of Bid in glutamate-induced neuronal cell death (Figure 6c). These data indicate that the initial increase in LOX-dependent lipid peroxidation at 6-8 h occurs upstream of Bid activation and is not affected by the Bid inhibitor. However, Bid activation seems to be an important link between the primary increase in lipid peroxides and the secondary boost of lipid peroxides which marks the "point of no return" that includes fatal mitochondrial damage and AIF-dependent execution of cell death. Notably, we could not detect Bid cleavage in the HT-22 cells indicating that either only a very small amount of Bid was cleaved or a different way of Bid activation that involved mitochondrial translocation of full length Bid independent of caspase-8 cleavage as recently described (Figure 6d). [START_REF] Landshamer | Bidinduced release of AIF from mitochondria causes immediate neuronal cell death[END_REF][START_REF] Konig | Full length Bid is sufficient to induce apoptosis of cultured rat hippocampal neurons[END_REF] This role for Bid as an essential link between initial oxidative stress mediated by enhanced LOX activity and the secondary boost of lipid peroxidation associated with Bid-induced demise of mitochondrial function was further addressed using a tBid expression vector. The transfection of HT-22 cells with tBid induced significant cell death compared to cells transfected with the control plasmid (Figure 7a). In contrast to BI-6C9, both LOX inhibitors PD146176 and AA861 failed to protect the cells against tBid toxicity. It is important to note that over-expression of tBid may only partly reflect the mechanism of Bid-mediated death signaling, since Bid cleavage could not be detected in HT-22 cells after the glutamate challenge. However, BI-6C9 prevented both, translocation of full length Bid to mitochondria and tBid-mediated AIF release and cell death, suggesting that this model system is appropriate to mimic mechanisms mediating Bid neurotoxicity. [START_REF] Landshamer | Bidinduced release of AIF from mitochondria causes immediate neuronal cell death[END_REF] In addition, the vitamin E analogue Trolox, an established antioxidant acting on lipid peroxides could not prevent tBid-induced cell death, while glutamate-induced cell death was entirely abrogated (Figure 7b). These data strongly suggest that lipid peroxidation occurs upstream of Bid activation, whereas tBidinduced mitochondrial demise and the associated execution of cell death cannot be attenuated by radical scavengers or LOX inhibitors.

Discussion

In the present study we identify an important role for Bid acting as a key link between early ROS formation by 12/15-LOX and downstream mitochondrial damage and release of mitochondrial AIF to the nucleus that executes cell death after glutamate challenge in neuronal cells. In this model system of HT-22 cells glutamate induces lethal oxidative stress independently of glutamate receptor-mediated calcium influx by inhibition of cystine import through system xc-inhibition [START_REF] Bannai | Exchange of cystine and glutamate across plasma membrane of human fibroblasts[END_REF] and subsequent depletion of intracellular glutathione levels. [START_REF] Murphy | Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress[END_REF] Glutathione depletion leads to enhanced lipid peroxidation and eventually cell death mediated by the activation of LOX but not by cyclooxygenases. Accordingly, only the 12/15-LOX-inhibitors but not the general COX inhibitor indomethacin rescued HT22 cells from glutamate-induced cell death. In a recent study we identified the functional loss of glutathione peroxidase 4 (Gpx4) as the underlying mechanism that links reduced glutathione levels to increases in lipid peroxidation by 12/15-LOX. [START_REF] Seiler | Glutathione peroxidase 4 senses and translates oxidative stress into 12/15-lipoxygenase dependent-and AIF-mediated cell death[END_REF] In an inducible Gpx4 knockout fibroblast system we unraveled that loss of GPx4 sparks 12/15-LOX-derived lipid peroxidation and subsequent execution of caspase-independent cell death by mitochondrial release of AIF. Here, we show that glutamate-induced glutathione deprivation in neuronal cells triggers LOX-dependent lipid peroxidation that contributes to essential steps of glutamate-induced cell death. The 12/15-LOX inhibitors prevented the initial moderate rise in lipid peroxidation and consequently protected cells from further ROS-mediated cell death signaling triggered by glutamate. The central role for increased lipid peroxide generation in glutamate-mediated neuronal cell death was further substantiated by using the radical scavengers Trolox and NAC which efficiently protected the HT-22 cells when applied within a time window of 8 h. Both, reduced glutathione levels and increased ROS formation, are established mechanisms that contribute to neuronal death in models of chronic and acute neurodegeneration. [START_REF] De | Role of extracellular signal-regulated protein kinase in neuronal cell death induced by glutathione depletion in neuron/glia mesencephalic cultures[END_REF][START_REF] Khanna | Neuroprotective properties of the natural vitamin E alpha-tocotrienol[END_REF][START_REF] Lin | Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases[END_REF] Thus, the present finding in HT-22 cells showing a prominent role of 12/15-LOX for ROS formation in glutamate-induced cell death are relevant for mechanisms underlying neuronal injury and death in neurodegenerative diseases and after acute brain damage, such as trauma and stroke, where extracellular glutamate levels significantly increase after the respective injury. In addition, experiments in primary neurons also confirmed a major role for LOX-dependent mechanisms in glutamate-induced neuronal cell death in the presence of glutamate receptor ion channels. In these primary cells the LOX inhibitor PD146176 did not prevent the initial short increase in [Ca 2+ ] i after glutamate exposure, but significantly attenuated the secondary sustained increase in intracellular calcium levels and cell death (data not shown). These findings support the conclusion that the LOX inhibitors interfered with delayed downstream execution mechanisms of glutamate neurotoxicity, such as ROS formation, sustained increases in [Ca 2+ ] i and mitochondrial damage. Thus, lipoxygenases are promising targets for therapeutic strategies against glutamate-mediated death signaling that may occur in the presence or absence of glutamate receptor ion channels. It is important to note that NOX inhibition also protected against cell death in the present model system, suggesting that NOX also contributes to glutamate neurotoxicity (Supplementary Figure 3). This finding supplements recent reports indicating that NOX activation links NMDA-receptor mediated increases in [Ca] i levels and ROS formation. [START_REF] Brennan | NADPH oxidase is the primary source of superoxide induced by NMDA receptor activation[END_REF] Our data now suggest that NOX activity is also involved in glutamate-induced ROS formation and cell death in the absence of NMDA-receptors and independent of increased

[Ca] i .
It is interesting to note that ROS formation after glutamate exposure of HT-22 cells apparently occurred in a biphasic manner. We observed initial moderate increases in lipid peroxidation within 6-8 h followed by a boost of lipid peroxides detectable up to 18 h after glutamate administration. While the 12/15-LOX inhibitors fully abrogated the lipid peroxide accumulation, pharmacological inhibition of the pro-apoptotic BH3only death protein Bid only impeded sustained increase in lipid peroxides but did not interfere with the initial ROS formation within the first 8 h. In contrast to recent data suggesting a potential role of LOX alone for the release of cytochrome C in isolated mitochondria, [START_REF] Pallast | 12/15-Lipoxygenase targets neuronal mitochondria under oxidative stress[END_REF] our present findings in intact cells reveal a crucial role for Bid that is required for mitochondrial execution of cell death after the initial formation of lipid peroxides. Bid has been revealed as an important key mediator of cell death in different paradigms of neurodegeneration, including model systems of oxidative stress and excitotoxicity in vitro, and cerebral ischemia and brain trauma in vivo. [START_REF] Culmsee | Molecular insights into mechanisms of the cell death program: role in the progression of neurodegenerative disorders[END_REF] Accordingly, Bid knockout mice developed significantly reduced brain damage after cerebral ischemia [START_REF] Plesnila | BID mediates neuronal cell death after oxygen/ glucose deprivation and focal cerebral ischemia[END_REF][START_REF] Yin | Bid-mediated mitochondrial pathway is critical to ischemic neuronal apoptosis and focal cerebral ischemia[END_REF] and brain trauma. [START_REF] Bermpohl | Traumatic brain injury in mice deficient in Bid: effects on histopathology and functional outcome[END_REF] Similar neuroprotective results were obtained in cultured neurons from Bid-deficient mice when exposed to oxygen glucose deprivation (OGD). [START_REF] Plesnila | BID mediates neuronal cell death after oxygen/ glucose deprivation and focal cerebral ischemia[END_REF] Further, small molecule inhibitors of Bid provided protective effects against glutamate-induced excitotoxicity or OGD in cultured primary neurons [START_REF] Culmsee | Apoptosisinducing factor triggered by poly(ADP-ribose) polymerase and Bid mediates neuronal cell death after oxygen-glucose deprivation and focal cerebral ischemia[END_REF][START_REF] Becattini | Structure-activity relationships by interligand NOE-based design and synthesis of antiapoptotic compounds targeting Bid[END_REF] and prevented mitochondrial demise, AIF release and cell death in HT-22 cells exposed to oxidative stress induced by glutamate or amyloid-beta peptide. [START_REF] Landshamer | Bidinduced release of AIF from mitochondria causes immediate neuronal cell death[END_REF] These different paradigms of lethal stress induce activation of Bid which translocates to the mitochondria where it mediates mitochondrial membrane permeabilization and release of death proteins such as cytochrome c or AIF. [START_REF] Konig | Full length Bid is sufficient to induce apoptosis of cultured rat hippocampal neurons[END_REF][START_REF] Culmsee | Targeting Bid to prevent programmed cell death in neurons[END_REF] Therefore, Bid activation is a common feature of death signaling that can significantly amplify deadly stress signals through involvement of mitochondrial mechanisms in the execution of cell death.

Indeed, the proposed timing of transition from moderate to severe oxidative stress fits well with the timing of Bid translocation to mitochondria and indicators of mitochondrial damage such as loss of mitochondrial membrane potential and subsequent release of AIF as determined in our previous work. [START_REF] Landshamer | Bidinduced release of AIF from mitochondria causes immediate neuronal cell death[END_REF] These findings suggest a transition phase where Bid acts as a crucial link between the 12/15-LOXdependent initial increases in lipid peroxidation and the following mitochondrial damage. This conclusion is supported by our previous studies where the small molecule Bid inhibitor BI-6C9 or Bid siRNA prevented mitochondrial damage, AIF translocation and cell death in neurons. [START_REF] Landshamer | Bidinduced release of AIF from mitochondria causes immediate neuronal cell death[END_REF] Moreover, the therapeutic time window of 8-10 h identified in our previous study for the Bid inhibitor is in accordance with the 'point of no return' and the associated secondary boost of oxidative stress revealed in the present study. Here, the LOX inhibitor PD146176 showed a similar therapeutic time window of approximately 8 h after onset of the glutamate challenge. This supports the view that accumulating oxidative stress leads to Bid-mediated mitochondrial damage which marks the execution phase of cell death that cannot be blocked by LOX inhibitors or radical scavengers targeting the initiation phase.

The essential role for Bid mediating mitochondrial dysfunction and cell death downstream of 12/15-LOX activation in the present model of oxidative stress was further confirmed using tBid expression constructs that induce cell death by immediate mitochondrial translocation of tBid and subsequent induction of mitochondrial damage. [START_REF] Culmsee | Targeting Bid to prevent programmed cell death in neurons[END_REF][START_REF] Kazhdan | Targeted gene therapy for breast cancer with truncated Bid[END_REF] In this paradigm, only the Bid inhibitor, [START_REF] Landshamer | Bidinduced release of AIF from mitochondria causes immediate neuronal cell death[END_REF] but neither the 12/15-LOX inhibitors nor the radical scavenger Trolox could prevent tBid toxicity. These data strongly suggest that activation of 12/15-LOX and formation of ROS initiated cell death mechanisms after glutamate treatment, whereas activation of Bid, mitochondrial damage and the boost of ROS are hallmarks of downstream mechanisms that cannot be blocked by 12/15-LOX inhibitors or radical scavengers.

Both, mitochondrial translocation of full length Bid after the glutamate challenge and over-expression of tBid exerted similar effects on mitochondria

and AIF-dependent cell death in the applied model system of HT-22 cells. [START_REF] Landshamer | Bidinduced release of AIF from mitochondria causes immediate neuronal cell death[END_REF] Here, we could not detect Bid cleavage after exposure to glutamate, suggesting that full length Bid translocated to the mitochondria and/or only a small part of Bid was cleaved to tBid. This is in line with our previous observations [START_REF] Landshamer | Bidinduced release of AIF from mitochondria causes immediate neuronal cell death[END_REF] and reports by others [START_REF] Konig | Full length Bid is sufficient to induce apoptosis of cultured rat hippocampal neurons[END_REF] A role for 12/15-LOX as an upstream trigger of mitochondrial execution mechanisms of cell death was further supported by analysis of mitochondrial morphology and AIF translocation after the glutamate challenge. Mitochondria are dynamic organelles that undergo permanent fission and fusion under physiological conditions. In damaged neurons, the disturbance of this dynamic process may lead to excessive fragmentation of mitochondria thereby promoting cell death progression. Although the mechanisms controlling mitochondrial morphology under pathological conditions are only partly known, increasing evidence suggests a potential role for oxidative stress and impaired bioenergetics as potential triggers of mitochondrial fission in the cell death program. [START_REF] Cho | S-nitrosylation of Drp1 mediates beta-amyloid-related mitochondrial fission and neuronal injury[END_REF][START_REF] Knott | Mitochondrial fragmentation in neurodegeneration[END_REF][START_REF] Yuan | Mitochondrial fission is an upstream and required event for bax foci formation in response to nitric oxide in cortical neurons[END_REF] Here, we demonstrate that glutamate-induced cell death is associated with mitochondrial fission. Inhibition of 12/15-LOX prevented such glutamate-induced disruption of the mitochondrial morphology and also blocked the mitochondrial release of AIF to the nucleus. These data suggest that glutamate neurotoxicity involves enhanced mitochondrial fission that promotes the loss of mitochondrial integrity and progression of AIF-dependent cell death, and this process is triggered by glutamate-induced activation of 12/15-LOX. The translocation of AIF from mitochondria to the nucleus is a key feature of caspase-independent neuronal death as shown previously in models of glutamate toxicity, [START_REF] Culmsee | Apoptosisinducing factor triggered by poly(ADP-ribose) polymerase and Bid mediates neuronal cell death after oxygen-glucose deprivation and focal cerebral ischemia[END_REF][START_REF] Landshamer | Bidinduced release of AIF from mitochondria causes immediate neuronal cell death[END_REF] oxygen glucose deprivation [START_REF] Culmsee | Apoptosisinducing factor triggered by poly(ADP-ribose) polymerase and Bid mediates neuronal cell death after oxygen-glucose deprivation and focal cerebral ischemia[END_REF] and axonal stretch injury. [START_REF] Slemmer | Causal role of apoptosis-inducing factor for neuronal cell death following traumatic brain injury[END_REF] Upstream mechanisms of AIF release from mitochondria are not well understood but may involve activation of PARP [START_REF] Yu | Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor[END_REF] and calpains. [START_REF] Polster | Calpain I induces cleavage and release of apoptosis-inducing factor from isolated mitochondria[END_REF] In addition, mitochondrial translocation of the BH3-only death agonists Bid [START_REF] Culmsee | Apoptosisinducing factor triggered by poly(ADP-ribose) polymerase and Bid mediates neuronal cell death after oxygen-glucose deprivation and focal cerebral ischemia[END_REF][START_REF] Landshamer | Bidinduced release of AIF from mitochondria causes immediate neuronal cell death[END_REF] , Bim 36 or BNIP3 [START_REF] Xu | Necrostatin-1 protects against glutamate-induced glutathione depletion and caspase-independent cell death in HT-22 cells[END_REF] have been associated with AIF translocation in neurons.

Here, the 12/15-LOX inhibitor PD146176 prevented AIF translocation to the nucleus, supporting a role for 12/15-LOX activation upstream of this mitochondrial mechanism of cell death. Notably, we also identified a therapeutic time window for interference with LOX activation in paradigms of neuronal cell death that may be relevant for therapeutic strategies in related neurological diseases. Recent work in models of cerebral ischemia exposed LOX as a potential target for neuroprotective strategies in stroke treatment. [START_REF] Van Lk | Baicalein and 12/15-lipoxygenase in the ischemic brain[END_REF][START_REF] Van Lk | Novel lipoxygenase inhibitors as neuroprotective reagents[END_REF] In these studies, genetic 12/15-LOX deletion significantly reduced the infarct size in a mouse model of transient cerebral ischemia as compared to wild type mice, and similar protective effects against ischemic brain damage were also achieved by treatment of wild type mice with the LOX inhibitor baicalein.

In summary, our data identify 12/15-LOX as a key trigger in glutamate-induced oxidative stress that is initiated by glutathione depletion, significantly amplified by Biddependent mechanisms of mitochondrial damage, and executed by mitochondrial AIF release to the nucleus (Figure 8). The delineated sequences of glutamate-induced cell death signaling in HT-22 cells is highly relevant for neurodegenerative diseases and acute neurological disorders such as ischemic stroke or brain trauma, since these mechanisms have been identified as key features of neuronal death in related experimental models in vitro and in vivo.

Methods

Cell culture and viability assays -HT-22 cells were cultured in Dulbecco's modified

Eagle medium (DMEM, Invitrogen, Karlsruhe, Germany) supplemented with 10% heat-inactivated fetal calf serum, 100 U/mL penicillin, 100 µg/mL streptomycin and 2 mM glutamine. The LOX inhibitors PD146176 and AA861 (Sigma, Deisenhofen, Germany) were dissolved in DMSO (Sigma, Deisenhofen, Germany). PD146176 was diluted to a concentration of 50 mM and AA861 to a concentration of 10 mM and these stock solutions were diluted with cell culture medium for each experiment up to final concentrations of 0.5 µM PD146176 and 0.1 µM AA861. Glutamate (2 -5 mM) was added to the medium, and cell viability was evaluated 18 h later. Quantification of cell viability in HT-22 cells was performed in 96-well plates by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction at 0.25 mg/mL for 1.5 h. The reaction was terminated by removing the media and freezing the plate at -80 °C for at least 1 h. Absorbance was then determined after solving MTT dye in DMSO at 570 nm versus 630 nm (FluoStar, BMG Labtech, Offenburg, Germany). In addition, real time detection of cellular viability was performed by measurements of cellular impedance by the xCELLigence system (Roche, Penzberg, Germany). For annexin-V/propidium iodide-staining, HT-22 cells were cultured in 24-well plates and damaged with glutamate (3 and 5mM). The cells were harvested 12-15 hours after glutamate treatment by using Trypsin/EDTA, washed once in PBS and resuspended in binding buffer (PromoKine). Propidium iodide and annexin-V-FITC were added at 1µl/100µl (PromoKine Annexin V-FITC Detection Kit) and incubated for 5 minutes at room temperature. Apoptotic and necrotic cells were detected using a FACScan (Becton, Dickinson and Company). Annexin-V and propidium iodode fluorescence was exited at a wavelength of 488 nm. Emission was detected at 530±40 nm for annexin-V and at 680±30 nm for propidium iodide. 10000 cells per sample were collected. Plasmids and gene transfer -Plasmid pCDNA 3.1+ was obtained from Invitrogen (Karlsruhe, Germany). The tBid vector and control vectors were generated as previously described. [START_REF] Kazhdan | Targeted gene therapy for breast cancer with truncated Bid[END_REF] For plasmid transfections 8 x 10 4 HT-22 cells were seeded in 24-well plates. Antibiotic containing growth medium was removed and replaced with 900 µl antibiotic-free growth medium. Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany) and ptBID plasmide or empty vector pCDNA 3.1+ were dissolved separately in Optimem I (Invitrogen, Karslruhe, Germany). After 10 min of incubation at room temperature each DNA solution was combined with the respective volume of the Lipofectamine solution, mixed gently, and allowed to form plasmid liposomes for further 20 min at room temperature. The transfection mixture was added to the antibiotic-free cell culture medium to a final concentration of 1µg DNA, and 1.5 µl/mL Lipofectamine 2000 in HT-22 cells. Controls were treated with 100 µl/mL Optimem only, and vehicle controls with 1.5 µl/mL Lipofectamine 2000.

Embryonic cortical cultures:

Detection of oxidative stress -Intracellular reactive oxygen species (ROS) were

detected by Dichlorodihydrofluoresceine-diacetate (DCF). Within 6 -17 h after glutamate treatment HT-22 cells were loaded with 1 µM CM-H2DCFDA (Invitrogen, Karlsruhe, Germany) for 30 min and fluorescence at 530 nm was monitored using a CyanTM MLE flow cytometer (DakoCytomation, Copenhagen, Denmark) at an excitation wavelength of 488 nm. For detection of cellular lipid peroxidation, cells were loaded with 2 µM BODIPY 581/591 C11 for 60 min in standard medium 6 -17 h after glutamate treatment. Cells were then collected, washed and resuspended in phosphate-buffered saline (PBS) and flow cytometry was performed using 488 nm UV line argon laser for excitation and BODIPY emission was recorded on channels FL1 at 530 nm (green) and FL2 at 585 nm (red). Data were collected from at least 20,000 cells.

Immunocytochemistry -For immunocytochemistry HT-22 cells were fixed with 4% PFA after their respective treatment. The cells were permeabilized by exposure for 5 min to 0.4% Triton X-100, and cells were placed in blocking solution [3% horse serum] for 30 min. Cells were then exposed to a polyclonal anti-AIF antibody (1:100 in block solution), overnight at 4°C, followed by an incubation for 1 h with biotinylated anti-goat IgG antibody and 30 minutes in the presence of streptavidin oregon green 514 conjugate according to the manufacturers protocol. The specificity of AIF immunoreactivity was controlled by omission of the primary antibody. Nuclei were counterstained with DAPI. Images were acquired using a fluorescence microscope. Western blot analysis was performed as previously described. [START_REF] Plesnila | Nuclear translocation of apoptosis-inducing factor after focal cerebral ischemia[END_REF] Briefly, the blot was probed with an anti-AIF goat polyclonal antibody (sc-9416, 1 : 1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-Bid (Cell signaling, Danvers, Massachusetts, USA) at 4°C overnight. Membranes were then exposed to the appropriate HRP-conjugated rabbit anti-goat secondary antibody (1:2500, Vector Laboratories, Burlingame, CA, USA) or anti-rabbit secondary antibody (1:2500, Vector Laboratories, Burlingame, CA, USA) followed by a chemiluminescence detection of antibody binding. Equal protein loading and purity of the extracts was controlled by re-probing the membrane with a monoclonal anti-αtubulin antibody (T9026, 1 : 20 000, Sigma, data not shown) or an anti-HDAC1 antibody (dianova, 1 : 1000). Chemidoc ® software (Bio -Rad®) was used for quantification of western blot signals.

Immunoblots

Statistical analysis -All data are given as means ± standard deviation (SD). For statistical comparisons between two groups Mann-Whitney U-test was used; multiple comparisons were performed by analysis of variance (ANOVA) followed by Scheffé's post hoc test. Calculations were performed with the Winstat standard statistical software package.

  that suggested activation and mitochondrial translocation of full length Bid before Bid cleavage and execution of mitochondrial death pathways. It is important to note that the Bid inhibitor prevented the translocation of activated full length Bid and tBid to mitochondria and the according detrimental effects of mitochondria, suggesting that the effects of both forms of activated Bid on mitochondria are very similar. The exact mechanisms of Bid activation in the present model system are currently unknown and matter of ongoing studies.

Cortices were removed from embryonic day 16 Sprague-

 16 Dawley rats (Janvier, France) or Alox15-mice (Jackson Laboratory, USA) and dissociated by trypsinization and trituation as followed: Isolated cortices were treated with trypsin (Sigma-Aldrich, Germany) (1mg/ml) for 15 minutes at 37°C. The cortices were washed with Hank's balanced salt solution (HBSS) (made from 10x HBSS, Invitrogen, Germany) and mixed with 1mg/ml trypsininhibitor (Sigma-Aldrich, Germany) and incubated for 2 minutes at room temperature. Afterwards cortices were washed two times with HBSS, and trituated in MEM+ obtained from Eagle's minimum essential medium (Invitrogen, Germany) by addition of 1 mM HEPES (Biomol, Hamburg, Germany), 26 mM NaHCO3, 40 mM glucose, 20 mM KCl, 1.2 mM L-glutamine (each Sigma-Aldrich, Germany), 1 mM sodium pyruvate (Biochrom, Berlin, Germany), 10% (v/v) fetal calf serum (FCS) (Invitrogen, Karlsruhe, Germany) and 10 mg/l gentamicin sulfate (Sigma-Aldrich, Germany). Cells were seeded in 35-mm culture dishes coated with polyethylenimine (Sigma-Aldrich, Germany). After 4 hours the MEM+ was replaced by Neurobasal medium (Invitrogen, Germany). Cells were treated on day 6 after preparation with 20 µM of glutamate. After 18 to 24 hours the neurons were fixed with paraformaldehyde (4 %) and stained with the fluorescent DNA-binding dye 4', 6-diamidino-2phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, Germany). Living and pycnotic cells were counted using a fluorescence microscope (Leica, Germany) without knowledge of the treatment history. In total we counted 200 cells per cell culture dish (n=5).

  -HT-22 cells were treated as described above. Nuclear and cytosolic extracts were obtained by using a Nuclear extraction kit (Active Motif). For total cell protein extracts HT-22 cells were seeded in 24 well cell culture plates. At least 4 wells per condition were pooled. Cells were washed in PBS and lysed with 100 µl lysis buffer (Mannitol 0.25 M, Tris 0.05 M, EDTA 1M, EGTA 1M, DTT 1mM, Triton-X 1% (each Sigma-Aldrich, Germany), supplemented with 1 tablet per 10 ml Complete Mini protease inhibitor cocktail (Roche)). Protein extracts were kept on ice. To remove insoluble membrane fragments, extracts were centrifuged at 15,000 x g for 15 min at 4° C. The supernatants were stored at -80° C until further use.

Figure 1

 1 Figure 1 Glutamate leads to a time-dependent damage in HT-22 cells. (a) HT-22

Figure 2

 2 Figure 2 LOX inhibitors prevent peroxide formation in HT-cells exposed to glutamate.

Figure 3

 3 Figure 3 LOX-inhibitors protect HT-22-cells against glutamate induced cell death.

Figure 4

 4 Figure 4 (a) LOX inhibitor PD146176 protects HT-22-cells when applied up to 8 h

Figure 5

 5 Figure 5 LOX inhibitor PD146176 0.5 µM prevents mitochondrial fragmentation and

Figure 6

 6 Figure 6 Bid-inhibitor BI-6C9 protects cells against glutamate toxicity and prevents

Figure 7

 7 Figure 7 LOX inhibitors or Trolox do not attenuate the cytotoxic effect of tBid.

  Figure 1

Acknowledgements:

The authors gratefully acknowledge excellent technical support by Miriam Hoehn, Sandra Engel and Renate Hartmannsgruber. We would like to thank Dr. Cornelia Brendel, Thorsten Volkmann and Gavin Giel for the technical support with the FACS analyses. We would also like to thank Roche Applied Science for providing the xCELLigence System. Part of this work was supported by a grant from the Michael J. Fox-Foundation to CC.

Conflict of interest

The authors declare no conflict of interest.