Francesco Cecconi
email: francesco.cecconi@uniroma2.it

Daniela De Zio

Matteo Bordi

Elisa Tino

Chiara Lanzuolo

Eugenio Mora

Fabiola Ciccosanti

Gian Maria Fimia

Valerio Orlando

Elisabetta Ferraro

Dulbecco

The DNA repair complex Ku70/86 modulates Apaf1 expression upon DNA damage

Keywords: apoptosis/apoptosome/transcriptional regulation Apaf1, apoptotic protease activating factor, Bac, bacterial artificial chromosome, ChIP, chromatin immunoprecipitation, CMV, Cytomegalovirus, DNA-PKcs, DNA-dependent protein kinase catalytic subunit, DSB, double strand-break, EMSA electrophoretic mobility shift assay, ERBB2, erythrocyte blastosis virus B2, ETNA, Embryonic Telencephalic Naïve Apaf1 cells, G6pdx, glucose-6-phosphate dehydrogenase Xlinked, HDAC, histone deacetylase, NHEJ, non-homologous end-joining, 8-OHdG, 8-hydroxydeoxyguanosine, Pol II, RNA polymerase II, STS, staurosporine, TSP,

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Introduction

Apaf1 is a crucial factor in regulating the mitochondrial intrinsic pathway of apoptosis. Apaf1 normally being in an inactive conformation, its oligomerization is caused by cytochrome c release from mitochondria, after apoptotic stimuli, in the presence of dATP/ATP [START_REF] Kim | Formation of apoptosome is initiated by cytochrome c-induced dATP hydrolysis and subsequent nucleotide exchange on Apaf-1[END_REF]. Seven Apaf1 molecules generate the apoptosome [START_REF] Acehan | Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding, and activation[END_REF][START_REF] Reubold | A new model for the transition of APAF-1 from inactive monomer to caspase-activating apoptosome[END_REF], which is able to recruit the procaspase 9 and allow its activation [START_REF] Bao | Apoptosome: a platform for the activation of initiator caspases[END_REF]. In such a multimeric complex, the apoptosome activates executioner caspases, e.g. caspase 3, which orchestrate the "safe" destruction of the cell [START_REF] Boatright | Mechanisms of caspase activation[END_REF].

Apaf1 is regulated at several levels. As regards the transcriptional control, it is well established as being positively regulated by p53 [START_REF] Fortin | APAF1 is a key transcriptional target for p53 in the regulation of neuronal cell death[END_REF][START_REF] Kannan | DNA microarray analysis of genes involved in p53 mediated apoptosis: activation of Apaf-1[END_REF], and E2F1 [START_REF] Moroni | Apaf-1 is a transcriptional target for E2F and p53[END_REF], factors involved in apoptosis and cell cycle regulation. It has been demonstrated that p53 recognizes and binds two sites on the human promoter of Apaf1 [START_REF] Moroni | Apaf-1 is a transcriptional target for E2F and p53[END_REF], and that this interaction seems to be enhanced by Zac-1, a transcription factor involved in the induction of G1 arrest [START_REF] Rozenfeld-Granot | A positive feedback mechanism in the transcriptional activation of Apaf-1 by p53 and the coactivator Zac-1[END_REF]. E2F is a family of transcription factors controlling the G1/S transition during the cell cycle. Among these factors the member E2F1 has been demonstrated to induce apoptosis by directly activating Apaf1 [START_REF] Furukawa | Apaf-1 is a mediator of E2F-1-induced apoptosis[END_REF]. Regarding epigenetic regulation, Apaf1 promoter seems to be silenced by methylation in several tumours (albeit this finding is still controversial in some cases) [START_REF] Furukawa | Methylation silencing of the Apaf-1 gene in acute leukemia[END_REF][START_REF] Peltenburg | Expression and function of the apoptosis effector Apaf-1 in melanoma[END_REF][START_REF] Soengas | Inactivation of the apoptosis effector Apaf-1 in malignant melanoma[END_REF][START_REF] Teodoridis | CpG island methylation of DNA damage response genes in advanced ovarian cancer[END_REF]. The inhibition of histone deacetylase (HDAC) activity restores the Apaf1 expression in mouse developing retina [START_REF] Wallace | Histone deacetylase activity in conjunction with E2F-1 and p53 regulates Apaf-1 expression in 661W cells and the retina[END_REF]; this was also observed in developing but not in mature neurons, unless E2F1 was concomitantly expressed [START_REF] Wright | Chromatin modification of Apaf-1 restricts the apoptotic pathway in mature neurons[END_REF]. In particular, HDAC inhibition resulted in an increased binding of E2F1 and p53 to the Apaf1 mouse promoter [START_REF] Wallace | Histone deacetylase activity in conjunction with E2F-1 and p53 regulates Apaf-1 expression in 661W cells and the retina[END_REF]. Moreover, different Apaf1 levels correlated with differences in the levels of E2F1 and were found to be critical for allowing cytochrome c to induce apoptosis in brain tumours but not in normal tissues [START_REF] Johnson | Differential Apaf-1 levels allow cytochrome c to induce apoptosis in brain tumors but not in normal neural tissues[END_REF].

Being a positive regulator of the apoptotic cell death, Apaf1 transcriptional regulation is crucial under the various conditions where apoptosis occurs, such as in driving development, controlling tissue homeostasis, and preventing the propagation of DNAdamaged cells.

The main damage to DNA helices is the double strand-break (DSB), which is essentially repaired by the non-homologous end-joining (NHEJ) pathway [START_REF] Lieber | Mechanism and regulation of human nonhomologous DNA end-joining[END_REF]. This repairs the DNA by a homology-independent mechanism rejoining broken ends irrespective of the sequence. DSB can be produced after exposure to different genotoxic insults. When it occurs, the Ku70/86 heterodimer binds to the DNA free ends and recruits the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). The formation of this complex results in the subsequent recruitment and phosphorylation of other proteins, such as XRCC4, DNA Ligase IV, Cernunnos/XLF and Artemis so as to ligate the DNA broken ends [START_REF] Downs | A means to a DNA end: the many roles of Ku[END_REF]. The Ku complex seems to be critical for cell survival, since the failure of the cells to carry out DNA repair results in an apoptotic response. Ku70 and Ku86 proteins were originally found as a major target of auto-antibodies taken from Japanese patients with scleroma-polymyositis overlap syndrome [START_REF] Mimori | Characterization of a high molecular weight acidic nuclear protein recognized by autoantibodies in sera from patients with polymyositis-scleroderma overlap[END_REF]. Current knowledge on Ku deals with its fundamental role in DNA repair, but the Ku complex is also implicated in other cellular processes, including telomere maintenance, antigen receptor gene arrangements (VDJ recombination), regulation of specific gene transcription and apoptosis (reviewed by [START_REF] Downs | A means to a DNA end: the many roles of Ku[END_REF]. Indeed, Ku70 can sequester Bax from the mitochondria and mediate Bax de-ubiquitylation [START_REF] Amsel | Regulation of the proapoptotic factor Bax by Ku70-dependent deubiquitylation[END_REF]. Regarding its transcriptional function, the Ku complex has been reported to act in both a sequence-nonspecific and -specific manner. This factor has been found to be associated with RNA polymerase II elongation sites without a sequence-specific DNA binding [START_REF] Mo | Subnuclear localization of Ku protein: functional association with RNA polymerase II elongation sites[END_REF]. On the other hand, there are several reports indicating that the Ku complex acts as a transcription factor: e.g. in the repression of the human -myosin heavy-chain promoter during heart failure [START_REF] Sucharov | The Ku protein complex interacts with YY1, is up-regulated in human heart failure, and represses alpha myosin heavy-chain gene expression[END_REF]; or in contributing the oncogene ERBB2 overexpression in breast cancer cells by interacting with activator protein-2 transcription factors [START_REF] Nolens | Ku proteins interact with activator protein-2 transcription factors and contribute to ERBB2 overexpression in breast cancer cell lines[END_REF].

In this report we analyse the transcriptional regulation of murine Apaf1 and we demonstrate a functional interaction between Ku70/86 heterodimer and a specific region of Apaf1 promoter. We find that Ku acts as transcription repressor, leading to a down-regulation of Apaf1 expression. In particular, our findings suggest that, upon DNA damage, the Ku complex exerts a pro-survival role not only through repairing DNA damage, but also contributing to modulate Apaf1 expression.

Results

Analysis of murine Apaf1 promoter activity

To investigate the transcriptional modulation of murine Apaf1, we focused on a 7kb region of the Apaf1 promoter (-4210) (Figure 1A). We generated three deletion constructs ( -1509;

-589; +305) (Figure 1A) and placed them upstream of the firefly luciferase cDNA, in order to compare their activity and to identify relevant regions for transcriptional regulation. These constructs were transfected in different cell types (B 16 F 12 , murine melanoma cells; C 3 H 10 T 1/2 , murine fibroblasts; ImimPc-2, human pancreatic primary solid tumor cells;

and ETNA, Embryonic Telencephalic Naïve Apaf1 cells), and the relative promoter activities were measured. As reported in figures 1B-E, one of the constructs ( -589) shows a significant increase in promoter activity when compared to the whole promoter in each cell line analysed. The constructs -1509 and -4210 have comparable activities whereas, as expected, the construct +305 shows an activity comparable with that of the pG13-basic empty vector, since it lacks the minimal regions required for transcription. Based on this observation, we hypothesized the existence of a promoter regulatory element in a region of about 1000bp. This element is located between nucleotides -1509 and -589 and is able to repress Apaf1 promoter activity.

Identification of the repressing element on Apaf1 promoter

In order to identify the smallest region able to suppress Apaf1 expression, we set out to study this region in more detail. We generated an additional series of deletion constructs (referred as 1 to 7) (Suppl. Figure 1) and transfected them in the ImimPc-2 and ETNA cell lines. We found that a region of about 110bp, located between -1509 and -1402, was involved in promoter repression. Indeed, all the constructs that did not contain this fragment, displayed a higher activity than that observed for -4210 and -1509 (Suppl. Figures 1B,C).

Therefore, this region should reasonably include the site able to repress Apaf1 promoter activity. We divided the fragment between -1509 and -1402 into two regions, E1 and E2, each about of 50bp, and cloned them upstream of the construct -589 (Figure 2A) to verify their ability to modulate normal promoter activity. Region E1 significantly decreased -589 promoter activity in ImimPc-2 and ETNA cells, whereas E2 only partially reduced such activity in ETNA (Figures 2B,C).

In addition, we cloned E1 upstream of a control promoter, pCMV (E1pCMV). We compared the activity of the two constructs (pCMV and E1pCMV) so as to test the ability of this region to act as an autonomous repressor element. Our analyses showed that the presence of E1 significantly decreased pCMV activity in both the cell lines analysed (Figure 2D,E).

Moreover, in silico analysis performed by the multiple alignment of the E1 region against Apaf1 promoter sequences of different organisms revealed a high conservation of this sequence in mammals (Suppl. Figure 2). Taken together, these data indicate that E1 could represent a novel repressing element involved in Apaf1 transcriptional regulation.

Ku70/86 binds Apaf1 promoter in vitro

To identify factors involved in the activity of this repressor, we performed an electrophoretic mobility shift assay (EMSA). By incubating nuclear extracts from ImimPc-2 and ETNA cells with a radioactively labelled E1 oligonucleotide, we observed a supershift band in both samples (Figure 3A). This suggests that the E1 fragment is recognized and bound by some nuclear factors, which probably act as repressors of Apaf1 transcription. In order to detect these putative factors, we carried out a DNA pull-down assay. Biotinylated double-stranded oligonucleotides containing the E1 sequence or an unrelated one, as a negative control, were associated to streptavidine-coated beads and allowed to react with nuclear extracts from ETNA cells. Bound proteins were eluted using an excess of free oligonucleotides and analysed by SDS-PAGE. After SYPRO Ruby staining, two clear bands, corresponding to proteins with apparent molecular weights of 72 and 90kDa, were detected as specific for E1 binding (see arrows in Figure 3B). The protein bands were excised from the gel, digested with trypsin, and subjected to MALDI-MS/MS for protein identification. As described in Table 1, mass spectrometry analysis identified the two proteins as Ku70 and Ku86.

Ku70/86 binds Apaf1 promoter in vivo and represses its transcription

To confirm the interaction between Ku70/86 and Apaf1 promoter in vivo, we performed a chromatin immunoprecipitation (ChIP) assay. Cross-linked chromatin from ETNA cells was immunoprecipitated with antibodies against the heterodimer Ku and the RNA polymerase II (Pol II), as a positive control. We found that Ku specifically binds the E1 region of Apaf1 promoter at variance with the negative control G6pdx, as shown in the anti-Ku immunoprecipitated sample subjected to quantitative real time PCR using Apaf1 promoter oligonucleotides (Figure 3C).

On the basis of these results, we set out to establish the functional role of this interaction. Since these proteins function synergically, we analysed the effect of Ku70 silencing on Apaf1 promoter activity (Figure 3D,E). In fact, it has been reported that Ku70deficient mice exhibit decreased levels of Ku86, and viceversa [START_REF] Errami | Ku86 defines the genetic defect and restores X-ray resistance and V(D)J recombination to complementation group 5 hamster cell mutants[END_REF][START_REF] Gu | Alt FW Ku70-deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination[END_REF][START_REF] Singleton | Molecular and biochemical characterization of xrs mutants defective in Ku80[END_REF]. We found that 72 hours of transfection are sufficient to efficiently knockdown Ku70/86 (Figure 3E).

Quantitative real time PCR and immunoblotting analyses showed that a significant increase of Apaf1 mRNA and protein levels occurred both in ImimPc-2 and ETNA cells when compared to cells 72 h-transfected with an unrelated control oligonucleotide (Figure 3D,E).

Conversely, when we transfected ETNA cells with both Ku70 and Ku86 expressing vectors (pKu70/86), we observed a decrease of Apaf1 expression (Figure 3F). Taken together, these data show that Ku70/86 binds Apaf1 promoter and represses Apaf1 expression.

Ku70/86 binds dynamically the Apaf1 promoter and modulates its expression upon DNA damage

Since the Ku complex is known to be involved in DNA damage repair, we decided to explore whether Ku/Apaf1 promoter interaction is modulated following induction of DNA damage by hydrogen peroxide or etoposide in ETNA cells. Fluorescence images and immunoblotting analyses displayed the occurrence of histone H2A.X phosphorylation (Figure 4 A-C) and the oxidative modification of deoxyguanosine (8-OHdG) (Suppl. Figure 3 A, B), which are well-established markers of DNA lesion [START_REF] Bonner | GammaH2AX and cancer[END_REF][START_REF] Lan | In situ analysis of repair processes for oxidative DNA damage in mammalian cells[END_REF], already after one hour of hydrogen peroxide or etoposide incubation. This event well correlates with Ku70 accumulation within the nuclei either upon hydrogen peroxide or etoposide treatment (Figure 4B, C), suggesting that Ku70 had a crucial role in DNA repair process also under our experimental conditions. We then assessed whether this nuclear accumulation was associated with the modulation of Apaf1 expression, focusing on a time window of 6 hours when apoptosis was not still executed (Suppl. Figure 4). We found that Apaf1 mRNA and protein levels were significantly decreased by about 30% with respect to the control in both H 2 O 2and etoposide-treated ETNA cells (Figures 5A,B). Interestingly, after 6 hours of treatment Apaf1 levels increased to reach values higher than control cells (Figures 5A,B). In order to explain these dynamical changes of Apaf1 [START_REF] Margaritis | Poised RNA polymerase II gives pause for thought[END_REF][START_REF] Saunders | Breaking barriers to transcription elongation[END_REF], this result could be explained by the fact that Pol II has to be ready for the subsequent activation of Apaf1 transcription.

Moreover, Apaf1 repression is exclusively dependent on DNA damage, since other apoptotic stimuli, such as protein kinase C-inhibition by staurosporine (STS), did not affect Apaf1 transcription via Ku/Apaf1 promoter binding (Suppl. Figure 5).

These data indicate that, upon DNA damage, Apaf1 transcription is dynamically modulated by the Ku complex.

Ku70/86-mediated Apaf1 regulation modulates cell death upon DNA damage

In order to detect whether Apaf1 modulation mediated by Ku70/86 binding concurred to cell death control, we analysed during a time course the events underlying the mitochondrial pathway of apoptosis after hydrogen peroxide treatment. Analysis of cytochrome c subcellular distribution indicated that cytochrome c was released from mitochondria as soon as 1 hour after hydrogen peroxide administration (Figure 6A).

However, caspase 3 was activated only at 6 hours of treatment, time at which Apaf1 resulted to be no more inhibited (Figure 6A), suggesting that the down-regulation of Apaf1, occurring early upon DNA damage, had a role in the inhibition of caspase 3 cleavage, even in the presence of cytosolic cytochrome c.

Moreover, under conditions of Ku complex down-regulation by siKu70 transfection in ETNA cells, the decrease of Apaf1 mRNA and protein levels treatment was not observed upon hydrogen peroxide treatment (Figure 6B-D). By contrast, the protein levels of Apaf1 and cleaved caspase 3 were greatly enhanced, when comparing siKu70versus siCtrtransfected ETNA cells already after 1 hour of treatment (Figure 6C,D). Likewise, under these conditions, cytochrome c was released into the cytosol at the same extent both in Ku70proficient and -deficient ETNA cells (Figure 6C,D). Also, caspase 3 was activated even in the absence of hydrogen peroxide (Figure 6C,D). This suggests that Ku depletion can induce an up-regulation of Apaf1 expression, which may be sufficient to activate caspase 3.

To further corroborate this assumption, we transfected an Apaf1 expressing vector (pApaf1) lacking E1 regulatory region recognized by Ku70/86, in Apaf1-deficient cells named ETNA -/-cells (32) (Figure 6E, F). Cleaved caspase 3 was already detected as soon as

Apaf1 was re-introduced in these cells. Moreover, no significant change of Apaf1 protein and mRNA levels were observed in pApaf1-transfected cells upon hydrogen peroxide treatment, although this resulted in DNA damage and, in turn, in the phosphorylation of histone H2A.X.

This finding suggests the down-regulation of Apaf1 expression, observed upon DNA damage, mainly depends on the effect of the Ku complex on Apaf1 transcription.

Taken together these data indicate that Ku70/86-dependent modulation of Apaf1 expression contributes to the regulation of the apoptotic pathway occurring upon DNA damage induction.

Discussion

Here we demonstrate that a specific sequence of Apaf1 promoter functions as a repressing element of its activity. In fact, the removal of this sequence is enough to increase Apaf1 promoter activity. Mass spectrometry, chromatin immunoprecipitation assay and silencing experiments have revealed that the heterodimer Ku, composed of Ku70 and Ku86 subunits, can bind to this specific sequence of Apaf1 promoter, negatively regulating its expression.

To date, two transcription factors, E2F and p53, have been revealed to regulate Apaf1 in response to several stress factors, thus controlling cell cycle progression [START_REF] Fortin | APAF1 is a key transcriptional target for p53 in the regulation of neuronal cell death[END_REF][START_REF] Kannan | DNA microarray analysis of genes involved in p53 mediated apoptosis: activation of Apaf-1[END_REF][START_REF] Moroni | Apaf-1 is a transcriptional target for E2F and p53[END_REF]. E2F1 has been also suggested as having a differential capacity to induce Apaf1 transcription, on the basis of Apaf1 promoter association or not with active chromatin [START_REF] Wright | Chromatin modification of Apaf-1 restricts the apoptotic pathway in mature neurons[END_REF]. In particular, it has been demonstrated that Apaf1 promoter is associated with active chromatin in developing neurons and repressed chromatin in mature ones [START_REF] Wright | Chromatin modification of Apaf-1 restricts the apoptotic pathway in mature neurons[END_REF]. Apaf1 expression drops dramatically during neuronal differentiation and allows mature neurons to resist cytochrome c-dependent apoptosis [START_REF] Wright | Chromatin modification of Apaf-1 restricts the apoptotic pathway in mature neurons[END_REF][START_REF] Johnson | Differential Apaf-1 levels allow cytochrome c to induce apoptosis in brain tumors but not in normal neural tissues[END_REF]. Our finding that Apaf1 can be repressed by Ku could provide a further explanation to this event, since Ku has been found to be implicated in telomeric silencing by anchoring telomeres to repressed chromatin [START_REF] Hediger | Live imaging of telomeres: yKu and Sir proteins define redundant telomere-anchoring pathways in yeast[END_REF]. Ku could bind the Apaf1 promoter far more in differentiated cells than in proliferating ones, a hypothesis which our experiments in neuronal precursor cells (ETNA cells) support. In fact, we did not observe a strong Ku/Apaf1

promoter interaction in physiological conditions, as Apaf1 has to be expressed at high levels in neuronal progenitors, in order to guarantee the potential of displaying apoptosis. ETNA cells derive, indeed, from the telencephalon of developing brain at embryonic stage 14.5, when massive apoptosis occurs in proliferating neuronal precursors [START_REF] Cecconi | Gruss P Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development[END_REF]. Nevertheless, in Ku70/86 double knockout mice, a dramatic increase of cell death has been detected in developing embryonic neurons [START_REF] Gu | Defective embryonic neurogenesis in Ku-deficient but not DNA-dependent protein kinase catalytic subunit-deficient mice[END_REF]. This could be in part due to the enhancement of Apaf1 expression in the absence of Ku/Apaf1 promoter interaction, which might be crucial for having Apaf1 at a threshold level needed for physiological apoptosis in developing neurons.

This work investigated the interaction between Ku and Apaf1 promoter during DNA damage, when cell survival response of repairing DNA is activated. Indeed, Ku has been reported as being involved in several processes, such as in the DNA repair pathway of NHEJ recombination, in silencing telomeres, in apoptosis and in gene transcription regulation (reviewed by [START_REF] Downs | A means to a DNA end: the many roles of Ku[END_REF]. As regards the DNA repair process, it is well-known that Ku directly binds the free ends of double stranded DNA and recruits the DNA-PKcs and other repair factors, so promoting numerous signalling pathways (reviewed by [START_REF] Downs | A means to a DNA end: the many roles of Ku[END_REF]. The finding that Ku heterodimer and Apaf1 promoter could interact with the result of Apaf1 repression led us to hypothesise a possible function of this binding during the DNA repair process. We thus also explored the dynamics of Ku/Apaf1 promoter binding when DNA damage is induced upon genotoxic stress, this by using chromatin immunoprecipitation assay coupled to quantitative PCR. We discovered that Ku binds far more to the target sequence of Apaf1 promoter as soon as DNA damage is induced, when caspase 3 and apoptosis are not yet activated. This results in decreased Apaf1 levels, and suggests that the cell response to DNA damage is not only to repair the damage, but also to maintain lower the levels of at least one of the main apoptotic genes, Apaf1. Indeed, Apaf1 is fundamental for the formation of the apoptosome, which is the core of the apoptotic programme and serves to activate executioner caspases, such as caspase 3 [START_REF] Boatright | Mechanisms of caspase activation[END_REF]. It can be assumed that, when the damage to DNA becomes more severe and irreversibly unrepaired, Ku70/86 moves from Apaf1 promoter thus contributing to the apoptotic response. This idea is corroborated by the kinetics of Ku/Apaf1 promoter interaction that turns out to be comparable to the untreated cells at longer time points.

Moreover, fluorescence microscopy analysis of nuclear 8-hydroxydeoxyguanosine (8-OHdG) content, a well-enstablished oxidative marker of DNA damage, indicated that it significantly increased to reach a plateau at 6 hours of hydrogen peroxide treatment, without further rise at longer time points (see Suppl. Figure 3). Thus, the temporary association between 8-OHdG increase and Apaf1 up-regulation argues for a causal relationship between the occurrence of an irreversible DNA damage and the release of Ku70/86 from Apaf1 promoter. This suggests that Apaf1 should be transcriptionally up-regulated in order to promote apoptosome formation and caspase 3 activation.

Indeed, in such a stress-conditions, other players in the apoptosis pathway could be regulated by the cytosolic protein Ku70, such as the Bcl2-family member Bax. Bax is, in fact, sequestered from the mitochondria and deubiquitylated by Ku70 under normal conditions.

Upon induction of apoptosis, Ku70 is acetylated thereby releasing Bax, which, in turn, translocates to the mitochondria and triggers cell death [START_REF] Amsel | Regulation of the proapoptotic factor Bax by Ku70-dependent deubiquitylation[END_REF]. Therefore, we can speculate that the prompt release of cytochrome c we observed upon hydrogen peroxide administration and in Ku-silenced cells, where both in untreated (Ctr) and treated cells (T1) is slightly more released into the cytosol (Figure 6A andC), is possibly due to Bax translocation to the mitochondria. In this perspective, Ku70/86-mediated modulation of Apaf1 expression upon DNA damage could mainly serve to delay the apoptotic process in order to allow DNA repair.

Also, we could hypothesize the possibility that p53, a well-known transcriptional activator of Apaf1 (6-8) and a DNA damage sensing protein [START_REF] Kruse | Modes of p53 regulation[END_REF], could play a role in this regulation. In particular, after DNA damage induction, Ku70 and Ku86 form the huge DNA-PK complex with the DNA-PKcs, which in turn activates several pathways by the direct or indirect phosphorylation of different substrates, such as p53 [START_REF] Kruse | Modes of p53 regulation[END_REF]. In this scenario, the upregulation of Apaf1 could be the result of both the Ku displacement from Apaf1 promoter and p53 binding.

The repression function of Ku on Apaf1 expression could reconcile the apparent conflicting observations, which indicate that Ku behaves as a tumour suppressor and oncoprotein (reviewed by 37). In fact, since the impairment of DNA repair process produces genetic instability and, in turn, cancer development, it has been suggested that Ku's loss-offunction induces tumorigenesis [START_REF] Li | Ku70: a candidate tumor suppressor gene for murine T cell lymphoma[END_REF][START_REF] Pucci | Tumor specific modulation of KU70/80 DNA binding activity in breast and bladder human tumor biopsies[END_REF][START_REF] Tong | Synergistic role of Ku80 and poly(ADP-ribose) polymerase in suppressing chromosomal aberrations and liver cancer formation[END_REF]. Conversely, its gain-of-function has been associated

with hyperproliferation and evading apoptosis of different tumour histotypes [START_REF] Klein | Chemosensitivity of B cell chronic lymphocytic leukemia and correlated expression of proteins regulating apoptosis, cell cycle and DNA repair[END_REF][START_REF] Lim | Expression of Ku70 and Ku80 mediated by NF-kappa B and cyclooxygenase-2 is related to proliferation of human gastric cancer cells[END_REF]. On the basis of the results obtained in this work, it could be speculated that the above-mentioned resistance phenomena are related to a Ku-dependent dysregulation of Apaf1 expression.

Indeed, Apaf1 has been reported to be a potent tumour suppressor and drug resistance inducer (reviewed by 43). Therefore, the identification of factors modulating Apaf1 expression, such as Ku itself, may be highly relevant in the development of novel anticancer therapeutic agents.

Materials and methods

Plasmids constructs

A region of about 7 Kb of the murine Apaf1 promoter was obtained with PCR from a Bac (bacterial artificial chromosome) clone as template, with the primers: 5'-AAGCTTCACCTTTAGTCCCAGCA-3' and 5'-AAGCTTTGTGCCTCAGGGTTTTCTC- CMV-10 vector. As regards pApaf1 expressing vector see [START_REF] Cozzolino | Apoptosome inactivation rescues proneural and neural cells from neurodegeneration[END_REF].

Cell cultures and transfections

The following cell lines were used in this study: ImimPc-2 (human pancreatic tumoral cells), ETNA (embryonic telencephalic naive Apaf1 cells), B 16 F 10 (murine melanoma cells)

and C 3 H 10 T 1/2 (murine fibroblasts). They were grown in DMEM (Sigma-Aldrich Inc., St.

Louis, MO) containing 10% fetal bovine serum (FBS, Sigma-Aldrich Inc., St. Louis, MO), 100 U/ml penicillin and 100 μg/ml streptomycin, in a humidified atmosphere of 5% CO 2 at 37°C (except for ETNA cells which were grown at 33°C). Transient siRNA transfection was performed with smart pool siRNA directed against four Ku70 mRNA sequences (siKu70), specific for mouse or human (Dharmacon RNA technologies, Lafayette, CO). Their sequences are available on request. Control cells were transfected with a scramble siRNA duplex (Ambion, Foster City, CA), which does not present homology with any other murine or human mRNAs. Transient expression of siRNA and of each vector was obtained with Lipofectamine2000 (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions.

After 4 h incubation with transfection reagents, the cells were cultured in normal growth medium for the indicated period of time.

Cell treatments

ETNA cells were treated with apoptotic stimulus etoposide (Sigma-Aldrich Inc., St. Louis, MO) at a concentration of 20μM for 1, 3, 6, 9, 12 and 24 hours; hydrogen peroxide (Sigma-Aldrich Inc., St. Louis, MO) at a concentration of 500μM for 1 hour and then replaced with fresh medium for 1, 3, 6, 12, and 24 hours; and staurosporine (Gibco, Carlsbad, CA) at a concentration of 250nM for 1, 3, 6 and 24 hours.

Luciferase assay

ImimPc-2, ETNA, B 16 F 10 and C 3 H 10 T 1/2 cells were co-transfected with one of the constructs, described above, containing the Firefly luciferase and a pRL-TK plasmid that expresses the Renilla luciferase, this used as an internal control of the transfection efficiency.

24 hours or 48 hours after transfection, cells were harvested and analysed by using Dualluciferase reporter assay system (Promega, Madison, WI). The relative luciferase activity was calculated as the ratio between the firefly luciferase activity and the renilla luciferase activity.

Real time RT-PCR

RNA was isolated by using RNeasy Micro Kit (Qiagen Inc., Valencia, CA), according to the manufacturer's instructions

EMSA

EMSA was performed using E1 double-stranded oligonucleotide (5'-ATTTACACTAATATATCACTAATATATGCTTGGTTTCAAAGCTACAAAT-3'), corresponding to the region located between -1509 and -1402 nucleotides of the Apaf1 promoter. The oligonucleotide was end-labeled using [ -32 P]ATP. Nuclear extracts were prepared as follows: 10 7 cells were centrifuged at 1000 rpm for 5 min, the pellet was resuspended in buffer H (10 mM HEPES pH 7.9, 50 mM NaCl, 0.5 M sucrose, 0.1 mM EDTA, 0.5% Triton, 1 mM DTT, 1 mM PMSF) and incubated on ice for 5 min. After centrifugation at 1000 rpm for 10 min, pellet was washed in buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT). Then it was resuspended in buffer C (10 mM HEPES pH 7.9, 500 mM NaCl, 0.1 mM EDTA, 0.1% IGEPAL, 1 mM DTT) and vortexed for 10 min at 4°C. The sample was centrifuged at 13000 rpm for 5 min and the supernatant was stored at -80°C. Protein concentration was determined by Bradford protein assay (Bio-Rad, Hercules, CA). The extracts were preincubated for 5 min at RT in presence of poly (dI-dC) (Amersham, Buckinghamshire, UK) to reduce aspecific bindings.

The reaction was performed at RT for 20 min in binding buffer (50% glycerol, 250 mM KCl, 100 mM HEPES, 1 mM DTT, 5 mg/ml BSA) using 5μg of nuclear extracts and 20 cpm oligonucleotides. Samples were loaded on a non-denaturing 6% polyacrylamide gel in 0.25x TBE (Tris-borate-EDTA buffer) for 2h at 4°C. The radioactive signals were detected using the Storm Imager (Amersham, Buckinghamshire, UK).

DNA pull-down assay

Streptavidin magnetic beads (Promega, Madison, WI) were washed three times with

In-gel tryptic digestion and MALDI-TOF/TOF analysis

Bands were excised from the gels with an automatic spot picker (Investigator ProPic, Genomic Solutions Inc.) and subjected to in-gel tryptic digestion. In brief, gel bands were destained with 50%ACN in 50 mM ammonium bicarbonate. Then, the gel bands were shrunk by addition of 100%ACN. The dried gel pieces were reswollen with 4. Immunoblots reported are from one experiment representative of three that gave similar results. Density of immunoreactive bands was calculated using the software AlphaEaseFC (Alpha-Innotech), normalized for actin and reported as arbitrary units (shown as the mean ± S.D.). Each value was normalized for actin or SOD1 and reported as % of siCtr. (E, F) ETNA -/- cells were transfected with pApaf1 expressing vector or with the empty vector (-) and after 24 h they were left untreated (-) or treated with hydrogen peroxide (500μM) for 1, 3 and 6 hours.

3 '.

 3 This region was cloned into the HindIII site of pGL3b-asic vector (expressing Firefly luciferase) (Promega, Madison, WI), generating the -4210 construct, from which the -1509, -589, and +305 deletion constructs were created using the sites for NheI-EcoRV, XhoI and KpnI, respectively. A series of successive deletions ( -1402, -1351, -1287, -1257, -1202, -1027, -903) was generated by PCR using -4210 construct as template and cloning them into the NheI-AatII sites.For the deletion constructs the reverse oligonucleotide was 5'-AGTGAGACGTCTG CTCCT-3'; while the forward oligonucleotides were the following: 1) 5'-ATG CTAGCTGGGTTGTATGTGTCATAC-3', 2) 5'-ATGCTAGCAGTACTAGGGAGGT GGAGGC-3', 3) 5'-ATGCTAGCTGAGTTCAAGGACAGCC-3', 4) 5'-ATGCTAGCAC AGGAGAAACCCTGTATCAAA-3', 5) 5'-ATGCTAGCAATGTTTAGTCTTAAGGGA-3-pGL3-basic was generated cloning the CMV promoter upstream the luciferase cDNA into the BglII-SpeI sites.The constructs E1 -589, E2 -589 and E1pCMV were generated cloning the doublestranded oligonucleotides (obtained annealing 5 min at 95° and o.n. at RT) E1 and E2 (5'-ATTTACACTAATATATCACTAATATATGCTTGGTTTCAAAGCTACAAAT-3' and 5'-GATATCCAGAGTCTTTTCTGAATTCTATTAGCTCTCTTATTCCAC-3') upstream of the promoters in -589 and pCMV-pGL3-basic constructs, using the sites for BglII and BglII-SpeI, respectively.The plasmid containing the full-length form of mouse Ku70 (pKu70), cloned upstream the p3XFLAG-CMV-10 (Sigma-Aldrich Inc., St. Louis, MO), was a kind gift of Dr Yasushi Enokido from Tokyo University. pKu86 was obtained by PCR cloning of the full-length mouse Ku86 cDNA between the sites NotI and BamHI (modified to blunt end) of p3XFLAG-

  Anti-H2A.X antibody (clone JBW301, Millipore, Billerica, MA) was used at a concentration of 1:1000. Anti-cytochrome c monoclonal antibody (clone 7H8.2C12, BD Biosciences, San Jose, CA) and anti-Cu/Zn SOD (SOD1) polyclonal antibody (Assay Designs, Ann Arbor, MI) were used at concentration of 1:1000. Anti-OxPhos Complex I 39 kDa subunit antibody (clone 20C11, Molecular Probes-Invitrogen, Carlsbad, CA) was used at concentration of 1:2000. Anti--actin (clone AC15) antibody was purchased from Sigma-Aldrich Inc. (St. Louis, MO) (1:3000 diluted).

Buffer A ( 5 mM

 5 Tris pH 8, 0.5 mM EDTA, 1 M NaCl). Annealed E1 oligonucleotide (biotinlabelled) was incubated with beads for 15 min at RT in buffer A. Beads were then washed twice with buffer A and resuspended in buffer C (5 mM Tris pH8, 1 mM EDTA, 1 mM DTT, 50 mM NaCl). Nuclear proteins, extracted by a nuclear extraction kit (Active Motif, Carlsbad, CA), were incubated with beads bound to the oligonucleotide E1 or to a negative control oligonucleotide (5'-GGGGTTGTATGTGTCATACAGCCAGGTT-3'), washed twice with buffer C and eluted in 1 M NaCl or by using oligonucleotide competition. Eluted proteins were analysed by SDS-PAGE and stained with SYPRO Ruby (Bio-Rad, Hercules, CA).

Figure 1

 1 Figure 1 Analysis of murine Apaf1 promoter activity. (A) A schematic representation of the

Figure 2

 2 Figure 2 Identification of E1 as the minimal region able to reduce both Apaf1 and CMV

Figure 3

 3 Figure 3 In vitro and in vivo interaction between Ku70/86 and murine Apaf1 promoter. (A)

Figure 4

 4 Figure 4 DNA damage induction by hydrogen peroxide and etoposide. (A) ETNA cells were

Figure 5

 5 Figure 5 Dynamics of Ku/Apaf1 promoter binding upon DNA damage. (A-D) ETNA cells

Figure 6

 6 Figure 6 Cell death regulation under Ku and Apaf1 modulation and DNA damage. (A)

(

  E) Apaf1, cleaved caspase 3 and H2A.X were assayed in the total, cytosolic and nuclear extracts, respectively. Actin and H2B were used as loading controls. Immunoblots reported are from one experiment representative of three that gave similar results. Density of Apaf1 immunoreactive bands was calculated using the software AlphaEaseFC, normalized for actin and reported as arbitrary units (shown as the mean ± S.D.). (F) The mRNA levels of Apaf1 were evaluated at each time point by quantitative real time PCR using oligonucleotides specific for the C-terminus of Apaf1 mRNA, which do not anneal to the endogenous Apaf1/genetrap fused transcript produced in ETNA -/-cells. Data display the fold-changes of Apaf1 mRNA relative to control cells, which is arbitrary set as 1, and were shown as the mean ± S.E.M.; n = 3, * p 0.05, ** p 0.01.

Figure 1 pGL3mApaf

 1 Figure 1

  Figure 3

  Figure 4

Table 1 .

 1 MALDI-TOF/TOF identifications

		Swiss		No.	No.	%
		Prot	Mascot	Masses	Masses Not	Sequence	MW	MW
	Protein Name	Entry	Score	Matched	Matched	Coverage	Calculated	Observed
	ATP-dependent DNA
	helicase II, 80 kDa	P13010	158	27	40	39	83091,5	90000
	subunit (Ku86)
	ATP-dependent DNA
	helicase II, 70 kDa	P12956	166	28	40	47	69953,1
	subunit (Ku70)

actin

Acknowledgements: F. Cecconi is supported by Grants from the Telethon Foundation, AIRC, the Italian Ministry of Health and The Italian Ministry of University and Research. D. De Zio is supported by a fellowship from the Telethon Foundation. The authors thank M. Acuna Villa and M. W. Bennett for editorial and secretarial work, F. Florenzano and G. Filomeni for research assistance, A. Nobili for technical support.

ethanol precipitation and resuspended in 30 μl TE.

Quantitative real time PCR was performed by using 1μl DNA. Data were normalized to the input and calculated as 2 -CT , and the obtained values were related to the no-antibody condition ( 2 -CT ). All reactions were performed in triplicate. Primers used to amplify the Apaf1 repressing region are forward: 5'-CCTGGAGGATATCCAGAGTC-3' or 5'-TGTCTGGTGTCTTACAACCATTG-3', reverse: 5'-CATCCCGCTCAACATTTGTA-3' or 5'-GTTCGTTGTTGCTGAGCGATAAA-3'. Primers used to amplify the transcription start point (TSP) region are forward: 5'-CTGGGCGAGTGAATTTGC-3' and reverse: 5'-AGACGTCTGCTCCTCTAGCTG-3'. Primers used to amplify the glucose-6-phosphate dehydrogenase X-linked (G6pdx) gene are forward: 5'-AGACTAGCCCGAAGCTCCTC-3' and reverse: 5'-TCAGACCACCCCTTTACCTG-3'.

Analysis of cell viability and apoptosis

Adherent and detached cells were combined and stained with 50 μg/ml propidium iodide prior to analysis by a FACScalibur instrument (BD Biosciences, San Josè, KA). Apoptotic cells were evaluated by calculating peak areas of hypodiploid nuclei in previously premeabilized cells (sub-G1).

Immunofluorescence

ETNA cells, grown in 35-mm Petri dishes, were washed in PBS and then fixed with 4% paraformaldehyde in PBS for 30 min. After permeabilization with 0.4% Triton X-100 in PBS for 5 min, cells were blocked in 3% bovine serum albumin (BSA) or 10% normal goat serum (NGS) in PBS for 30 min and incubated over night at 4°C with anti-H2A.X or with anti-8-OHdG primary antibodies (clone JBW301, Millipore, Billerica, MA; clone 15A3, QED Biosciences Inc., San Diego, CA), in 1% BSA or 2% NGS and 0,4% Triton X-100.

Cells were then washed in PBS and incubated for 1 hour with Alexa Fluor 488-conjugated goat anti-mouse (Molecular Probes, Eugene, OR). After rinsing in PBS, cell nuclei were stained with 1 μg/ml DAPI and observed by confocal microscopy with a Leica TSC-SPE confocal microscope equipped with a 63x/1.15 Olympus objective and a Leica Application Suite (LAS) software (Leica Microsystems). Fluorescence images were adjusted for brightness, contrast and colour balance by using Adobe Photoshop CS (San Jose, CA). Quantification was done on 30 cells per field, at least 6 fields (n = 6) for each time point.

Densitometric analyses of fluorescence images

Data presentation

All experiments were performed at least three different times, unless otherwise indicated. Data are expressed as the means ± S.D. or S.E.M. and significance was assessed by Student's t test. Differences with p values < 0.05 were considered as significant.


MD Umberto Aguglia

MD Ettore Beghi

MD Angelo Labate
email: a.labate@isn.cnr.it

PhD Francesca Condino

MD Vittoria Cianci

MD Laura Mumoli

Sara Gasperini

MD Aldo Quattrone

MD. Antonio Gambardella

MD Sara Gasparini

Cianci, Gambardella, Gasparini, Labate, Mumoli Aguglia

Condino

Labate, Quattrone Gambardella

Age At Onset Predicts Good Seizure Outcome In Sporadic Non-Lesional and Mesial Temporal Sclerosis Based Temporal Lobe Epilepsy

Keywords: Temporal Lobe Epilepsy, Prognosis, Mesial Temporal Sclerosis, Partial epilepsy

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Temporal lobe epilepsy (TLE) represents the most common type of partial epilepsy. From a neuropathological point of view, TLE may be divided into "lesional" and "non lesional" subtypes.

In epilepsy surgery series, the most frequent causes of lesional TLE are tumors, vascular malformations, cortical dysplasia or gliosis/scar due to remote etiologies, traumatic lesions and less commonly immune mediated condition such as limbic encephalitis. [START_REF] Wolf | Surgical pathology of chronic epileptic seizure disorders[END_REF][START_REF] Soeder | Causes, presentation and outcome of lesional adult onset mediotemporal lobe epilepsy[END_REF][START_REF] Malter | Antibodies to glutamic acid decarboxylase define a form of limbic encephalitis[END_REF] Conversely, non lesional TLE represents the most common subtype and comprises patients with mesial temporal sclerosis (MTS). [START_REF] Jack | Intractable non-lesional epilepsy of temporal lobe origin: lateralization by interictal SPECT versus MRI[END_REF][START_REF] Labate | MRI evidence of mesial temporal sclerosis in sporadic "benign" temporal lobe epilepsy[END_REF] It is a heterogeneous disorder since there are patients with a severe refractory form, while many others have a mild epileptic disorder and they enter remission with or without antiepileptic medication. [START_REF] Labate | MRI evidence of mesial temporal sclerosis in sporadic "benign" temporal lobe epilepsy[END_REF][START_REF] Aguglia | Mild Non-lesional Temporal Lobe Epilepsy. A Common, Unrecognized Disorder with Onset in Adulthood[END_REF] Indeed, in this type of epilepsy the prediction of the clinical course would help both physicians and patients. Because many studies on TLE have originated from groups with a special interest in surgical treatment, the available information mostly concerns patients with refractory non lesional TLE. [START_REF] Jack | Intractable non-lesional epilepsy of temporal lobe origin: lateralization by interictal SPECT versus MRI[END_REF][START_REF] Jackson | Hippocampal sclerosis can be reliably detected by magnetic resonance imaging[END_REF][START_REF] Berkovic | Hippocampal sclerosis in temporal lobe epilepsy demonstrated by magnetic resonance imaging[END_REF][START_REF] French | Characteristics of medial temporal lobe epilepsy: I. Results of history and physical examination[END_REF][START_REF] Williamson | Characteristics of medial temporal lobe epilepsy: II Interictal and ictal scalp electroencephalography, neuropsychological testing, neuroimaging, surgical results, and pathology[END_REF] In such cases, antecedent factors for epilepsy, such as febrile seizures (FS) or cerebral infections are very common, [START_REF] French | Characteristics of medial temporal lobe epilepsy: I. Results of history and physical examination[END_REF] and MTS represents the most common substrate as demonstrated by magnetic resonance imaging (MRI) studies. [START_REF] Jack | Intractable non-lesional epilepsy of temporal lobe origin: lateralization by interictal SPECT versus MRI[END_REF][START_REF] Jackson | Hippocampal sclerosis can be reliably detected by magnetic resonance imaging[END_REF][START_REF] Berkovic | Hippocampal sclerosis in temporal lobe epilepsy demonstrated by magnetic resonance imaging[END_REF][START_REF] French | Characteristics of medial temporal lobe epilepsy: I. Results of history and physical examination[END_REF][START_REF] Williamson | Characteristics of medial temporal lobe epilepsy: II Interictal and ictal scalp electroencephalography, neuropsychological testing, neuroimaging, surgical results, and pathology[END_REF] In a previous cross-sectional study comparing mild to severe non-lesional TLE, [START_REF] Aguglia | Mild Non-lesional Temporal Lobe Epilepsy. A Common, Unrecognized Disorder with Onset in Adulthood[END_REF] we found that mild non-lesional TLE is an unrecognized disorder characterized by onset in adulthood and frequent family history of epilepsy, including TLE. Familial TLEs include autosomal dominant lateral TLE (ADLTLE) and familial mesial TLE (FMTLE). Most patients with familial TLE have a benign course, [START_REF] Ottman | LGI1 mutations in autosomal dominant partial epilepsy with auditory features[END_REF][START_REF] Striano | Familial mesial temporal lobe epilepsy (FMTLE): a clinical and genetic study of 15 Italian families[END_REF][START_REF] Gambardella | Familial mesial temporal lobe epilepsies: clinical and genetic features[END_REF] and refractory seizures have been reported only in a minority of patients. [START_REF] Kobayashi | Hippocampal atrophy and T2 weighted signal changes in familial mesial temporal lobe epilepsy[END_REF] The prognosis of sporadic non-lesional TLE has not been investigated by prospective studies; moreover, a recent longitudinal study conducted in children with new-onset epilepsy demonstrated that MRI lesions predict poor seizure outcome. [START_REF] Spooner | New-onset temporal lobe epilepsy in children: lesion on MRI predicts poor seizure outcome[END_REF] Based on this knowledge, our aim was to evaluate predictive prognostic factors in a cohort of patients with well defined non-lesional, sporadic TLE regardless of the age of onset. Criteria for inclusion in the study were a history of two or more unprovoked seizures of temporal lobe origin, regardless of the age of onset, being resident in Calabria, and having a follow-up of two years or longer. Data collected at enrolment included patient's demographics, perinatal factors (encephalitis, brain anoxia, trauma, ischemia or hemorrhage), family history of non-TLE or febrile seizures (FS), personal history of FS, age at onset of TLE, seizure semiology and frequency, neurological and cognitive findings, antiepileptic drugs (AEDs), interictal EEG and imaging. Ictal phenomena were coded with reference to the 1981 international classification. [START_REF]Commission on Classification and Terminology of the International League Against Epilepsy. Proposal for revised clinical and electroencephalographic classification of epileptic seizures[END_REF] In subjects with normal neuroimaging and interictal EEG, the following semiological criteria were considered reliable to make a diagnosis of TLE: déjà-vù, stereotyped flashbacks or olfactory hallucination and a rising epigastric aura. Three of us (A.G., A.L., U.A.) reviewed, double blind, the interictal EEGs acquired in each patient at first investigation. The interictal EEGs were recorded according the 10-20 International system with supplementary T1 and T2 electrodes. The EEG abnormalities (spikes, spike-waves, sharp theta/delta waves, runs of temporal intermittent rhythmic delta activity over one temporal region) were labeled as unilateral if confined to one temporal side at least 95% of the time. MTS was diagnosed by MRI and coded as present or absent. Excluded were patients without TLE, patients with familial TLE, patients with incomplete clinical history, poor compliance, psychogenic seizures, and patients with only EEG or imaging documentation suggestive of TLE. MRI evidence of MTS, simultaneous presence of FLAIR hyperintensity and IR atrophy or scattered T2-hyperintense spots on hemispheric white matter were not considered reasons for exclusion. [START_REF] Labate | Temporal lobe abnormalities on brain MRI in healthy volunteers. A prospective case-control study[END_REF] In the whole group carbamazepine (CBZ) or oxcarbazepine (OXC) were the most utilised AEDs and they were usually in monotherapy (51% of the patients) in the seizure-free group and in polytherapy (74% of the patients) in the non seizure-free group, in combination with topiramate (36% of these patients), phenobarbital (28%), levetiracetam (10%) or other AEDs (3%).

Methods

Initial

Follow-up study. Follow-up examinations were performed every 2-12 months only in patients who fulfilled the inclusion criteria and were managed in person by trained epileptologists (A.G., A.L., U.A.) of the two University Centers. Follow-up data included family history of epilepsy, seizure frequency and semiology, behavioral and clinical findings. MRI study was performed at the Institute of Neurologic Science of National Research Council, Piano Lago, Cosenza, following a methodology extensively described by our group elsewhere. [START_REF] Labate | MRI evidence of mesial temporal sclerosis in sporadic "benign" temporal lobe epilepsy[END_REF] Imaging done before 2000 was repeated. All imaging was evaluated by a neuroradiologist blind to patient identity, initial imaging findings, seizure localization and outcome. The last follow-up examination was performed in May 2006.

Diagnosis of patients.

All diagnoses were re-evaluated every 3-6 months by a panel consisting of two epileptologists who were not blinded to previous reviews. Although EEG and imaging data were not obligatory, the diagnosis was changed according to different clinical, EEG or imaging findings achieved during follow up.

Statistical analysis. The end point was time to 24-month seizure freedom (no seizures with or without AED treatment). We classified patients according to tertiles of age at onset of epilepsy(<13.7 years, 13.7 to 32.0 years, >32.0 years) and then determined the effect of the tertiles on the endpoint. For continuous variables, mean, SD and range were calculated and the Student's t test for unpaired samples was used to assess differences between groups. Categorical variables were expressed as percentages and differences among group distributions were assessed using the chisquare test. Survival curves were generated according to the Kaplan-Meier (KM) method and compared with the log-rank test. With reference to 24-month seizure freedom, prognosis was coded into two categories (0=good; 1=poor) and assessed by univariate and multivariate analysis. Tested covariates included age at onset of epilepsy, gender, family history of epilepsy, FS, perinatal factors, ictal manifestations, MTS, and interictal EEG (Table 1). First, we identified by univariate analysis variables that resulted associated to poor prognosis at a 5% level of significance. These variables were then included into a Cox proportional-hazards regression model. Data have been expressed as hazard ratios with 95% confidence intervals. All calculations were done by SPSS version 13 (Chicago, Illinois, USA).

Results

Patients' recruitment and follow-up. A total of 473 patients were assessed. Two hundred fourteen (45%) fulfilled our inclusion criteria whereas 259 were excluded for one or more of the following Clinical and instrumental findings. The sample included 104 women and 86 men. Mean age at study entry was 45.2+17.6 years, and age at onset of epilepsy was 33.0+19.7 years. Family history of epilepsy was identified in 58 patients (30%) whereas 33 (17%) had febrile seizures and nine (4.7%) had perinatal events. MRI scans at enrollment showed signs of MTS in 57 patients (30%) that were confirmed on unblinded re-evaluation in 51 (26.8%). One hundred and 13 patients (53.5%) had epigastric aura, 67 (35.3%) autonomic phenomena (flushing, whitening, tachycardia, diaphoresis, hyperthermia, or pilomotion), 41 (21.6%) unilateral dystonic movements, 23 (12%) dysfasia (impaired comprehension, anomia or paraphasia), 19 (10%) drop attacks, 17 (8.9%) auditory features, 14 (7.4%) dejà/jamais or vu-veçu, and seven (3.7%) olfactory seizures. Complex partial seizures (loss of consciousness or unresponsiveness preceded by aura and/or associated with oro-alimentary or hand automatisms) were found in 145 patients (73.3%), and secondarily generalization (major motor seizures) was present in 115 (60.5%). Patients with MTS differed from those without MTS in terms of history of febrile seizures (47.1 vs. 6.5%), mean age at onset of epilepsy (15.7 vs. 29.5 years), epigastric aura (70.6 vs. 55.4%), dysphasic seizures (19.6 vs. 9.4%), and interictal EEG abnormalities (94.1 vs. 78.4%). Epileptiform abnormalities were found in 147 patients (77.4%) unilaterally and in ten patients (5.2%) bilaterally. Normal interictal EEG was found in 33 patients (17.4%). A strong correlation between EEG epileptiform activity and MRI features of MTS has been previously reported elsewhere. [START_REF] Labate | MRI evidence of mesial temporal sclerosis in sporadic "benign" temporal lobe epilepsy[END_REF] Carbamazepine (CBZ) or oxcarbazepine (OXC) were the commonest AEDs given as monotherapy in seizure-free patients (51%) or in polytherapy in non seizure-free patients (74%) in combination with topiramate (36%), phenobarbital (28%), levetiracetam (10%), or other drugs (3%).

Cumulative probability of remission and prognostic predictors. As shown in Table 1, 100 patients (53%) achieved 24-month seizure freedom. The cumulative probability of remission was 42.2% at 2 years, 48.1% at 5 years, and 49.4% at 10 years. The chance of remission was unaffected by seizure type (data not shown), but was directly correlated to the age at onset of seizures (Figure 1) and inversely correlated to a history of febrile seizures and to the presence of MTS (Table 2).

Compared to patients with normal interictal EEG, those with abnormal tracings had a lower, nonsignificant chance of remission. However, only age at disease onset was confirmed by multivariate analysis to be an independent predictor of seizure remission (Table 3).

Discussion

TLE is the commonest type of partial epilepsy and MTS the commonest underlying pathology. Indeed, TLE is considered predominantly intractable and seizures are very often physically and socially disabling. In such cases, an abnormal EEG, the presence of MTS, and an unsuccessful response to first AED treatment predict long-term prognosis and seizure relapse. [START_REF] Hauser | Seizure recurrence after a 1st unprovoked seizure: an extended follow-up[END_REF][START_REF] Berg | Relapse following discontinuation of antiepileptic drugs: a metaanalysis[END_REF] Furthermore, in approximately one-third of children or adults with TLE, MRI lesions, specially MTS or dual pathology, are predictors of intractable epilepsy and pharmacoresistance. [START_REF] Spooner | New-onset temporal lobe epilepsy in children: lesion on MRI predicts poor seizure outcome[END_REF][START_REF] Semah | Is the underlying cause of epilepsy a major prognostic factor for recurrence?[END_REF][START_REF] Briellmann | Hippocampal sclerosis: MR prediction of seizure intractability[END_REF] Conversely, many patients with sporadic or familial TLE have well controlled epilepsy, regardless of seizure semeiology and presence of MTS. [START_REF] Labate | MRI evidence of mesial temporal sclerosis in sporadic "benign" temporal lobe epilepsy[END_REF][START_REF] Aguglia | Mild Non-lesional Temporal Lobe Epilepsy. A Common, Unrecognized Disorder with Onset in Adulthood[END_REF][START_REF] Gambardella | Familial mesial temporal lobe epilepsies: clinical and genetic features[END_REF][START_REF] Labate | Temporal lobe abnormalities on brain MRI in healthy volunteers. A prospective case-control study[END_REF][START_REF] Blom | Incidence of epilepsy in children: a follow-up study three years after the first seizure[END_REF] However, early prognostic predictors are still limited to acquired refractory TLE, with little attention to the more benign drug-responsive TLE variants. [START_REF] Labate | MRI evidence of mesial temporal sclerosis in sporadic "benign" temporal lobe epilepsy[END_REF][START_REF] Aguglia | Mild Non-lesional Temporal Lobe Epilepsy. A Common, Unrecognized Disorder with Onset in Adulthood[END_REF][START_REF] Labate | Hippocampal and thalamic atrophy in mild temporal lobe epilepsy: a VBM study[END_REF] Our group has recently described clinical, genetic, neuroimaging features and outcome of benign sporadic TLE who were not selected for surgical purposes. In a previous cross-sectional study of 73 nonsurgical adult patients with non-lesional TLE (with or without MTS), about two-thirds were found to have a good prognosis after a follow-up ranging from 36 to 96 months. [START_REF] Aguglia | Mild Non-lesional Temporal Lobe Epilepsy. A Common, Unrecognized Disorder with Onset in Adulthood[END_REF] The identification of MRI evidence of MTS in many of these cases has shown that MTS itself does not necessarily mean intractable epilepsy and bad outcome as previously thought. [START_REF] Labate | MRI evidence of mesial temporal sclerosis in sporadic "benign" temporal lobe epilepsy[END_REF][START_REF] Labate | Hippocampal and thalamic atrophy in mild temporal lobe epilepsy: a VBM study[END_REF] Interestingly, in the present study we found that epilepsy remitted in approximately half (100 out of 190; 53%) of patients with non-lesional, sporadic TLE, after a mean follow-up of 11 years.

Although at univariate analysis predictors of seizure freedom included older age at onset (33 vs 17 years), lower occurrence of FS (11% vs 24%), and MTS (19% vs 35%), only age at seizure onset was an independent prognostic predictor. Our data cannot be entirely explained by selection bias nor by defects in the study design. In fact, although one may expect that only patients with persisting seizures tend to remain under surveillance, subjects with difficult-to-treat epilepsy are frequently seen in secondary and tertiary epilepsy centers regardless of their age at onset of seizures.

Other possible explanations include the lower tendency of seizures to relapse in adult and elderly individuals compared to children. Seizure recurrence has been correlated to younger age at onset by several investigators. [START_REF] Costeff | Convulsions in childhood: their natural history and indications for treatment[END_REF][START_REF] Blom | Incidence of epilepsy in children: a follow-up study three years after the first seizure[END_REF][START_REF] Hirtz | The risk of recurrence of non-febrile seizures in children[END_REF][START_REF] Beghi | Prognosis of epilepsy in newly referred patients: a multicenter prospective study. Collaborative group for the study of epilepsy[END_REF][START_REF] Hart | National general practice study of epilepsy: recurrence after a first seizure[END_REF] In a study of mostly adult surgical patients, more than half of participants had their onset of epilepsy during childhood and early adolescence. [START_REF] Berg | How long does it take for partial epilepsy to become intractable?[END_REF] In another study in patients undergoing temporal lobectomy, younger age at seizure onset was significantly correlated to the presence and grade of hippocampal sclerosis even after controlling for disease duration and history of febrile seizures. [START_REF] Davies | Relationship of hippocampal sclerosis to duration and age of onset of epilepsy, and childhood febrile seizures in temporal lobectomy patients[END_REF] In this series, the relationship between hippocampal sclerosis and history of febrile seizures was no longer significant when controlling for age at onset. On this basis, the authors concluded that an earlier age at onset of epilepsy predisposes to the development of childhood febrile seizures and the younger hippocampus is more susceptible to insults causing hippocampal sclerosis than that of more mature individuals. The same explanation can be offered for our findings.

We did not find seizure type or focal abnormal EEG to predict prognosis in our sample. Déjà vu was a very common ictal phenomenon in family members with benign mesial TLE mapping to chromosome 4q13.2-q21.3. [START_REF] Hedera | Familial mesial temporal lobe epilepsy maps to chromosome 4q13.2-q21.3[END_REF] Intriguingly, in our study experiential phenomena, such as déjàjamais vu/veçu, were slightly (but non significantly) more frequent in patients who achieved seizure freedom. As déjà vu experience is also common in normal subjects, this ictal phenomenon may have been misinterpreted and underestimated. Some of our patients also developed drop attacks, usually long after the onset of epilepsy, a finding that was already described in patients with TLE. [START_REF] Gambardella | Late-onset drop attacks in temporal lobe epilepsy: a reevaluation of the concept of temporal lobe syncope[END_REF] The fact that about 7% of our patients reported auditory auras, which are suggestive of a lateral temporal origin, further reinforces the view that idiopathic TLE with auditory features represents a benign clinical entity within the larger group of non-lesional TLE. [START_REF] Bisulli | Idiopathic partial epilepsy with auditory features (IPEAF): a clinical and genetic study of 53 sporadic cases[END_REF] Accordingly, also in these patients with mild lateral TLE, seizures started around the age of 20 years of age, which is later than that observed in refractory TLE.

Of interest, in our series this type of aura was much more rare than viscero-sensory or affective auras, a finding that is strikingly similar to what seen in autosomal dominant familial TLE. [START_REF] Striano | Familial mesial temporal lobe epilepsy (FMTLE): a clinical and genetic study of 15 Italian families[END_REF] It is therefore reasonable to speculate that benign sporadic and familial TLE are part of a biological continuum in which familial TLE inherited in an autosomal dominant manner is much more rare.

[33] This is not surprising, since TLE is mainly a heterogeneous disorder with complex genetics where putative susceptibility genes and environmental factors are believed to act together to produce the disease phenotype. We also found that the presence of EEG abnormalities, since the vast majority of patients (regardless of seizure freedom) showed focal interictal EEG abnormalities. Moreover, EEG abnormalities were usually unilateral (more than 70%) and located over one temporal region. Thus, the interictal EEG was not helpful to predict benign versus intractable course of TLE.

Our study has some limitations. First of all, the diagnosis of sporadic non lesional TLE was based predominantly on seizure description and interictal EEGs; however the diagnosis was made by trained epileptologists and confirmed through repeated follow-up assessment. Second, only 25 patients with frequent seizures underwent video-EEG monitoring thus preventing exclusion of extratemporal TLE in other cases. It must be addressed, however, that in all our patients the diagnosis of TLE was made on the basis of the constellation of clinical, MRI and EEG criteria, which are considered to be reliable interictal indicators of TLE. [START_REF]Commission on Classification and Terminology of the International League Against Epilepsy. Proposal for revised clinical and electroencephalographic classification of epileptic seizures[END_REF] Third, during the period of patients' enrolment (1987)(1988)(1989)(1990)(1991)(1992)(1993)(1994)(1995)(1996)(1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004), major improvements in MR technology occurred and it is possible that non-lesional patients (ie, without MTS) were more rarely encountered in the last years of recruitment.

In conclusion, this study provides new information in support of clinical decision making because we showed that older age at onset predicts good seizure outcome and more likely remission in patients with sporadic non-lesional TLE despite the presence of complex partial or secondary generalized seizures, focal EEG abnormalities and signs of MTS. Few questions still remain unsolved: Can patients with sporadic non-lesional TLE spontaneously remit? How long AEDs should be continued? Can genetic predisposition and susceptibility contribute to determine remission? For example, we recently observed that the A/G genotype of the GABA B receptor was associated with a 6.45 odds ratio to develop drug resistance in patients with TLE, [START_REF] Gambardella | GABA(B) receptor 1 polymorphism (G1465A) is associated with temporal lobe epilepsy[END_REF] findings not replicated in different ethnic populations. Prospective epidemiological population based studies are needed to address these questions.

  assessment. 473 patients with clinical diagnosis of partial seizures of temporal lobe origin were consecutively seen in from April 1987 to April 2004 in two twin University Epilepsy Centers located in Reggio Calabria and Catanzaro, Calabria, Southern Italy, sharing protocol, study design, and research objectives. Patients were ascertained from different sources, including general practitioners, pediatricians, emergency departments (Reggio Calabria hospital), and patients.

  reasons: familial TLE (n= 32); extratemporal seizures or uncertain seizure localization (n=103); psychogenic or other nonepileptic seizures (n=26), poor compliance (n=20); unavailable MRI study (n=10); brain lesions diagnosed by CT or MRI (n=95). Twenty-four additional patients were excluded during follow-up because of extra-temporal epilepsy (n=13), major stroke (n=2), generalized epilepsy (n=4), or drop-out (n=5). Thus, 190 patients were ultimately diagnosed with sporadic non-lesional TLE. The sample was followed for 2116.5 person-years (mean 11.1 years; range 2-75 years). Follow-up interictal EEGs were performed in 98 patients, video-EEG monitoring in 25 with daily seizures, and neuropsychological examination in 84. Follow-up MRI scans were performed in 25 patients and reviewed with knowledge of seizure localization and previous MRI findings.

Table 1 . Patient data Clinical and laboratory data

 1

	Seizure-free	Non seizure-free	p-value
	n=100	n=90

Table 2 . Cumulative time-dependent 24-month seizure freedom at 2, 5 and 10 years by selected prognostic predictors.

 2

	Results of univariate analysis
	______________________________________________________________________________
	_________________________
	Variable	24-month seizure freedom (%)
		2 years	5 years	10 years	P
	______________________________________________________________________________
	_________________________
	Gender
	Women	42.6	47.8	48.9
	Men	41.9	48.6	50.2
	Age, years (tertiles)
	<13.7	17.5	20.8	20.8
	13.7-32.0	47.4	52.8	55.0
	>32.0	61.9	71.3	73.5
	Family history of epilepsy
	No	41.0	47.9	48.9
	Yes	45.0	48.5	50.3
	Perinatal factors
	No	42.2	48.3	49.7
	Yes	44.4	44.4	44.4
	History of febrile seizures
	No	47.4	52.3	54.0
	Yes	18.2	27.6	27.6
	EEG
	Normal	63.6	63.6	63.6
	Abnormal, unilateral	38.3	44.3	45.9
	Abnormal, bilateral	30.0	50.0	50.0