PhD Bénédicte Mousson De Camaret

Jan-Willem Taanman

Sylvie Padet

Maïté Chassagne

Martine Mayençon

Pascale Clerc-Renaud

Ginette Mandon

Marie-Thérèse Zabot

Alain Lachaux

Dominique Bozon

Marie- Thérèse Zabot

Dominique Bozon ||

Kinetic properties of mutant deoxyguanosine kinase in a case of reversible hepatic mtDNA depletion

Keywords: Deoxyguanosine kinase, mutations, enzymatic assay, skin fibroblasts, mtDNA depletion, revertant phenotype mitochondrial DNA depletion syndrome, mtDNA, mitochondrial DNA, MRI, magnetic resonance imaging, ND2, NADH dehydrogenase 2, nt, nucleotide, q-PCR, quantitative PCR, TK2, thymidine

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

Introduction

Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is a quantitative disorder characterized by a variable tissue-specific reduction in mtDNA copy number. Myopathic (OMIM 609560) and hepatocerebral (OMIM 251880) forms are the main clinical presentations. The onset usually occurs in infancy or early childhood, but may be delayed into childhood with prolonged survival [START_REF] Ducluzeau | Progressive reversion of clinical and molecular phenotype in a child with liver mitochondrial DNA depletion[END_REF]. MDS patients with liver as the most or only affected organ usually die of severe liver failure before 1 year of age [START_REF] Ducluzeau | Depletion of mitochondrial DNA associated with infantile cholestasis and progressive liver fibrosis[END_REF][START_REF] Salviati | Mitochondrial DNA depletion and dGK gene mutations[END_REF][START_REF] Slama | Deoxyguanosine kinase mutations and combined deficiencies of the mitochondrial respiratory chain in patients with hepatic involvement[END_REF]. We reported previously the first case of a progressive reversion of the clinical and molecular phenotype in a child (patient 1) with liver-restricted MDS [START_REF] Ducluzeau | Progressive reversion of clinical and molecular phenotype in a child with liver mitochondrial DNA depletion[END_REF]. MDS is an autosomal recessive disorder and five nuclear genes are known to be involved in the pathogenesis of MDS in infancy or childhood: deoxyguanosine kinase (DGUOK) [START_REF] Mandel | The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA[END_REF], thymidine kinase 2 (TK2) [START_REF] Saada | Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy[END_REF] and SUCLA2 [START_REF] Elpeleg | Deficiency of the ADPforming succinyl-CoA synthase activity is associated with encephalomyopathy and mitochondrial DNA depletion[END_REF] involved in the maintenance of the mitochondrial dNTP pool, POLG involved in mtDNA replication [START_REF] Naviaux | POLG mutations associated with Alpers'syndrome and mitochondrial DNA depletion[END_REF] and MPV17 controlling mtDNA maintenance [START_REF] Spinazzola | MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion[END_REF]. In mammal cells and tissues, DGUOK and TK2 are the two rate-limiting enzymes involved in the first step of the mitochondrial salvage pathway of the purine and pyrimidine deoxyribonucleosides, respectively. Mutations in the DGUOK gene have been identified in patients with hepatic or hepatocerebral forms of MDS [START_REF] Salviati | Mitochondrial DNA depletion and dGK gene mutations[END_REF][START_REF] Slama | Deoxyguanosine kinase mutations and combined deficiencies of the mitochondrial respiratory chain in patients with hepatic involvement[END_REF][START_REF] Mandel | The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA[END_REF][START_REF] Taanman | A novel mutation in the deoxyguanosine kinase gene causing depletion of mitochondrial DNA[END_REF]. For this reason, we decided to study this gene in order to identify possible molecular anomalies in patient 1.

The human DGUOK is localized to chromosome band 2q13 and spans nearly 32 kb with 7 exons. Five alternative splice variants encoding four different protein isoforms have been described for this gene. Variant 1 represents the longest transcript (NM_0890916) and encodes the longest 277 aa isoform. Human DGUOK (nucleoside triphosphate deoxyguanosine 5'-phosphotransferase, EC 2.7.1.113) is a dimer of two identical subunits of 30 kDa. The three-dimensional structure of the human DGUOK monomer has been reported [START_REF] Johansson | Structural basis for substrate specificities of cellular deoxyribonucleoside kinases[END_REF], providing an explanation for the substrate specificity of the enzyme. DGUOK selectively phosphorylates purine deoxynucleosides and their analogs, with the highest efficiency for dG, using ATP and UTP as the most efficient phosphate donors. DGUOK belongs to the family of deoxynucleoside kinases, which includes mammal deoxycytidine kinase (dCK) and thymidine kinase 2 (TK2) and the Drosophila melanogaster deoxynucleoside kinase (Dm-dNK). The high sequence homology between DGUOK and cytosolic dCK (~45%) [START_REF] Johansson | Structural basis for substrate specificities of cellular deoxyribonucleoside kinases[END_REF] explains why dCK has an overlapping substrate specificity with DGUOK [START_REF] Wang | Substrate specifity of mitochondrial 2'-deoxyguanosine kinase. Efficient phosphorylation of 2chlorodeoxyadenosine[END_REF][START_REF] Arnér | Mammalian deoxyribonucleoside kinases[END_REF]. DGUOK activity measurements have been described in cell and tissue extracts, including human skin fibroblasts [START_REF] Wang | Substrate specifity of mitochondrial 2'-deoxyguanosine kinase. Efficient phosphorylation of 2chlorodeoxyadenosine[END_REF]. Kinetic parameters of the enzyme have been Copyright 2006 Biochemical Society assessed with DGUOK purified from bovine liver [START_REF] Park | Properties of a highly purified mitochondrial deoxyguanosine kinase[END_REF][15] or brain [START_REF] Wang | Substrate specifity of mitochondrial 2'-deoxyguanosine kinase. Efficient phosphorylation of 2chlorodeoxyadenosine[END_REF] and with human recombinant DGUOK [START_REF] Herrström Sjöberg | Substrate specificity of human recombinant mitochondrial deoxyguanosine kinase with cytostatic and antiviral purine and pyrimine analogs[END_REF].

To date, ten different DGUOK mutations have been identified in 17 MDS families with one or more affected siblings [3-5, 10, 17-18]. A partial defect of DGUOK activity has been reported in the liver of one patient who presented with isolated liver failure and carried two compound heterozygous missense mutations [START_REF] Salviati | Mitochondrial DNA depletion and dGK gene mutations[END_REF]. Site-directed mutagenesis has been used to study the consequences of these two missense mutations on the activity of recombinant enzymes [START_REF] Wang | Mitochondrial deoxyguanosine kinase mutations and mitochondrial DNA depletion syndrome[END_REF].

In this study, two different missense mutations were identified in patient 1. To find out if these mutant enzymes could be involved in the mechanism responsible for the reversal of the MDS phenotype, we optimised the DGUOK activity assay for cultured skin fibroblast mitochondria. We determined the kinetic parameters of the wild-type and mutant enzymes in organello and showed that the DGUOK mutant enzymes from patient 1 have a markedly higher residual activity compared to the severe deficiency of two other patients with splicing defects or premature stop codon. DGUOK activity in mitochondria from skin fibroblasts correlated with the course of the disease and the type of mutations in DGUOK.

Subjects and methods

Case reports

Patient 1. This boy was born to unrelated French parents with no clinical relevant family history. The initial patient history until age 28 months was previously reported [START_REF] Ducluzeau | Depletion of mitochondrial DNA associated with infantile cholestasis and progressive liver fibrosis[END_REF]. He presented with infantile cholestasis and progressive liver fibrosis due to mtDNA depletion (85 % at 12 months of age). For this liver MDS without extra-hepatic clinical involvement at age 28 months, a liver transplantation was initially discussed to correct the progressive liver failure in case of worsening. Surprisingly however, the patient displayed a spontaneous gradual improvement of his liver function with continuous increment of clotting factors since 32 months of age. At the age of 46 months, clinical, biochemical and molecular follow-up demonstrated an almost complete reversion of the MDS [START_REF] Ducluzeau | Progressive reversion of clinical and molecular phenotype in a child with liver mitochondrial DNA depletion[END_REF].

During the following months, liver tests continued to improve [START_REF] Ducluzeau | Progressive reversion of clinical and molecular phenotype in a child with liver mitochondrial DNA depletion[END_REF]. At 10 years of age, the child is now clinically healthy without any treatment or special regimen, yet hepatic ultrasonography showed the same heterogeneous liver compared to the previous examinations. Kidney and cardiac function, and cerebral magnetic resonance imaging (MRI) showed no abnormalities. Clotting factors remained stable (II 64%, V 73%, VII 73%) as well as prothrombine time (70% of the normal value). The liver-specific enzyme, alanine aminotransferase (ALAT) remained slighly increased (104 U/l; normal: 10-65). Lactate level was normal after fasting and feeding (1.1 and 2.0 mmol/l, respectively; normal <2.2).

Lactate/pyruvate ratios were also normal (<12; normal <20). A second child, a boy, was born unaffected. Informed consent for this study was obtained from the parents.

Patient 2. This boy presented with a typical form of hepatocerebral MDS. He was the second child of healthy young unrelated French parents without family history. A first child, a boy, died of a hemorrhagic syndrome of unknown etiology at 10 days of life. At birth, the baby presented with sub-ictere, hypoglycemia and lactic acidosis. At 2 months of age, he became hypotrophic and developed progressive axial hypotonia and roving eye movements. Visual evoked potentials were markedly abnormal. MRI did not show evident abnormality of the white matter. Electron microscopy of the liver revealed steatosis and an accumulation of abnormal pleomorphic mitochondria with abnormal cristae. Enzymatic measurements of the respiratory chain complexes revealed a specific severe defect of the activities of the complexes I, III and IV (5, 7 and 7% of the mean control values, respectively) related to mtDNA depletion (85%) ("other case" in [START_REF] Ducluzeau | Depletion of mitochondrial DNA associated with infantile cholestasis and progressive liver fibrosis[END_REF]). In contrast, complex II activity was at the upper limit of the normal range. Subsequently, liver function rapidly worsened. Neurological Informed consent for the current studies was obtained from the parents.

Cell cultures and mitochondria preparation

Fibroblasts from forearm skin explants obtained from patients, family members and controls were cultured in Ham F10 medium supplemented with 12% of fetal calf serum, with penicilin, streptomycin and amphotericin B and 50 mg/L of uridine. Cells were routinely cultured in plastic flasks (T175) at 37°C in a humidified atmosphere of 5% CO 2 in air. Cultures were checked for mycoplasma infection prior to all experiments. Mitochondria-enriched preparations from cultured fibroblasts were obtained as described previously [START_REF] Procaccio | Nuclear DNA origin of mitochondrial complex I deficiency in fatal infantile lactic acidosis evidenced by transnuclear complementation of cultured fibroblasts[END_REF][21]. For liver, the method for preparation of mitochondria was adapted from Rustin et al [START_REF] Rustin | Biochemical and molecular investigations in respiratory chain deficiencies[END_REF], using a homogenization buffer containing 40 mM KCl, 2 mM EGTA, 20 mM Tris-HCl pH 7.4 and 1 mg/mL of BSA. Mitochondria were washed once in their respective buffers and pelleted for 15 min at 11,000 g. Pellets were resuspended in a minimal volume of buffer and kept in aliquots at -80°C. Protein concentration was measured by the bicinchoninic acid method.

Enzymatic measurements

Enzymatic activities of the respiratory chain complexes were measured in liver homogenates and in mitochondria-enriched preparations from cultured fibroblasts as reported previously Tris-HCl, pH 8, 5/6 mM ATP/MgCl 2 , 0.25 mg/mL of BSA, 2 mM DTT, and 50 µM [ 3 H] labeled nucleoside (SA 50 GBq/mmol) in a total volume of 50 µL. To exclude possible interference by cytosolic deoxycytidine kinase (dCK), reactions were conducted in the presence of 2 mM deoxycytidine (dC). One unit is defined as the formation of 1 pmol product (dAMP or dGMP) per hour per mg of protein. Assays were initiated by addition of 2.5 to 7.5 µg of mitochondrial protein per assay and conducted at 37°C during 16 h for the dA substrate and 18 h for the dG substrate. The amount of deoxyribonucleotides formed was determined by spotting aliquots of 40 µl on Whatman DE-81 anion-exchange papers (20x25 mm) (Fisher Bioblock, USA). The papers were then washed 1×15 min and 1×5 min in 1 mM ammonioformate (Carlo Erba, Italy). The papers were put in 1 mL elution buffer (0.1 M HCl + 0.4 M KCl); 4 mL Ultima Gold Safe scintillation fluid (Perkin Elmer, USA) was added and the radioactive solutions were counted in a Tri-Carb scintillation counter (Perkin Elmer, USA). For the measurement of the phosphate acceptor constants, a wide concentration range of each deoxynucleoside was used (0.3 to 50 µM for dG and 0.6 to 50 µM for dA). For the measurement of phosphate donor constants, ATP/MgCl 2 was varied (0.25/0.30-7.5/9 mM), while dG or dA was kept at 20 µM. For mitochondria-enriched preparations from liver, the same procedure was used with the following modifications: 1/1.2 mM ATP/MgCl 2 in the standard reaction mixture, 15 µg of protein per assay and assays were conducted during 6 h for the dA substrate and 4h for the dG substrate. The data were analyzed by the Sigma Plot 9.0 Enzyme Kinetic Module (Systat Software, Inc).

Molecular investigations

For mtDNA quantification, continuous real-time quantitative PCR (q-PCR), with the fluorescent SYBR green I double strand DNA-specific dye, was used to determine the copy number for two mitochondrial genes (ND2 and 16S) and a nuclear gene (ATPsynβ), as described previously [START_REF] Chabi | Quantification of mitochondrial DNA deletion, depletion and overreplication : application to diagnosis[END_REF]. For each gene tested, a standard curve was generated using a serial dilution of the target gene standard PCR product. Controls and patients were included in the same run. The mean of the results obtained from the two different mtDNA probes was used to calculate the mtDNA/nuclear DNA ratio.

For DGUOK mutation analysis, the human chromosome 2 working draft sequence segment (accession number NT_025651) was used to sequence each of the 7 exons and their intron/exon boundaries of the propositi and their parents as described by Mandel et al [START_REF] Mandel | The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA[END_REF].

For analysis of the DGUOK transcript of patient 2, total RNA was isolated from cultured fibroblasts of the patient and a control as described earlier [START_REF] Taanman | Molecular mechanisms in mitochondrial DNA depletion syndrome[END_REF]. In a thin-walled tube, 20 Copyright 2006 Biochemical Society pmol of oligo dT anchor primer (from the 5'/3' RACE kit of Roche) was annealed with 1 µg of total RNA in a volume of 5 µL by incubation at 70°C for 5 min, followed by a quick chill at 4°C for 5 min and a hold on ice. To obtain single-stranded cDNA, reverse transcription of RNA were performed with the ImProm-II Reverse Transcriptase kit (Promega) in a volume of 20 µL, as recommended by the supplier. Subsequently, 5 µL of the single-stranded cDNA product was used to amplify DGUOK cDNA with the Thermo-Start PCR Master Mix from ABgene. The PCRs were performed in a volume of 50 µL with either 50 pmol of the exon 3 (forward) primer 5'-ACTGCCCAAAGTCTTGGAAAC or 50 pmol of the exon 4 (forward) primer 5'-TGGTTCCCTCAGTGACATCGA and 50 pmol of the exon 5-6 junction (reverse) primer 5'-GTGGAGCTTCGTTGTCTTGTG (nt 262-282, 474-494 and nt 694-714 of the DGUOK cDNA, respectively; numbering according to [START_REF] Johansson | Cloning and expression of human deoxyguanosine kinase cDNA[END_REF]. After an initial denaturation step at 95°C for 15 min, the samples were subjected to 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and polymerisation at 72°C for 30 s, and a final polymerisation step at 72°C for 7 min. PCR products were resolved by agarose gel electrophoresis and extracted from the gel with the QIAquick Gel Extraction kit (Qiagen). The gel-extracted products of the PCR with the exon 3 and exon 5-6 junction primers were re-amplified with the same primers. Products of the second PCR were purified with the QIAquick PCR Purification kit (Qiagen) and subjected to DNA sequencing, using the exon 3 primer.

Results

Molecular investigations DGUOK mutations and RT-PCR analysis

-Patient 1 is a compound heterozygote for two missense mutations. The c.137A>G mutation in exon 1 changes a highly conserved asparagine to a serine (N46S) in the P-loop of the protein. The other mutation, c.797T>G in exon 6, changes a leucine to an arginine (L266R) in the C-terminal α-9 helical domain. The mother was carrier of the N46S mutation and the father of the L266R mutation (Supplementary Figure 1A). The younger unaffected brother was heterozygous for the N46S mutation. Testing of 100 control subjects did not identify any other carrier of these missense mutations.

-Patient 2: the DGUOK gene of patient 2 harboured three new heterozygous base changes (Supplementary Figure 1B): To examine whether the G→A mutation at the 3'-end of exon 4 had an effect on intron 4 splicing, we amplified exons 3-5 from fibroblast cDNA of patient 2 and a control with primers corresponding to sequences of exon 3 and the exon 5-6 junction. Two different amplification products were revealed: a ~450-bp band of expected size in the control sample and a ~300-bp band in the patient sample (Supplementary Figure 2A). DNA sequencing of these amplification products demonstrated that the 450-bp fragment from the control sample corresponded to DGUOK exons 3-4-5, while the shorter one from the patient sample corresponded to DGUOK exon 3 directly linked to exon 5 (Supplementary Figure 2A). Thus, it appears that the c.591G>A mutation in patient 2 causes skipping of exon 4. To further exclude the presence of a small amount of correctly processed mRNA, we tried to amplify exons 4-5 from the patient cDNA with a primer corresponding to sequences of exon 4 and the Copyright 2006 Biochemical Society exon 5-6 junction primer. Although amplification of control cDNA yielded a DNA fragment with a size expected for the exon 4-5 fragment, the cDNA sample of the patient did not yield any product (Supplementary Figure 2B). Furthermore, these results also show that the transcript from the other allele with the c.142+1G >A is undetectable.

-Patient 3 was homozygous for the previously reported 4 bp duplication in exon 6 (c.763_766dupGATT) [START_REF] Salviati | Mitochondrial DNA depletion and dGK gene mutations[END_REF], creating a premature stop codon resulting in a truncated 255 aa non-functional protein [START_REF] Wang | Mitochondrial deoxyguanosine kinase mutations and mitochondrial DNA depletion syndrome[END_REF]. Both parents are carriers of this duplication (Supplementary Figure 1C).

Quantification of mtDNA in fibroblast cultures

Levels of mtDNA were determined by q-PCR in fibroblast cultures of the three patients and compared to the levels of three control cultures. Cells were collected at the same passage number. In exponentially growing fibroblast cultures, a relatively mild depletion of mtDNA was observed in the three patients. Residual mtDNA levels in the cultures of patients 1, 2 and 3 were 67, 48 and 65% of the mean of the three control cultures (mtDNA/nDNA= 100±12%, range: 77-108%), respectively.

DGUOK enzymatic measurements

To assess the pathogenicity of the DGUOK gene mutations in patient 1 compared to the two other patients, we could not use liver directly as the specimen from patient 1 was no longer available. Consequently, we developed an enzymatic assay for mitochondria-enriched preparations from cultured fibroblasts, using a radiochemical analysis. Reaction progress curves showed that the assay was linear in controls for up to 20 h for dA and dG, in the range of 2.5-7.5 µg of mitochondrial protein per assay. Between 2.5 and 5 µg, and 5 and 7.5 µg of mitochondrial protein per assay, the same decrease of the specific activities (U/µg) was observed (12% and 13 % with dA and dG, respectively). Nonlinear reaction velocity versus amount of enzyme preparation per assay can result from a number of factors [START_REF] Segel | Enzyme kinetics. Behavior and analysis of rapid equilibrium and steady-state enzyme systems[END_REF], such as the presence of contaminating inhibitors or instability of the enzyme under the assay conditions. All data are shown for an input of 5 µg of protein per assay. Reproducibility was 11% between days (n=12), using replicate lysed samples, frozen in small aliquots and assayed on different days over a period of two months. DGUOK activity was stable for at least one year at -80°C. As a second step, we measured the specific activities and we characterized the kinetic properties of the mutant enzymes compared to the wild-type enzyme with dG or dA as phosphate acceptors and ATP as phosphate donor.

Specific activities:

patients: mitochondria of exponentially growing fibroblasts from patient 3 (c.763_766dupGATT) showed a very low activity with both dG and dA. Residual activity was 2.6% and 1% of the mean control value with dG and dA as the phosphate acceptors, respectively (Table 1). Also for patient 2 with splice mutations, a severe decrease of the catalytic rate was observed: 2.7% and 1.2% of the mean control activity with dG and dA as the phosphate acceptors, respectively (Table 1). In contrast, for patient 1 (N46S/L266R) the DGUOK residual activity was 14 % and 10 % of the mean control activity with dG and dA as the phosphate acceptors, respectively (Table 1). DGUOK activity in mitochondria from liver of patients 2 and 3 was reduced by 98% compared to two normal controls. As mentioned earlier, liver samples were no longer available from patient 1.

carriers: the DGUOK activity was also determined for family members of patient 1. The activity levels of the mother, the father and the brother of patient 1 were all below the low end of the control range (61%, 42% and 45% of the mean control value for dG, and 59%, 41% and 44% of the mean control value for dA, respectively). Similarly, the activity levels of the mother of patient 3 were 44% and 42% of the mean control values for dG and dA, respectively (Table 1).

Kinetic properties: in control fibroblasts, the kinetic relationship between velocity and substrate concentration regarding the two deoxynucleoside substrates, dG and dA, was investigated and results were plotted according the Hanes equation (s/v versus s), as well as the Eadie-Hofstee (v versus v/s) and Hill (log v/V-v versus log s) methods. Apparent K m (K m app ) and V max values were calculated with the Hanes equation (Figure 1). The phosphorylation of both dG and dA did not follow Michaelis-Menten kinetics but showed apparent negative cooperativity as indicated by nonlinear Eadie-Hofstee plots (Figure 2) and the Hill constants n<1 (n: 0.53 and 0.75 for dG and dA, respectively) (Table 2). An apparent negative cooperativity was also found in mitochondria from two control livers (Hill constant mean: 0.52 and 0.70 for dG and dA, respectively).

For patients 2 and 3, the fibroblast and liver samples showed no kinetic behaviors compared to the controls and no kinetic properties could be characterized. For the N46S/L266R mutant enzymes of patient 1, the K m app values for both dG and dA were about 3 µM and in the same range to that of the controls (1.8 and 6.8 µM for dG and dA, respectively) (Table 2). The mutant enzymes had 9 % of the V max /K m app value with dG and 25% with dA as compared Copyright 2006 Biochemical Society with the wild-type DGUOK enzyme. Negative cooperativity was also found (Hill constants: 0.61 and 0.68 for dG and dA, respectively). Negative cooperativity was furthermore observed for both dG and dA in all carriers (Table 2). The V max /K m app values with dG or dA as the phosphate acceptors ranged from 32% to 51% with dG and from 55% to 78% of the mean control value with dA, respectively.

The mutant enzymes were also characterized with regard to the phosphate donor properties with ATP. The K m app values for wild-type DGUOK were 1.5 and 0.95 mM when dG and dA were the phosphate acceptors, respectively (Table 3). The values of Hill constants (0.80 and 0.77 with ATP/dG and ATP/dA, respectively) indicate that wild-type DGUOK has a negative cooperativity with ATP as substrate. Negative cooperativity was also shown for the carriers.

In contrast, the N46S/L266R mutant enzymes (patient 1) exhibited two phases of the velocity curve when v values were plotted versus ATP/dA or ATP/dG concentrations compared to the controls or the carriers (Figure 3). A transition in the velocity curve is clearly observed at 2.5 mM ATP. Consequently, kinetic data showed bimodal plots using the Hanes equation.

Apparent K m and V max values were calculated separately for the two distinct regions. At low concentrations, ATP appears to be a more efficient phosphate donor for dG and dA, having higher efficiencies (V max /K m app ) and lower K m app values as compared to the situation at concentrations above the transition point (Table 3).

Discussion

We identified the nuclear gene involved in the liver-restricted MDS of patient 1, a 10-year-old who presented with a favourable course of the disease. The patient had two missense mutations (N46S/L266R) in his DGUOK gene. DGUOK mutations were initially reported in the hepatocerebral form of mtDNA depletion [START_REF] Mandel | The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA[END_REF][START_REF] Taanman | A novel mutation in the deoxyguanosine kinase gene causing depletion of mitochondrial DNA[END_REF] but can cause also isolated liver disease [START_REF] Salviati | Mitochondrial DNA depletion and dGK gene mutations[END_REF]. To date, the DGUOK mutations reported in patients with liver or hepatocerebral MDS result either in a premature stop codon and truncation of the DGUOK protein [START_REF] Salviati | Mitochondrial DNA depletion and dGK gene mutations[END_REF][START_REF] Slama | Deoxyguanosine kinase mutations and combined deficiencies of the mitochondrial respiratory chain in patients with hepatic involvement[END_REF][START_REF] Mandel | The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA[END_REF][START_REF] Taanman | A novel mutation in the deoxyguanosine kinase gene causing depletion of mitochondrial DNA[END_REF][START_REF] Labarthe | Clinical, biochemical and morphological features of hepatocerebral syndrome with mitochondrial DNA depletion due to deoxyguanosine kinase deficiency[END_REF] or in amino acid substitutions [START_REF] Salviati | Mitochondrial DNA depletion and dGK gene mutations[END_REF][START_REF] Slama | Deoxyguanosine kinase mutations and combined deficiencies of the mitochondrial respiratory chain in patients with hepatic involvement[END_REF]. In patient 2 with the hepatocerebral form of MDS, we identified the first splice mutations in DGUOK leading to an absence of correctly processed DGUOK transcript. For patient 3, who died at 2 months of age, the isolated hepatic form was related to a 4-bp duplication in exon 6 resulting in a premature stop codon at amino acid 255 [START_REF] Salviati | Mitochondrial DNA depletion and dGK gene mutations[END_REF]. A recombinant enzyme, generated with the same C-terminal 22 amino acid truncation was inactive and deletions of the C-terminal 11 or 18 amino acids resulted also in inactive enzymes suggesting that the C-terminal α-9 α-helical domain of DGUOK plays a critical role in catalysis [START_REF] Wang | Mitochondrial deoxyguanosine kinase mutations and mitochondrial DNA depletion syndrome[END_REF].

In contrast, patient 1 with the reversal form of MDS had two missense mutations, one of them (N46S) yet unreported. Residue N46 is one of the four highly conserved residues (positions 44 to 47) in the P-loop of the protein family of deoxynucleoside kinases. The P-loop is essential for the binding of the phosphates of ATP by the residues Ala48, Lys51 and Ser52.

Interactions of the triphosphate part of ATP involve also the LID region (Arg206 and Arg208) and the Arg142 residue present in the loop preceding helix α5. Using Swiss-Pdb Viewer (http://www.expasy.ch/spdbv) [29] to visualize the 3D-structures of mutant enzymes compared to the wild-type model [START_REF] Johansson | Structural basis for substrate specificities of cellular deoxyribonucleoside kinases[END_REF], it appears that the N46S mutation induces a drastic change in the close interactions with amino acid 46. In the S46 protein, the interactions with five residues disappear, four of them (the conserved Tyr218, Glu220, Gln221 and Gly224) belonging to the helix α8 and one residue (the conserved Pro196) belonging to the helix α7 (Supplementary Figure 3A). This predicted change probably impairs the phosphate bonds between ATP and both the P-loop and the LID region and, consequently, causes destabilization of the DGUOK-ATP complex essential for an optimum catalytic transfer of a phosphate group to dG or dA. The other mutation, L266R, was reported in another patient recently [START_REF] Slama | Deoxyguanosine kinase mutations and combined deficiencies of the mitochondrial respiratory chain in patients with hepatic involvement[END_REF]. The mutation affects a residue in the C-terminal α-9 α-helical domain which is not conserved throughout the family of deoxynucleoside kinases [START_REF] Johansson | Structural basis for substrate specificities of cellular deoxyribonucleoside kinases[END_REF]. Substitution of the aliphatic residue Leu for the basic residue Arg introduces abnormal and tight interactions The N46S/L266R enzymes showed a decreased catalytic rate with dG and dA (14% and 10% of the mean control activity with dG and dA, respectively) but results indicated no alteration of substrate binding capacity as revealed by the apparent K m values and no change in apparent negative cooperativity with dG and dA as substrates. Indeed, the predicted N46S/L266R DGUOK 3-D structure models do not affect the main residues involved in the active site, especially Arg118 and Asp147 [START_REF] Johansson | Structural basis for substrate specificities of cellular deoxyribonucleoside kinases[END_REF]. We cannot conclude that catalytic efficiency (V max /K m app ) for dG or dA phosphorylation is reduced as V max is also dependent on amounts of the mutant DGUOK protein. In contrast, the velocity was reduced by more than 97% in the patients 2 and 3 with the splice mutations and the GATT duplication, respectively, and kinetic behaviors were absent. Phosphorylation of dG and dA over the long incubation time by even a low level of contaminating cytosolic dCK, may explain these residual activities despite the presence of excess dC (2 mM) which is not only the more efficient substrate for dCK but also a strong inhibitor of the purine phosphorylation carried out by dCK [START_REF] Arnér | Mammalian deoxyribonucleoside kinases[END_REF][START_REF] Bohman | Deoxycytidine kinase from human leukemic spleen: Preparation and characterization of the homogeneous enzyme[END_REF].

When ATP was used as a limiting phosphate donor, patient 1 mutant enzymes showed abnormal kinetics. The two distinct regions of the velocity curve separated by an obvious transition indicate the presence of two different kinetic behaviors, one for the lower ATP concentrations, the other for the higher ones. At the low concentration range, a severely decreased catalytic rate (V max1 reduced by more than 90%) was observed rather than an alteration of substrate binding capacity as revealed by the K m app values. With increasing ATP concentrations, the mutant undergoes an increase in apparent K m for ATP (3 to 10-fold the control values with dG and dA, respectively), with the transition to higher K m app being accompanied by a shift to greater maximum velocity (V max2 only 5-fold reduced compared to the control V max ). Biphasic velocity curves have been described in preparations containing two enzymes (or two forms of the same enzyme) catalyzing the same reaction [START_REF] Segel | Enzyme kinetics. Behavior and analysis of rapid equilibrium and steady-state enzyme systems[END_REF]. We have to keep in mind that three different mutant dimers of the enzyme theorically coexist (N46S/N46S, L266R/L266R and N46S/L266R) in mitochondria of patient 1, if some of them are not degraded. In such a system, the observed velocity would be the sum of the velocities contributed by each mutant dimer. In addition to the conformational change of the P-loop predicted in the mutant S46, the stability of the P-loop 3-D structure may be compromised by the unusual interactions of the C-terminal domain and the β4 and β5 helix predicted in the mutant R266. As a result, the two mutations may both contribute to the impairment of the binding mode of ATP at the phosphate donor site, decreasing both the intrinsic affinity of the phosphate donor site and the rate of the phosphate transfer. We demonstrated a low DGUOK enzyme activity in the fibroblasts of a patient who displayed a spontaneous improvement of his liver MDS. The mtDNA content of his exponentially growing fibroblasts was only partially decreased. It is well established that during S-phase when both the cytosolic de novo synthesis of deoxynucleotides and the dCK are high, the import of purine deoxynucleotides sustains mtDNA synthesis in DGUOK deficient cells [START_REF] Taanman | Mitochondrial DNA depletion can be prevented by dGMP and dAMP supplementation in a resting culture of deoxyguanosine kinase-deficient fibroblasts[END_REF].

In liver of patient 1, the DGUOK mutations caused a severe mtDNA depletion (85% and 80% at 12 and 21 months of age, respectively) responsible for an extensive liver fibrosis [START_REF] Ducluzeau | Depletion of mitochondrial DNA associated with infantile cholestasis and progressive liver fibrosis[END_REF].

Although we assume that mutant DGUOK showed also residual activity in liver, this was apparently not enough to supply sufficient mitochondrial purine deoxynucleotides for normal mtDNA replication. Unfortunately, no liver samples were available from the patient to assess liver DGUOK activity directly. On the other hand, the absence of brain dysfunction in patient 1 argues for a residual DGUOK activity compatible with a normal neurological development.

In the neonatal period, liver and brain are tissues relying mostly on oxidative phosphorylation for ATP production. MtDNA replication is highly dependent on DGUOK without the compensatory effect of both cytosolic de novo dNTP synthesis, which is limited in postmitotic tissues, and dCK activity, which is known to be very low in these tissues [START_REF] Eriksson | Structure and function of cellular deoxyribonucleoside kinases[END_REF].

We hypothesize that the residual DGUOK activity accounts for the highly unusual phenotypic reversal. At 32 months of age, the patient displayed a spontaneous gradual improvement of his liver function with no extrahepatic manifestations. It appears that after the neonatal period, the mtDNA needs were lowered and the DGUOK activity became less critical. Even a low DGUOK activity may prevent further disease development and allow regeneration of the damaged liver. Moreover, the special regimen since 3 months of age, including daily fractional meals and enteral nutrition during the night supplemented with medium chain triglycerides, was probably effective to limit the liver energy requirements. In the literature, only one heterozygous patient (R142K/E227K) had a documented residual DGUOK activity in liver (27% as compared to control individuals) [START_REF] Salviati | Mitochondrial DNA depletion and dGK gene mutations[END_REF]. As he underwent liver transplantation at the age of 17 months, the natural course of his liver MDS is unknown.

Alternatively, the reversion of the phenotype may be explained by a somatic nuclear mosaicism confined to liver. Reversion by mosaicism may occur, early in development, in one somatic cell, either from a de novo mutation which could rescue the mutant allele or by creating a normal allele from a mutant by gene conversion [START_REF] Pasmooij | Multiple correcting COL17A1 mutations in patients with revertant mosaicism of epidermolysis bullosa[END_REF]. In these cases, the DGUOK activity would be higher in liver than in fibroblasts. To test this hypothesis, a liver biopsy is needed. Such a mosaicism could promote a functioning cell pool and may prevent liver failure mainly from accelerated cell regeneration. Whatever the molecular mechanism of a Copyright 2006 Biochemical Society hypothetic reversion event, reversion clusters with normal hepatocytes on light microscopy and fully restored function coexist in the patient's liver with some areas of residual fibrosis [START_REF] Ducluzeau | Progressive reversion of clinical and molecular phenotype in a child with liver mitochondrial DNA depletion[END_REF].

The optimized protocol of DGUOK activity measurement we developed for mitochondria from fibroblasts (and liver) allowed us to get a deeper understanding of the kinetic properties of the DGUOK enzyme and to evaluate the kinetic behavior of mutant enzymes in organello.

Experimental kinetic results obtained in organello reflect the enzymatic function of DGUOK, which depends on specific conditions of processing, folding and monomeric interactions. In the N46S/L266R mutant, even though the kinetic properties of each mutant enzyme cannot be resolved by this experimental approach, the characterization of a residual DGUOK activity in organello provides a general view of the mechanism leading to a reduced purine deoxynucleotide pool through the salvage pathway.

Table 1. Specific activities of human wild-type and mutant DGUOK enzymes with the phosphate acceptors

Samples containing 5 µg of mitochondrial protein were assayed under standard conditions (see Material and Methods). The phosphate acceptor concentrations were 50 µM for both dG and dA. The ATP/MgCl 2 concentration was fixed at 5/6 mM. For patients and carriers, assays

[

  21]. The DGUOK activity was measured radiochemically in mitochondria-enriched preparations from fibroblasts with a procedure based on that described by Ives and Wang [23], using 2'-[8-3 H]deoxyguanosine (dG) or 2'-[8-3 H]deoxyadenosine (dA) (MP Biomedicals Inc., USA) as substrates. Thawed mitochondria were lysed in 50 mM Tris, pH 7.4, 3 mM DTT, 1.5 mM PMSF and 0.5% Nonidet P-40 (2:1, v/v). The standard reaction mixture contained 75 mM Biochemical Journal Immediate Publication. Published on 31 Oct 2006 as manuscript BJ20060705 Copyright 2006 Biochemical Society

  (i) a G to T base change (c.4G>T) immediately downstream of the first ATG initiation codon. The second codon will be TCC instead of GCC, coding a Ser instead of Ala (A2S). Ala to Ser is quite a conservative change. Considering that this change affects the N-terminal part of the import sequence, this change will have no major effect on the import of the protein into mitochondria (ExPASy proteomics tools Mitoprot http://www.expasy.org/tools). Nevertheless, a G base change immediately after the start codon is likely to affect start codon recognition by the ribosome [27]. The ribosome may use the ATG codon further downstream as an alternative, (ii) a G to A base change at the consensus GT splice donor site of intron 1 (c.142+1G>A), and (iii) an G to A base change at the 3'-end of exon 4 (c.591G>A). This substitution does not change the amino acid but may induce splicing anomaly. Mutation analysis of the parent's DNA showed that the patient 2 inherited the A2S and the c.591G>A from his father and the c.142+1G >A from his mother.

  were performed on two different mitochondria-enriched preparations and mean values are shown with the range in parentheses. V is given in picomoles of dG or dA phosphorylated per hour per mg of protein. (%) percentage of the mean control values.

Biochemical Journal Immediate Publication. Published on 31 Oct 2006 as manuscript BJ20060705

Biochemical Journal Immediate Publication. Published on 31 Oct 2006 as manuscript BJ20060705 Copyright 2006 Biochemical Society status

  deteriorated rapidly with major hypotonia and the patient died at 3 months of age.Informed parental consent was obtained for this study.

	Patient 3. The third child of consanguineous Moroccan parents presented immediately after
	birth with hypoglycemia and lactic acidosis. Liver function tests revealed cholestasis and mild
	cytolysis. Neurological and cardiac functions were normal. At 1 month of age, there was a
	rapid evolution towards cytolysis, coagulopathy, fasting intolerance and severe lactic acidosis.
	Depletion of mtDNA in liver (80%) was demonstrated, associated with defects of mtDNA-
	related respiratory chain complexes (15, 20 and 30% of the mean control values for the
	complexes I, III and IV, respectively). The patient died at 2 months of age of liver failure.

Biochemical Journal Immediate Publication. Published on 31 Oct 2006 as manuscript BJ20060705

Biochemical Journal Immediate Publication. Published on 31 Oct 2006 as manuscript BJ20060705

Biochemical Journal Immediate Publication. Published on 31 Oct 2006 as manuscript BJ20060705

Biochemical Journal Immediate Publication. Published on 31 Oct 2006 as manuscript BJ20060705

Biochemical Journal Immediate Publication. Published on 31 Oct 2006 as manuscript BJ20060705

Biochemical Journal Immediate Publication. Published on 31 Oct 2006 as manuscript BJ20060705 Copyright 2006 Biochemical Society Figure 3 Mousson de Camaret B, et al.

		0.07								0.04
		0.06
		0.05								0.03
	ATP [mM] / v	0.02 0.03 0.04							ATP [mM] / v	0.01 0.02
		0.01
		0.00								0.00
		0	1	2	3	4	5	6	7	0	1	2	3	4	5	6	7
				ATP [mM]							ATP [mM]
							dG			dA
							V 50µM		V 50µM
	Controls (up to 1 year of age)
	mean					769		2145
	range (n)					670-905 (6)	1580-2825 (6)
	Patients
	patient 1 (N46S, L266R)			107 (105-110) (14%) 214 (197-231) (10%)
	patient 2 (c.142+1G>A, c.591G>A) 21 (16-27) (2.7%)	26 (25-27) (1.2%)
	patient 3 (c.763_766dupGATT)	20 (4-36) (2.6%)	22 (17-27) (1%)
	Controls (36-42 years of age)
	mean					808		2035
	(range) (n)					655-975 (6)	1546-2682 (6)
	Carriers
	Mother (N46S)				495 (475-515) (61%) 1201 (1128-1274) (59%)
	Father (L266R)				337 (314-361) (42%) 834 (807-861) (41%)
	Brother (N46S)				361 (344-378) (45%) 895 (864-926) (44%)
	Mother (c.763_766dupGATT)	355 (336-374) (44%) 855 (817-893) (42%)

Biochemical Journal Immediate Publication. Published on 31 Oct 2006 as manuscript BJ20060705 Copyright 2006 Biochemical Society

Acknowledgments

We gratefully acknowledge Prof Guibaud and Drs Till and Guffon for sending us the patient 2 and 3 samples. The paper is dedicated to Mrs J. Mousson.

between the C-terminal α-9 domain and both the β4 and β5 helix via the two conserved residues Tyr191 and Leu192, and the non-conserved residues Gln193, Val249 and Leu250 (Supplementary Figure 3B). Such interactions are likely to alter not only the proper folding of the C-terminal domain but also the conformation of domains close to the C-terminus.

In order to evaluate the consequences of these two missense mutations on the catalytic activity, an enzymatic assay was developed for mitochondria-enriched preparations from cultured fibroblasts. This assay was optimized for a detailed characterization of the kinetic parameters of the enzyme: the apparent K m (K m app ) values for the phosphate acceptors (dG and dA) and for the phosphate donor (ATP), the efficiency (V max /K m app ) and the Hill constants. In normal mitochondria, the reaction kinetics of DGUOK do not follow the Michaelis-Menten model but show apparent negative cooperativity with the phosphate acceptors (dG or dA) and donor (ATP) as indicated by nonlinear Eadie-Hofstee plots and Hill constants below unity. Such a mechanism was previously reported for the phosphorylation of dC and deoxythymidine (dT) by human dCK [START_REF] Ives | Deoxycytidine kinase. III. Kinetics and allosteric regulation of the calf thymus enzyme[END_REF] and TK2 [START_REF] Munch-Petersen | Diverging substrate specificity of pure human thymidine kinases 1 and 2 against antiviral dideoxynucleosides[END_REF], respectively. For purified DGUOK from beef liver mitochondria, a random bi-kinetic mechanism has been reported with the formation of a dead-end complex but without clear information about cooperativity [15]. In organello, negative cooperativity would tend to limit the initial velocity of the dimeric enzyme with increasing substrate concentrations and would contribute together with the feedback inhibition of the end-products [15] to regulate the purine dNTP pool. In mitochondria from control fibroblasts, the K m app values with dG and dA were 1.8 and 6.8 µM, respectively, but the V max was 3-fold higher with dA reducing the efficiency of dA phosphorylation by only 23%. The K m app value for dG was in the same range as the values reported for the purified bovine enzyme (4.7-7.6 µM) [START_REF] Wang | Substrate specifity of mitochondrial 2'-deoxyguanosine kinase. Efficient phosphorylation of 2chlorodeoxyadenosine[END_REF][START_REF] Park | Properties of a highly purified mitochondrial deoxyguanosine kinase[END_REF] and human recombinant enzyme (4 µM) [START_REF] Herrström Sjöberg | Substrate specificity of human recombinant mitochondrial deoxyguanosine kinase with cytostatic and antiviral purine and pyrimine analogs[END_REF][START_REF] Wang | Mitochondrial deoxyguanosine kinase mutations and mitochondrial DNA depletion syndrome[END_REF]. In contrast, the K m app value for dA was lower compared to those reported previously (60 µM and 467 µM for purified bovine and human recombinant enzyme, respectively). In our mitochondria-enriched preparations from fibroblasts, the product of dA phosphorylation is in part d-IMP, the deamination occuring at the nucleoside level by a contaminating adenosine deaminase activity (not shown). As DGUOK phosphorylates also deoxyinosine (dI) with a efficiency higher than for dG or dA [START_REF] Wang | Substrate specifity of mitochondrial 2'-deoxyguanosine kinase. Efficient phosphorylation of 2chlorodeoxyadenosine[END_REF][START_REF] Herrström Sjöberg | Substrate specificity of human recombinant mitochondrial deoxyguanosine kinase with cytostatic and antiviral purine and pyrimine analogs[END_REF], the K m value and efficiency for dA we measured in fibroblast mitochondria may reflect in part the phophorylation rate of the enzyme for dI. This may explain why the efficiency for dG was not several-fold higher than for dA as described for the purified bovine and human recombinant enzyme (12-fold) [START_REF] Wang | Substrate specifity of mitochondrial 2'-deoxyguanosine kinase. Efficient phosphorylation of 2chlorodeoxyadenosine[END_REF][START_REF] Herrström Sjöberg | Substrate specificity of human recombinant mitochondrial deoxyguanosine kinase with cytostatic and antiviral purine and pyrimine analogs[END_REF].

Table 2. Kinetic parameters of human wild-type and mutant DGUOK enzymes with the phosphate acceptors

Samples containing 5 µg of mitochondrial protein were assayed under standard conditions (see Material and Methods). The phophate acceptor concentrations were varied from 0.3 to 50 µM and to 0.6 to 50 µM for dG and dA, respectively. The ATP/MgCl 2 concentration was fixed at 5/6 mM. The three controls were within the age range of patients. For the patients, the values are from three different experiments. The apparent K m and V max values were calculated using the Hanes equation (s/v versus s). K m is given in µM and V max is given in picomoles of dG or dA phosphorylated per hour per mg of protein


Maria Isabel Cerezo-Guisado

Natalia García-Román

Luis Jesús García-Marín

Alberto Álvarez-Barrientos

Maria Julia Bragado

Maria Jesús Lorenzo
email: mjlorenzo@unex.es

María Jesús Lorenzo

Lovastatin inhibits the extracellular signal-regulated kinases pathway in immortalized rat brain neuroblasts

Keywords: statins, apoptosis, neuroblasts, ERK1/2, PD98059, LY294002 HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A, CNS, central nervous system, MEK, ERK kinase, ERK, extracellular signal-regulated kinase, CREB, cAMP response element binding protein, RBD, Ras-

We have previously shown that lovastatin, an HMG-CoA reductase inhibitor, induces apoptosis in spontaneously immortalized rat brain neuroblasts. In the present study, we analyzed the intracellular signal transduction pathways by which lovastatin induces neuroblast apoptosis. We showed that lovastatin efficiently inhibited Ras activation which was associated with a significant decrease in ERK1/2 phosphorylation.

Lovastatin also decreased CREB phosphorylation and CREB-mediated gene expression.

The effects of lovastatin on the Ras/ERK1/2/CREB pathway were time-and concentration dependent and fully prevented by mevalonate. In addition, we showed that two MEK inhibitors, PD98059 and PD184352, were poor inducers of apoptosis in serum-treated neuroblasts. However, these inhibitors significantly increased apoptosis induced by lovastatin treatment. Furthermore, we showed that pharmacological inhibition of both MEK and PI3K activities was able to induce neuroblast apoptosis with similar efficacy as lovastatin. Our results suggest that lovastatin triggers neuroblast apoptosis by regulating several signalling pathways, including the Ras/ERK1/2 pathway. These findings might also contribute to elucidate the intracellular mechanisms involved in the central nervous system side effects associated with statin therapy.

Introduction

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (HMG-CoA reductase inhibitors or "statins") are widely used in the treatment of lipid disorders, especially hypercholesterolemia. They decrease the risk for vascular disease including vascular death, myocardial infarction and stroke. The statin family is composed of six members: lovastatin, simvastatin, atorvastatin, cerivastatin, pravastatin and fluvastatin.

The last two are hydrophilic, whereas the others are lipid soluble, and each member differs in its specificity for HMG-CoA reductase [START_REF] Blumenthal | Statins: effective antiatherosclerotic therapy[END_REF][START_REF] Chong | Clinically relevant differences between the statins: implications for therapeutic selection[END_REF][START_REF] Graaf | Effect of statins and farnesyltransferase inhibitors on the development and progression of cancer[END_REF].

HMG-CoA reductase is a rate-limiting enzyme that catalyses the conversion of HMG-CoA into mevalonate [START_REF] Graaf | Effect of statins and farnesyltransferase inhibitors on the development and progression of cancer[END_REF][START_REF] Goldstein | Regulation of the mevalonate pathway[END_REF]. It is well established that the mevalonate pathway plays an important role in cell growth and survival [START_REF] Goldstein | Regulation of the mevalonate pathway[END_REF]. Several studies show that statins not only block the biosynthesis of mevalonate but, in addition, inhibit the proliferation and induce apoptosis of both normal and tumour cells [START_REF] Graaf | Effect of statins and farnesyltransferase inhibitors on the development and progression of cancer[END_REF][START_REF] Padayatty | Lovastatin-induced apoptosis in prostate stromal cells[END_REF][START_REF] Blanco-Colio | 3-Hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitors, atorvastatin and simvastatin, induce apoptosis of vascular smooth muscle cells by downregulation of Bcl-2 expression and Rho A prenylation[END_REF][START_REF] Hillyard | Inhibition of proliferation and signalling mechanisms in human lymphocytes by fluvastatin[END_REF][START_REF] Jaster | Inhibition of pancreatic stellate cell activation by the hydroxymethylglutaryl coenzyme A reductase inhibitor lovastatin[END_REF][START_REF] Koyuturk | Simvastatin induces proliferation inhibition and apoptosis in C6 glioma cells via c-jun N-terminal kinase[END_REF][START_REF] Takata | Cerivastatin-induced apoptosis of human aortic smooth muscle cells through partial inhibition of basal activation of extracellular signal-regulated kinases[END_REF][START_REF] Wu | Blocking the Raf/MEK/ERK pathway sensitizes acute myelogenous leukaemia cells to lovastatin-induced apoptosis[END_REF][START_REF] Nishida | Mevastatin induces apoptosis in HL60 cells dependently on decrease in phosphorylated ERK[END_REF]. These pleiotropic effects of statins are thought to be mediated by their ability to block the biosynthesis of isoprenoid intermediates of the cholesterol pathway, such as farnesyl pyrophosphate and/or geranylgeranyl pyrophosphate which serve as lipid attachments for the small GTP-binding proteins Ras, Rho and Rac [START_REF] Graaf | Effect of statins and farnesyltransferase inhibitors on the development and progression of cancer[END_REF][START_REF] Goldstein | Regulation of the mevalonate pathway[END_REF]. This post-translational prenylation step (either farnesylation or geranylgeranylation) is required both for membrane association and cell signalling, leading to cell survival and proliferation [START_REF] Graaf | Effect of statins and farnesyltransferase inhibitors on the development and progression of cancer[END_REF][START_REF] Zhang | Protein prenylation: molecular mechanisms and functional consequences[END_REF].

HMG-CoA reductase activity [START_REF] Maltese | Developmental changes in the distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase among subcellular fractions of rat brain[END_REF] and cholesterol biosynthesis [START_REF] Spady | Sterol synthesis in vivo in 18 tissues of the squirrell monkey, guinea pig, rabbit, hamster and rat[END_REF][START_REF] Cavender | Sterol metabolism in fetal, newborn and suckled lambs and their response to cholesterol after weaning[END_REF] are very high in the early phase of the ontogenetic development of the brain. These data indicate that the mevalonate pathway is essential to ensure normal growth, differentiation and maintenance of neuronal tissues. In agreement with this, recent reports show that the inhibition of this biosynthetic pathway during gestation by statin exposure is associated with severe central nervous system (CNS) defects [17,[START_REF] Edison | Mechanistic and epidemiologic considerations in the evaluation of adverse birth outcomes following gestational exposure to statins[END_REF]. On the other hand, studies in vitro show that statins at micro-and millimolar concentrations have neurotoxic effects on primary neuronal cultures of cerebral cortex [START_REF] Michikawa | Inhibition of cholesterol production but not of nonsterol isoprenoid products induces neuronal cell death[END_REF][START_REF] Tanaka | Geranylgeranylpirophosphate, an isoprenoid of mevalonate cascade, is a critical compound for rat primary cultured cortical neurons to protect the cell death induced by 3-hydroxy-3methylglutaryl-CoA reductase inhibition[END_REF][START_REF] Schulz | HMG-CoA reductase inhibition causes neurite loss by interfering with geranylgeranylpyrophosphate synthesis[END_REF] and we have shown that lovastatin induces apoptosis of immortalized rat brain neuroblasts, and that its effect is associated with a decreased prenylation of Ras [START_REF] García-Román | Lovastatin induces apoptosis of spontaneously immortalized rat brain neuroblasts: involvement of nonsterol isoprenoid biosynthesis inhibition[END_REF].

The Ras family of small GTPases are key regulators of signal transduction pathways that control cell proliferation, differentiation, survival and apoptosis. Ras and its relatives are activated in response to an extracellular or intracellular signal that generates the GTP-bound form and energizes the signal transducing ability. Hydrolysis of the bound GTP by an intrinsic GTPase activity relaxes the conformation and terminates the signal [START_REF] Denhardt | Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling[END_REF][24][START_REF] Mitin | Signaling interplay in ras superfamily function[END_REF]. Active Ras interacts with and modulates the activity of several downstream effectors. The best-characterized Ras effector pathway is the interaction with, and activation of, the three Raf serine/threonine kinases (A-Raf, B-Raf and Raf-1) leading to stimulation of the MEK1/MEK2 and ERK1/ERK2 (ERK1/2) mitogen-activated protein kinase (MAPK) cascade [START_REF] Denhardt | Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling[END_REF]24]. In the nervous system, ERK1/2 cascade is critical for neuronal differentiation, plasticity and neuronal survival [START_REF] Hetman | Role of extracellular signal regulated kinases 1 and 2 in neuronal survival[END_REF]. A role for ERK1/2 in neuronal survival was first indicated by experiments showing that overexpression of constitutively active mutants of MEK1 prevented the apoptosis induced by NGF withdrawal in neuronally differentiated PC12 cells [START_REF] Xia | Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis[END_REF].

Thereafter, although many studies supported a prosurvival activity of ERK1/2 in different neuronal cell types, several other studies suggested that ERK1/2 were not involved in the antiapoptotic signalling in neurons. Apart from those contradictory results, there is accumulating evidence that ERK1/2 may mediate the neuroprotective activity of several factors that neuroprotect against damaging insults and neuronal injury [START_REF] Hetman | Role of extracellular signal regulated kinases 1 and 2 in neuronal survival[END_REF]. The mechanism by which the Ras/ERK1/2 signalling pathway promotes neuronal survival is under study, although it has been suggested that activation of a transcription factor, cAMP response element binding protein (CREB) and/or a direct inhibition of Bad, a pro-apoptotic member of the Bcl-2 family, may mediate the prosurvival activity of ERK1/2 in trophic-deprived cerebellar granule neurons [START_REF] Bonni | Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and independent mechanisms[END_REF].

As has been previously mentioned, we have shown that lovastatin induces apoptosis in rat brain neuroblasts [START_REF] García-Román | Lovastatin induces apoptosis of spontaneously immortalized rat brain neuroblasts: involvement of nonsterol isoprenoid biosynthesis inhibition[END_REF]. The aim of the present work was to investigate the role of the Ras/ERK1/2 signalling pathway in regulating lovastatin-induced apoptosis in rat brain neuroblasts. The elucidation of the intracellular mechanisms affected by lovastatin will contribute to gain insight in the growth inhibition and apoptosis induced by lovastatin and other statins in neuronal cells, and additionally will contribute in establishing the important role that the mevalonate pathway plays in the development of the nervous system.

Materials and Methods

Reagents and antibodies

Lovastatin (Mevinolin, MK-803) was from Calbiochem (San Diego, CA, USA).

The inactive lactone of lovastatin was converted to the active form as previously described [START_REF] Kita | Feedback regulation of 3hydroxy-3-methylglutaryl coenzyme A reductase in livers of mice treated with mevilonin, a competitive inhibitor of the reductase[END_REF]. Mevalonic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA). Ham's F-12 medium, fetal calf serum, L-glutamine, streptomycin, penicillin and trypsin/EDTA solution were from PAN Biotech (Aidenbach, Germany, Europe). Tissue culture flasks and dishes were from TPP (Trasadingen, Switzerland, Europe). MEK1/2 inhibitor PD98059 (2´-amino-3´-methoxyflavone) and PI3K inhibitor LY294002 [2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] were from Calbiochem (San Diego, CA, USA). MEK1/2 inhibitor (PD184352) was kindly provided by Dr. Ana Cuenda (Departamento de Bioquímica y Biología Molecular, Universidad de Extremadura, Caceres, Spain). Complete protease inhibitor cocktail tablets were from Roche Molecular Biochemicals (Indianapolis, IN, USA). Glutathione-Sepharose 4B was from Pharmacia (Freiburg, Germany, Europe). The caspase substrate (Ac-DEVD-AMC), Ras antibody (cat. 610002), anti-phospho-Ser472/473/474 PKB (cat. 559029) and anti-PKB (cat. 559028) were from BD Biosciences Pharmigen (San Diego, CA, USA). ERK1/2 (cat. M3807) and phospho-ERK1/2 (Thr183 and Tyr185, cat. M8159) antibodies were from Sigma (St. Louis, MO, USA). CREB antibody (cat. 8977) was from Sigma, phospho-CREB (Ser 133, cat. 06-519) antibody was from Upstate Biotechnology (Lake Placid, NY, USA). Goat anti-rabbit (cat. 31460) and goat anti-mouse (cat. 31430) conjugated to horseradish peroxidase were from Pierce (Rockford, IL, USA). Other reagents were obtained from different commercial sources and were of the highest purity available.

Cell cultures and treatments

Spontaneously immortalized rat brain neuroblasts were used in this study. This cell line was obtained by spontaneous immortalization from cultures of 17-day fetal rat cerebral cortices [START_REF] Muñoz | Thyroid hormone receptor/c-erbA: Control of commitment and differentiation in the neuronal/chromaffin progenitor line PC12[END_REF] and was kindly provided by Dr. Alberto Muñoz (Instituto de Investigaciones Biomédicas, CSIC, Madrid, Spain). The cells represented primitive neuroblasts that expressed NF 68 and the primitive neuronal marker nestin, but lacked the astrocyte marker, glial fibrillary acidic protein (GFAP). After partial differentiation induction with dibutyryl -cAMP, the cells expressed further neuronal markers such as NF 145, NF 220, and neuron-specific enolase [START_REF] Muñoz | Thyroid hormone receptor/c-erbA: Control of commitment and differentiation in the neuronal/chromaffin progenitor line PC12[END_REF]. Cells were grown in Ham's F-12 supplemented with 10% fetal calf serum, L-glutamine (2 mM), streptomycin (100 µg/ml) and penicillin (100 U/ml). Cells were seeded at 5 x 10 5 in a 75-cm 2 tissue culture flask and incubated at 37ºC under a 5% CO 2 /95% air atmosphere.

Confluent cells were trypsinized and seeded in tissue culture dishes at a density of 2 x 10 4 cells/cm 2 . After 24 h, the medium was aspirated and replaced with fresh medium alone or containing the indicated concentrations of lovastatin, mevalonate, PD98059, PD184352 or LY294002 and the incubation was continued for a further 24 h. When indicated, cells were incubated with PD98059 or PD184352 for 1h before lovastatin addition. For the time course experiments, cells were incubated with 10 µΜ lovastatin for different periods of time.

Measurement of Ras activation

The capacity of Ras-GTP to bind to RBD (Ras-binding domain of Raf-1) was used to analyze the amount of active Ras as previously described [START_REF] Villalonga | Calmodulin prevents activation of Ras by PKC in 3T3 fibroblasts[END_REF]. Neuroblasts were seeded in 100 mm tissue culture dishes and after indicated treatments, cells were harvested in phosphate-buffered saline (PBS) and lysed for 15 min in 100 µl ice-cold lysis buffer containing 20 mM Tris (pH 7.4), 5 mM MgCl 2 , 1 mM EDTA, 0.2 mM sodium orthovanadate, 5 mM NaF, 10 % glycerol, 100 mM KCl, 1 % Triton X-100, 0.05 % 2-mercaptoethanol and 4 µg/ml complete protease inhibitor cocktail. Cell lysates were centrifuged at 20000 g for 10 min at 4ºC and protein concentrations in the supernatants were determined by using the Bio-Rad protein assay, according to the instructions of the manufacturer. Protein-equalized supernatants (250 µg) were incubated for 2 h at 4ºC with glutathione-Sepharose 4B beads pre-coupled with GST-RBD (1h at 4ºC). The beads were washed four times in the lysis buffer. Bound proteins were solubilized by the addition of 30 µl of Laemmli loading buffer and analyzed in 12 % SDS-PAGE gels. The amount of Ras in the bound fraction was analyzed by Western blot as described below.

Western blot analysis

Treated and control cells were harvested in PBS and lysed for 15 min in ice-cold lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1% Triton X-100, 10% glycerol, 5 mM MgCl 2 , 25 mM NaF, 10 mM sodium pyrophosphate, 1 mM EGTA, 0,5 mM sodium orthovanadate and 4 µg/ml complete protease inhibitor cocktail. After lysis, cell debris was removed by centrifugation at 20000 g for 10 min at 4 ºC and protein concentrations determined in the supernatants as indicated above. Equal amounts of protein (20 µg) were separated on 10 or 12% SDS-PAGE and blotted onto PVDF membranes (PolyScreen, NEN life Science Products, Inc. Boston, MA). The membranes were then blocked with 5% non-fat dry milk in Tris-buffered saline (pH 7.5) containing 0.5% tween 20 and incubated with appropriate primary and horseradish peroxidase (HRP)-conjugated secondary antibodies in antibody buffer [5% non-fat dry milk in Tris-buffered saline (pH 7.5) containing 0.05% tween 20 (TBS-T)]. After required washes with TBS-T, proteins were analyzed using an enhanced chemiluminescence detection system (Signal® West Pico Chemiluminescent Substrate, Pierce, Rockford, IL) and exposed to hyperfilm-ECL (Amersham, Pharmacia Biotech, Freiburg, Germany). Films were photographed and the intensity of each band was analyzed using a ChemiDoc System (Documentation and Analysis System, Bio-Rad, Hercules, CA, USA). Results were expressed as percentage of control.

Luciferase reporter construct and assay

The control β-galactosidase construct was pSV-β-Galactosidase (Promega). To construct the CRE response element luciferase reporter, the SV40 promoter in the vector pGL3-promoter (Promega) was excised with BglII and Hind III and replaced by a minimal HSV TK promoter (obtained by PCR amplification) containing nucleotides -109 to + 17 flanked by BglII and Hind III sites. A double stranded oligonucleotide containing the CRE response element from the somatostatin promoter (AGCCTGACGTCAGAGA) flanked by Asp718 and BglII sites was then cloned into this vector (pGL3-CRE). Neuroblasts were co-transfected with 1 µg of pGL3-CRE and 1 µg of β-Galactosidase reporter vector using GenePORTER transfection reagent (Genlantis, San Diego, CA, USA), as recommended by the manufacturer. Following transfection the cells were incubated for 24 h, the medium was changed, and the cells were incubated for an additional 24 h with 10 µM lovastatin in the presence or absence of 100 µM mevalonate. The cells were then lysed and luciferase and β-galactosidase assays performed using Promega reagents as recommended by the manufacturer.

Luciferase activity was normalized to β-galactosidase activity. Results were expressed as percentage of control.

Cell viability assay

Cell viability was evaluated by the crystal violet method [START_REF] Drysdale | Mechanism of macrophage-mediated cytotoxicity: production of a soluble cytotoxic factor[END_REF]. For these experiments 35 mm tissue culture dishes were used. At the end of each treatment, the medium was discarded, and the remaining viable adherent cells were stained with crystal violet (0.03% in 2% ethanol) for 5 min. Dishes were rinsed with tap water and 1 ml of 1% sodium dodecyl sulfate was added to each plate to solubilize the stained cells.

The absorbance of each plate was read at 560 nm. Dishes without cells were processed in parallel to correct the nonspecific adhesion of crystal violet to the plastic. Viable cells were calculated as percentage of absorbance with respect to untreated cells.

DNA Fragmentation Assay

Neuroblasts were plated on 100 mm culture dishes. After indicated treatments, floating cells were harvested by centrifugation at 20000 g for 1 min and adherent cells were detached mechanically with a rubber policeman. Adherent and floating cells were mixed, washed twice in ice-cold PBS and lysed in lysis buffer [10 mM Tris (pH 7.4), 5 mM EDTA and 0.5 % Triton X-100) for 60 min at 4ºC with agitation. After centrifugation at 20000 g for 30 min at 4ºC, supernatants were incubated with RNase A (0.1 mg/ml) at 37ºC for 45 min and then with Proteinase K (0.2 mg/ml) with 0.5% SDS solution at 37ºC for 45 min. Soluble DNA was then isolated by phenol-chloroformisoamylalcohol extraction and ethanol precipitation. DNA was collected by centrifugation at 20000 g for 20 min, dissolved in autoclaved water and resolved on 2% agarose-0.1 µg/ml ethidium bromide gels in TBE buffer [80 mM Tris-borate (pH 8.0), 2 mM EDTA]. DNA fragments were visualized under ultraviolet light and photographed using a ChemiDoc System (Documentation and Analysis System, Bio-Rad, USA).

Analysis of Nuclear DNA Content by Flow Cytometry

The ploidy determination of neuroblasts was estimated by flow cytometry DNA analysis. After culture under the indicated conditions, adherent cells were detached from dishes by the addition of a trypsin-EDTA solution. They were mixed with non-adherent cells and washed twice in ice-cold PBS without Ca 2+ and Mg 2+ . Cells were resuspended in ice-cold 70% ethanol and fixed for 5 min. After centrifugation at 500 g for 5 min, cells were resuspended in PBS containing 1 mg/ml of RNase A and incubated at 37ºC for 30 min. Cells were stained with 50 µg/ml propidium iodide for 30 min at room temperature and then flow cytometry analysis was done. DNA content per nucleus was evaluated in a CyAn MLE flow cytometer (DAKO, Gostrup, Denmark). This analysis was performed using a doublet discriminator system, which distinguishes between the signals coming from a single nucleus and the ones produced by two or more aggregated nuclei. For the computer analysis, only signals from single cells were considered (10000 cells/sample). This method of analysis of DNA content permits the identification and quantification of apoptotic cells, as revealed by the peak localized below the G0/G1 peak [33].

Analysis of caspase activity

Caspase activity was measured using the synthetic substrate Ac-DEVD-AMC.

Neuroblasts treated with different conditions were washed with cold PBS and lysed for 15 min in ice-cold lysis buffer containing 10 mM Tris (pH 7.5), 10 mM NaH 2 PO 4 , 130 mM NaCl, 1% Triton X-100 and 10 mM sodium pyrophosphate. Cell extracts were obtained after centrifugation at 20000 g for 15 min at 4ºC. For the assay, 20 µl of cell lysates were diluted in reaction buffer (20 mM HEPES pH 7.5, 10% glycerol, 2 mM ditiothreitol) and mixed with 20 µM of caspase substrate. The reactions were incubated at 37ºC for different period of times and the release of AMC from the substrate was monitored in a spectrofluorometer using excitation and emission wavelengths of 380 nm and 440nm, respectively. The results were corrected for protein content of the lysates and expressed as the increase of fluorescence for 1 h per mg protein.

Statistical analysis

Each experiment was repeated at least three times, with good agreement among the results of individual experiments. All data are expressed as the mean ± standard error of the mean. Results were analyzed by one-way analysis of variance (ANOVA) followed by Student´s t test. P values of less than 0.05 were considered significant.

Results

Effect of lovastatin on Ras activation

We have previously shown that lovastatin induced apoptosis of neuroblasts in a dose-and time-dependent manner and also that the lovastatin effect was associated with an inhibition of Ras prenylation evaluated by the decrease of the level of Ras in the membrane compartment [START_REF] García-Román | Lovastatin induces apoptosis of spontaneously immortalized rat brain neuroblasts: involvement of nonsterol isoprenoid biosynthesis inhibition[END_REF]. This post-translational prenylation step is required both for membrane association and Ras activation. Therefore, in the present work we first studied whether the inhibition of Ras prenylation by lovastatin was also associated with a decrease in its biological activity. To clarify this issue, time course and concentrationdependent experiments were done and Ras activation was analyzed by the GST-RBD-Raf-1 pull-down method. Time course experiments showed that in comparison to the control cells grown in 10% FCS media, lovastatin (10 µM) inhibited Ras activation in a time dependent manner (Fig. 1A). The lovastatin effect started to be significant after 3 hours of treatment and was markedly inhibited 24 hours after lovastatin treatment.

Inhibition of Ras activation occurred before induction of apoptosis that started to be significant after 12 hours of lovastatin treatment [START_REF] García-Román | Lovastatin induces apoptosis of spontaneously immortalized rat brain neuroblasts: involvement of nonsterol isoprenoid biosynthesis inhibition[END_REF]. Additionally, neuroblasts were treated with increasing concentrations of lovastatin for 24 hours and as shown in Fig. 1B, Ras activation decreased in a concentration-dependent manner after lovastatin treatment. The effect of lovastatin was evident at a concentration as low as 1 µM reaching the maximum effect at a concentration of 10 µM. In order to know whether the lovastatin effect on Ras activation was specific of HMG-CoA reductase inhibition, cells were incubated with lovastatin (10 µM) in presence or absence of mevalonate (100 µM) for 24 h. Co-treatment with mevalonate prevented the decrease in Ras activation induced by lovastatin (Fig. 1C). Treatment of cells with mevalonate alone did not modify Ras activation. In all the experiments, lovastatin decreased Ras prenylation evaluated by the presence of the unprocessed form of Ras (unprocessed Ras migrates more slowly that its processed form in SDS-PAGE). Taken together, these data suggest that the decrease of Ras activation induced by lovastatin may be due to a specific failure of Ras prenylation.

Effect of lovastatin on ERK1/2 phosphorylation

The best characterized Ras effector is the serine/threonine kinase Raf, which leads to the activation of the ERK1/2 pathway [START_REF] Denhardt | Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling[END_REF]24]. Therefore, we next studied the phosphorylation state of these kinases after lovastatin treatment by immunoblot analysis using phosphospecific antibodies. As shown in Fig. 2, lovastatin decreased ERK1/2 phosphorylation in a time-and concentration-dependent manner (Fig. 2A and Fig. 2B, respectively). Lovastatin (10 µM) produced a decrease in ERK1/2 phosphorylation that was significant at 3 hours and was maximum at 12 hours of treatment (Fig. 2A). On the other hand, the lovastatin effect on ERK1/2 phosphorylation was evident at a concentration of 1 µM, but the decrease in the phosphorylation of both kinases was only significant at higher concentrations (10 µM and 20 µM). Mevalonate (100 µM) prevented the decrease in ERK1/2 phosphorylation induced by lovastatin, indicating that the lovastatin effect was specific (Fig. 2C). Treatment of neuroblasts with mevalonate alone did not affect ERK1/2 phosphorylation. Expression levels of ERK1/2 remained unchanged throughout the experiment (Fig. 2), suggesting that ERK1/2 phosphorylation was specifically affected by exposure to lovastatin. It is noteworthy that the lovastatin effect on ERK1/2 phosphorylation was less potent than the effect on Ras activation. In fact, neuroblast incubation with lovastatin 10 µM for 24 hours reduced ERK1/2 phosphorylation by 45 % vs. control cells while the same treatment inhibited Ras activation by 90% vs. control.

Effect of lovastatin on CREB phosphorylation and CREB-mediated gene expression

It has been shown that one of the mechanisms by which the Ras/ERK1/2 signalling pathway promotes neuronal survival is by activating p90 rsk , which in turn phosphorylates the transcription factor CREB at serine 133. Activated CREB promotes cell survival, and inhibition of CREB phosphorylation triggers apoptosis. Therefore, we next evaluated the effect of lovastatin on the phosphorylation level of CREB. Treatment of neuroblasts with 10 µM lovastatin during different periods of time led to a reduction in the phosphorylation at Ser133 of CREB that was significant at 6 hours. The maximum effect was observed after 12 hours of treatment (Fig. 3A). As shown in Fig. 3B, lovastatin treatment induced a decrease in the phosphorylation of CREB in a concentration-dependent manner with respect to untreated control cells, but in these experiments the maximum decrease in CREB phosphorylation was detected at 20 µM lovastatin. Co-treatment with mevalonate (100 µM) prevented the decrease in CREB phosphorylation induced by lovastatin. Treatment of cells with mevalonate alone did not modify CREB phosphorylation (Fig. 3C). Lovastatin treatment did not modify CREB expression in any of the experimental conditions (Fig. 3).

To determinate whether the decrease in CREB phosphorylation induced by lovastatin was associated with an inhibition on CREB-mediated gene expression we next performed the luciferase reporter gene assay. As shown in Fig. 4, the incubation of the CRE-plasmid containing cells with 10 µM lovastatin for 24 hour resulted in an inhibition in luciferase expression. This effect was not observed in the cells transfected with the plasmid control lacking CREB binding sites (data not shown). Again, cotreatment with mevalonate prevented the effect of lovastatin and completely restored CREB-mediated gene expression to control levels. Taken together, these data suggest that lovastatin not only decreases CREB phosphorylation in neuroblasts but this action also correlates with decreased CREB capacity to activate transcription.

Effect of MEK inhibitors on neuroblast survival in absence or presence of lovastatin

Our results suggested that lovastatin might induce neuroblast apoptosis by its capacity to inhibit the Ras/ERK1/2 signalling pathway. Therefore, we next studied whether the pharmacological inhibition of this pathway was able to induce neuroblast apoptosis. Neuroblasts were incubated for 24 hours with different concentrations of PD98059 and PD184352 that inhibit MEK1/2, the MAP kinase kinase responsible for ERK1/2 phosphorylation and then the ability of these inhibitors to induce apoptosis was assessed by different assays, neuroblast viability, internucleosomal DNA fragmentation, quantification of neuroblasts undergoing apoptosis by flow cytometry and caspase activation. As shown in Fig 5, the treatment of neuroblasts with PD98059 alone was associated with a concentration-dependent decrease in cell viability (Fig. 5A), the appearance of internucleosomal DNA fragmentation (Fig. 5B) and an increase in the percentage of apoptotic neuroblasts (Fig. 5C). Moreover, PD98059 also enhanced caspase activity (Fig. 6A). Although PD98059 inhibited ERK1/2 phosphorylation in a concentration-dependent manner (Fig. 6B), the effects of this inhibitor were minimal when compared with control samples and never reached the parameters seen with lovastatin treatment (Fig. 5 and6). Similar results were obtained when PD184352, other MEK1/2 inhibitor, was used (Fig. 5 and6). These data suggest that the inactivation of the Ras/ ERK1/2 pathway may be necessary but not sufficient to evoke the lovastatin effect on neuroblast apoptosis.

It has been recently shown that the inhibition of the Raf/ERK pathway sensitizes cells to statins-induced apoptosis [START_REF] Takata | Cerivastatin-induced apoptosis of human aortic smooth muscle cells through partial inhibition of basal activation of extracellular signal-regulated kinases[END_REF][START_REF] Wu | Blocking the Raf/MEK/ERK pathway sensitizes acute myelogenous leukaemia cells to lovastatin-induced apoptosis[END_REF]. To evaluate whether direct inhibition of this pathway can increase the efficacy of lovastatin to trigger neuroblast apoptosis, cells were treated with lovastatin (10 µM) alone or in combination with PD 98059 (20 µM and 50 µM) or PD184352 (1 µM and 2 µM) for 24 hours. In these experiments, MEK inhibitors were added to the cells 1 hour before lovastatin treatment. As shown in Fig. 5 and 6, lovastatin effects were enhanced by pre-treatment with these inhibitors in a concentration-dependent manner. As expected, the down-regulation of ERK1/2 phosphorylation was also enhanced when lovastatin and PD98059 or PD184352 were used together (Fig. 6B).

Effect of the inhibition of Ras/ERK1/2 and phosphoinositide 3-kinase/protein kinase B (PI3K/PKB) pathways on neuroblast survival.

We have shown that Ras/ERK1/2 and PI3K/PKB signalling pathways are downregulated by lovastatin in neuroblasts and that the pharmacological inhibition of each of these pathways was not sufficient to induce the apoptosis degree elicited by lovastatin [START_REF] Cerezo-Guisado | Lovastatin inhibits the growth and survival pathway of phosphoinositide 3kinase/protein kinase B in immortalized rat brain neuroblasts[END_REF] and the present work. Therefore, we next studied the effect of co-incubation of PD98059 and PD184352 (MEK1/2 inhibitors) and LY294002 (PI3K inhibitor) on neuroblast survival and apoptosis. The treatment of neuroblasts with PD98059 (20 and 50 µM), PD184352 (1 and 2 µM) or LY294002 (20 µM) alone during 24 h was associated with a decrease in cell viability (Fig. 7A), a slight increase in the percentage of cells in the apoptotic sub-G1 population (Fig. 7B), and an increase in caspase activity (Fig. 7C) when compared with control. However, the effects of both inhibitors were additive on all the parameters studied (Fig. 7) and only the concomitant use of both inhibitors resembled the lovastatin efficacy in inducing neuroblasts apoptosis (Fig. 7A,B,C). As shown in Fig. 8, MEK inhibitors decreased ERK1/2 phosphorylation but did not modify PKB phosphorylation and LY294002 inhibited the phosphorylation of PKB without affecting ERK1/2 phosphorylation.

Discussion

It is known that the mevalonate pathway is essential to ensure normal growth, differentiation and maintenance of neuronal tissues [START_REF] Maltese | Developmental changes in the distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase among subcellular fractions of rat brain[END_REF][START_REF] Spady | Sterol synthesis in vivo in 18 tissues of the squirrell monkey, guinea pig, rabbit, hamster and rat[END_REF][START_REF] Cavender | Sterol metabolism in fetal, newborn and suckled lambs and their response to cholesterol after weaning[END_REF]. We have recently shown that mevalonate pathway inhibition by lovastatin induces apoptosis of spontaneously immortalized rat brain neuroblasts [START_REF] García-Román | Lovastatin induces apoptosis of spontaneously immortalized rat brain neuroblasts: involvement of nonsterol isoprenoid biosynthesis inhibition[END_REF]. In the present study, we investigated the molecular mechanisms underlying lovastatin-induced neuroblast apoptosis, focusing our work on the involvement of the Ras/ERK1/2/CREB signalling pathway.

In the present work, we have shown for the first time that lovastatin significantly decreased the activation of Ras stimulated by serum in spontaneously immortalized rat brain neuroblasts. The lovastatin effect on Ras activation was time-and concentration dependent and preceded neuroblast apoptosis [START_REF] García-Román | Lovastatin induces apoptosis of spontaneously immortalized rat brain neuroblasts: involvement of nonsterol isoprenoid biosynthesis inhibition[END_REF]. Our results are in agreement with previous studies that show that statins inhibit Ras prenylation and activation in different non-neuronal cell types [START_REF] Hillyard | Inhibition of proliferation and signalling mechanisms in human lymphocytes by fluvastatin[END_REF][START_REF] Nakagawa | HMG-CoA reductase inhibitor-induced L6 myoblast cell death: involvement of the phosphatidylinositol 3-kinase pathway[END_REF][START_REF] Bassa | Effect of inhibition of cholesterol synthetic pathway on the activation of Ras and MAP kinase in mesangial cells[END_REF][START_REF] Miura | Simvastatin suppresses coronary artery endotelial tube formation by disrupting Ras/Raf/ERK signaling[END_REF] and strongly suggest that lovastatin may induce neuroblast apoptosis by its capacity to disrupt Ras-mediated signal transduction in the cells.

Activation of Ras has been demonstrated to initiate activation of Raf and MEK1/2, resulting in ERK activation [START_REF] Denhardt | Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling[END_REF]24]. Since the Ras-MEK-ERK signalling pathway plays a key role in the transmission of cell survival signals, the phosphorylation of ERK 1/2 after lovastatin treatment was investigated. Our data show that lovastatin treatment led to a time-and concentration dependent decrease in the phosphorylation of ERK1/2 in neuroblasts that pointed to an inhibition of its kinase activity. These results suggest that the decrease in the phosphorylation of ERK1/2 may play a role in the apoptotic induction by lovastatin. Consistent with our findings, several reports show that statin-induced apoptosis and/or growth inhibition of various cell types correlates with down-regulation of ERK activity [START_REF] Hillyard | Inhibition of proliferation and signalling mechanisms in human lymphocytes by fluvastatin[END_REF][START_REF] Wu | Blocking the Raf/MEK/ERK pathway sensitizes acute myelogenous leukaemia cells to lovastatin-induced apoptosis[END_REF][START_REF] Nishida | Mevastatin induces apoptosis in HL60 cells dependently on decrease in phosphorylated ERK[END_REF][START_REF] Bassa | Effect of inhibition of cholesterol synthetic pathway on the activation of Ras and MAP kinase in mesangial cells[END_REF][START_REF] Miura | Simvastatin suppresses coronary artery endotelial tube formation by disrupting Ras/Raf/ERK signaling[END_REF][START_REF] Johnson | Lovastatin is a potent inhibitor of meningioma cell proliferation: Evidence for inhibition of a mitogen associated protein kinase[END_REF]. In contrast, another study reports that statin-induced growth inhibition of rat RBL-2H3 cells did not involve down-regulation of ERK activity [START_REF] Graham | MEK and ERK activation in ras-disabled RBL-2H3 mast cells and novel roles for geranylgeranylated and farnesylated proteins in Fc ecpsilonRI-mediated signaling[END_REF]. These controversial findings may be attributed to different biological actions of statins depending on the cell type. We have also shown that although the activation of Ras was almost completely inhibited by lovastatin, the serum-induced phosphorylation of the ERK1/2 was only partially inhibited in lovastatin-treated cells compared with control. The sustained phosphorylation of ERK1/2 in lovastatin-treated neuroblasts may be due to the activation of other upstream intracellular signalling molecules, such as PKC or Rac [START_REF] Skaletz-Rorowski | Lovastatin blocks basis fibroblast growth factor-induced mitogen-activated protein kinase signalling in coronary smooth muscle cells via phosphatase inhibition[END_REF][START_REF] Negre-Aminou | Differential effect of simvastatin on activation of Rac(1) vs. activation of the heat shock protein 27-mediated pathway upon oxidative stress, in human smooth muscle cells[END_REF] which in turn produce the activation of the MEK/ERK pathway.

It is accepted that ERK1/2 activation promotes neuronal survival by its capacity to activate p90 rsk , which in turn phosphorylates and activates CREB as well as increased CREB-mediated gene expression [START_REF] Bonni | Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and independent mechanisms[END_REF][START_REF] Riccio | Mediation by CREB family transcription factor of NGF-dependent survival of sympathetic neurons[END_REF]. In the present work, we have shown that the decrease in ERK1/2 phosphorylation/activity caused by lovastatin is associated with a reduction of both CREB phosphorylation and CREB-mediated gene expression. The turn off Ras/ERK/CREB signalling pathway has been related to apoptosis induced by other stimuli [START_REF] Bonni | Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and independent mechanisms[END_REF][START_REF] Rabelo | Inhibition of ERK1/2 and CREB phosphorylation by caspasedependent mechanism enhances apoptosis in fibrosarcoma cell line treated with butyrate[END_REF][START_REF] Hansen | Mechanisms leading to disseminated apoptosis following NMDA receptor blockade in the developing rat brain[END_REF] but, to our knowledge, this is the first report that demonstrates that lovastatin inhibits the activation of this survival signalling pathway in neuronal cells. We have previously shown that lovastatin decreased the expression of Bcl-2 in neuroblasts undergoing apoptosis [START_REF] García-Román | Lovastatin induces apoptosis of spontaneously immortalized rat brain neuroblasts: involvement of nonsterol isoprenoid biosynthesis inhibition[END_REF]. As this pro-survival gene is a target of CREB in neurons [START_REF] Bonni | Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and independent mechanisms[END_REF], our present data suggest that lovastatin may induce neuroblast apoptosis by decreasing the expression of Bcl-2 through the inhibition of the Ras/ERK/CREB signalling pathway. Our findings are in agreement with several works that indicate that CREB is essential for neuronal survival [START_REF] Walton | Is CREB a key to neuronal survival?[END_REF].

We have also shown that lovastatin effects were completely prevented by simultaneous exposure of cells to exogenous mevalonate, demonstrating that the inhibition of the Ras/ERK/CREB pathway induced by lovastatin was specifically due to a blockade of HMG-CoA reductase activity.

As said above, our results could suggest that lovastatin may induce neuroblast apoptosis by its capacity to inhibit the Ras/ERK/CREB pathway. Therefore, we investigated whether the pharmacological inhibition of this pathway was able to induce apoptosis in neuroblasts. Our results show that both PD98059 and PD184352, MEK inhibitors, were poor inducers of apoptosis in serum-treated neuroblasts. However, these inhibitors significantly increased apoptosis induced by lovastatin treatment. Taken together, these results suggest 1) that lovastatin does not trigger neuroblast apoptosis by down-regulating the Ras/ERK/CREB signalling cascade alone, and 2) that the sustained phosphorylation of ERK1/2 in lovastatin-treated neuroblasts may play a protective role [START_REF] Hetman | Role of extracellular signal regulated kinases 1 and 2 in neuronal survival[END_REF]. Our findings are in agreement with other reports where MEK inhibitors enhance the apoptotic actions of statins and other drugs in different non-neuronal cell types [START_REF] Wu | Blocking the Raf/MEK/ERK pathway sensitizes acute myelogenous leukaemia cells to lovastatin-induced apoptosis[END_REF][START_REF] Rabelo | Inhibition of ERK1/2 and CREB phosphorylation by caspasedependent mechanism enhances apoptosis in fibrosarcoma cell line treated with butyrate[END_REF][START_REF] Brantley-Finley | The JNK, ERK and p53 pathways play distinct roles in apoptosis mediated by the antitumor agents vinblastine, doxorubicin, and etoposide[END_REF]. Finally, in the present work we have shown that only the pharmacological inhibition of both MEK and PI3K activities was able to induce neuroblast apoptosis with similar efficacy as lovastatin. These data suggest that lovastatin may induce neuroblast apoptosis by inducing the simultaneous inactivation of both Ras/ERK1/2 and PI3K/PKB signalling pathways. Whether neuroblast death induced by lovastatin involves other intracellular pathways is under study.

In conclusion, we have shown for the first time that HMG-CoA reductase inhibition by lovastatin leads to an inhibition of the Ras/ERK/CREB signalling pathway in spontaneously immortalized rat brain neuroblasts which may contribute to explaining its apoptotic effect. The lovastatin effects were time-and concentrationdependent and prevented by adding mevalonate to the medium, which indicates that they were due to an inhibition of mevalonate synthesis. Our results also suggest that Ras/ERK pathway inactivation appears insufficient to induce the apoptosis evoked by lovastatin and that apoptosis of neuroblasts induced by this statin could require the simultaneous inhibition of both the Ras/ERK and PI3K-PKB signalling pathways.

These findings could contribute to elucidate the molecular mechanisms by which statins induce growth suppression and/or apoptosis in neuronal cells and may also help to explain the CNS side effects associated with statin therapy. Neuroblasts were treated as described in Fig. 1. At the end of each experiment, cells were lysed and total proteins (20 µg/lane) were separated by SDS-PAGE and analyzed by Western blotting using an anti-phospho-ERK1/2 specific antibody. As a control of the amount of protein, total ERK1/2 were also analyzed in the same samples. A representative blot of each experiment is shown with the densitometric analysis corresponding to the mean ± SE of at least three independent experiments. ns, not significant; **, p<0.01; ***, p<0.001; compared to untreated cells.

Figure 1 .

 1 Figure 1. Effect of lovastatin on Ras activation. A: Time-dependent effect: Neuroblasts initially cultured for 24 hours in Ham´s F12/10% FCS were incubated in culture media alone or with 10 µM lovastatin for different periods of time. B: Concentration-dependent effect: cells initially cultured for 24 hours in Ham´s F12/10% FCS were incubated for an additional 24 hours in the presence of media alone (0) or various concentrations of lovastatin. C: Effect of mevalonate treatment: Cells initially cultured for 24 hours in Ham´s F12/10% FCS were incubated with lovastatin (Lov 10 µM) in absence or presence of mevalonate (Mev 100 µM) for an additional 24 hours. Control cells were incubated in presence of media or mevalonate alone. At the end of each experiment, Ras activation was measured by GST-RBD pull-down followed by Western blot with anti-(pan)-Ras antibody. An aliquot of each lysate was also loaded in another gel to analyze total Ras protein levels. A representative blot of each experiment is shown with the densitometric analysis corresponding to the mean ± SE of at least three independent experiments. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; compared to untreated cells.

Figure 2 .

 2 Figure 2. Effect of lovastatin on ERK1/2 phosphorylation. A: Time-dependent effect. B: Concentration-dependent effect. C: Effect of mevalonate treatment.

Figure 3 .

 3 Figure 3. Effect of lovastatin on CREB phosphorylation. A: Time-dependent effect. B: Concentration-dependent effect. C: Effect of mevalonate treatment. Neuroblasts were treated as described in Fig. 1. At the end of each experiment, cells were lysed and total proteins (20 µg/lane) were separated by SDS-PAGE and analyzed by Western blotting using an anti-phospho-CREB specific antibody. As a control of the amount of protein,

Figure 4 .

 4 Figure 4. Effect of lovastatin on transcription from the CRE-response element. Neuroblasts initially cultured for 24 hours in Ham´s F12/10% FCS were co-transfected with pGL3-CRE-luciferase and pSV-β-Galactosidase reporter constructs. Following transfection, cells were incubated with lovastatin (Lov 10 µM) in absence or presence of mevalonate (Mev 100 µM) for 24 hours. Control cells were incubated in presence of media or mevalonate alone. Luciferase and β-Galactosidase activity were measured in the same extracts and the luciferase activity standardised for transfection efficiency by dividing the luciferase activity by β-Galactosidase activity. Results are expressed as the percentage relative to untreated cells. Each value represents the mean ± SE of four independent experiments performed in triplicate. ns, not significant; **, p<0.01; compared to untreated cells.

Figure 5 .

 5 Figure 5. Effect of MEK inhibitors alone or in combination with lovastatin on cell viability (A), internucleosomal DNA fragmentation (B) and percentage of apoptotic cells (C). Cells previously cultured for 24 hours in growth media were incubated with different concentrations of PD98059 (20 and 50 µM) or PD184352 (1 and 2 µM) in absence or presence of 10 µM lovastatin (Lov) for an additional 24 h. At the end of the experiment, cell viability was determined by the crystal violet method (A), internucleosomal DNA degradation was analyzed by using electrophoresis on 2% agarose gel (B) and the percentage of cells with hypodiploid DNA content was evaluated by flow cytometry (C). Each value represents the mean ± standard error of at least three independent experiments made in triplicate. ns, not significant; **, p<0.01; ***, p<0.001; compared to untreated cells. a, p<0.05; b, p<0.01; c, p<0.001; compared to lovastatin-treated cells (A and C). A representative photograph of 3 independent experiments is shown in B.

Figure 6 .

 6 Figure 6. Effect of MEK inhibitors alone or in combination with lovastatin on caspase activation (A) and ERK1/2 phosphorylation (B). Neuroblasts were treated as

  Figure 1

Biochemical Journal Immediate Publication. Published on 5 Sep 2006 as manuscript BJ20060731 Copyright 2006 Biochemical Society

Acknowledgments

The authors thank Ricardo Argent for his excellent technical assistance and Dr.

Ana Cuenda for kindly providing MEK1/2 inhibitor (PD184352). We also thank Mary Harper for the preparation of the manuscript. This work has been supported by Grants, SAF 2001-0154 from the Ministerio de Ciencia y Tecnologia, and 2PR01B007 from the Junta de Extremadura. M. I. Cerezo-Guisado is supported by a Ph D fellowship from Junta de Extremadura, Spain.