Peroxisome Proliferator-activated Receptor γ Is a Novel Target of the Nerve Growth Factor Signaling Pathway in PC12 Cells

Peroxisome proliferator-activated receptor γ (PPARγ), a member of the nuclear receptor superfamily, is subject to considerable interest because of its role in adipocyte differentiation, metabolic control, and anti-inflammatory action. PPARγ research in brain cells is presently focused on glial PPARγ because of its potential as a pharmacological target in the treatment of neurodegenerative diseases with an inflammatory component. In neurons PPARγ function is far from clear, and PPARγ agonist-dependent and -independent effects on cell survival or differentiation have been reported. We used PC12 cells, widely used to study neuronal signaling, such as nerve growth factor (NGF)-induced differentiation and survival or epidermal growth factor-dependent cell proliferation to dissect the possible involvement of PPARγ in these pathways. We show that NGF but not epidermal growth factor increases the transcriptional activity of PPARγ, and modulates the expression of this transcription factor. Because NGF signals through the tyrosine kinase (TrkA) NGF receptor and/or the p75NTR receptor, we used rescue experiments with a PC12 cell mutant lacking TrkA to show that NGF-induced PPARγ activation is dependent on TrkA activation. Our results point out PPARγ as a novel target of the TrkA-mediated neuronal cell survival and differentiating pathway and suggest a potential new inflammatory-independent therapeutic approach for pharmacological intervention in neurological disorders. Peroxisome proliferator-activated receptor γ (PPARγ), a member of the nuclear receptor superfamily, is subject to considerable interest because of its role in adipocyte differentiation, metabolic control, and anti-inflammatory action. PPARγ research in brain cells is presently focused on glial PPARγ because of its potential as a pharmacological target in the treatment of neurodegenerative diseases with an inflammatory component. In neurons PPARγ function is far from clear, and PPARγ agonist-dependent and -independent effects on cell survival or differentiation have been reported. We used PC12 cells, widely used to study neuronal signaling, such as nerve growth factor (NGF)-induced differentiation and survival or epidermal growth factor-dependent cell proliferation to dissect the possible involvement of PPARγ in these pathways. We show that NGF but not epidermal growth factor increases the transcriptional activity of PPARγ, and modulates the expression of this transcription factor. Because NGF signals through the tyrosine kinase (TrkA) NGF receptor and/or the p75NTR receptor, we used rescue experiments with a PC12 cell mutant lacking TrkA to show that NGF-induced PPARγ activation is dependent on TrkA activation. Our results point out PPARγ as a novel target of the TrkA-mediated neuronal cell survival and differentiating pathway and suggest a potential new inflammatory-independent therapeutic approach for pharmacological intervention in neurological disorders. Peroxisome proliferator-activated receptor-γ (PPARγ) 1The abbreviations used are: PPARγ, peroxisome proliferator-activated receptor γ; TZD, thiazolidinedione; NGF, nerve growth factor; Trk, tyrosine kinase; 15d-PGJ2, 15-deoxy-Δ-1,2,14-prostaglandin J2; MAP, mitogen-activated protein; EGF, epidermal growth factor; PPRE, peroxisomal proliferator response element; WT, wild type; DN, dominant negative; RGZ, rosiglitazone. is a nuclear receptor activated by insulin-sensitizing thiazolidinedione (TZD) drugs used to treat type II diabetes. Because PPARγ also exerts anti-inflammatory effects through modulation of glial cells, (1Combs C.K. Johnson D.E. Karlo J.C. Cannady S.B. Landreth G.E. J. Neurosci. 2000; 20: 558-567Crossref PubMed Google Scholar, 2Landreth G.E. Heneka M.T. Neurobiol. Aging. 2001; 22: 937-944Crossref PubMed Scopus (162) Google Scholar) the therapeutic interest in PPARγ activators is presently focused on their anti-inflammatory potential in the treatment of neurological disorders, such as Alzheimer's disease and multiple sclerosis (3Feinstein D.L. Diabetes Technol. Ther. 2003; 5: 67-73Crossref PubMed Scopus (105) Google Scholar). PPARs (α, β/δ, and γ) are also involved in biological processes as diverse as differentiation, homeostasis regulation, control of cell proliferation and survival, maintenance of insulin sensitivity, and cancer (for recent reviews, see Refs. 4Rosen E.D. Spiegelman B.M. J. Biol. Chem. 2001; 276: 37731-37734Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar, 5Desvergne B. Michalik L. Wahli W. Mol. Endocrinol. 2004; 18: 1321-1332Crossref PubMed Scopus (184) Google Scholar, 6Evans R.M. Barish G.D. Wang Y.X. Nat. Med. 2004; 10: 355-361Crossref PubMed Scopus (1283) Google Scholar, 7Michalik L. Desvergne B. Wahli W. Nat. Rev. Cancer. 2004; 4: 61-70Crossref PubMed Scopus (512) Google Scholar). PPAR possible direct participation in neuronal cell signaling has not been fully considered. In particular, the role of PPARγ, the most intensively studied PPAR isoform, is a controversial matter. In cortical neurons, the PPARγ ligand 15-deoxy-Δ-1,2,14-prostaglandin J2 (15d-PGJ2) was reported to contribute to neuronal apoptosis (8Rohn T.T. Wong S.M. Cotman C.W. Cribbs D.H. Neuroreport. 2001; 12: 839-843Crossref PubMed Scopus (100) Google Scholar), whereas in PC-12 neural cells 15d-PGJ2 promotes neurite extension in a PPARγ-independent manner (9Jung K.M. Park K.S. Oh J.H. Jung S.Y. Yang K.H. Song Y.S. Son D.J. Park Y.H. Yun Y.P. Lee M.K. Oh K.W. Hong J.T. Mol. Pharmacol. 2003; 63: 607-616Crossref PubMed Scopus (58) Google Scholar). Troglitazone, a thiazolidinedione drug and PPARγ agonist, induces survival in rat motoneurons even though its PPARγ agonist effect appears not to be involved (10Nishijima C. Kimoto K. Arakawa Y. J. Neurochem. 2001; 76: 383-390Crossref PubMed Scopus (32) Google Scholar). In HT-22 cells, a PPARγ-independent neuroprotective effect was reported for both troglitazone and 15d-PGJ2 (11Aoun P. Watson D.G. Simpkins J.W. Eur. J. Pharmacol. 2003; 472: 65-71Crossref PubMed Scopus (58) Google Scholar), whereas a PPARγ-dependent stimulation of embryonic midbrain cell differentiation into dopaminergic neuronal cells was reported for 15d-PGJ2 (12Park K.S. Lee R.D. Kang S.K. Han S.Y. Park K.L. Yang K.H. Song Y.S. Park H.J. Lee Y.M. Yun Y.P. Oh K.W. Kim D.J. Yun Y.W. Hwang S.J. Lee S.E. Hong J.T. Exp. Cell Res. 2004; 297: 424-433Crossref PubMed Scopus (92) Google Scholar). In fact, many actions of 15d-PGJ2, which has been ascribed to PPARγ activation, have been shown to be mediated by inhibition of the NF-κB pathway (13Rossi A. Kapahi P. Natoli G. Takahashi T. Chen Y. Karin M. Santoro M.G. Nature. 2000; 403: 103-108Crossref PubMed Scopus (1203) Google Scholar, 14Straus D.S. Pascual G. Li M. Welch J.S. Ricote M. Hsiang C.H. Sengchanthalangsy L.L. Ghosh G. Glass C.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4844-4849Crossref PubMed Scopus (948) Google Scholar). Because most PPARγ agonists have low affinity for PPARγ, relatively high agonist concentrations are usually used, making it difficult to truly asses PPARγ-dependent effects. Nevertheless, compelling evidence shows that PPARγ is indeed involved in cell differentiation in non-neural tissues, especially in adipocytes (5Desvergne B. Michalik L. Wahli W. Mol. Endocrinol. 2004; 18: 1321-1332Crossref PubMed Scopus (184) Google Scholar, 6Evans R.M. Barish G.D. Wang Y.X. Nat. Med. 2004; 10: 355-361Crossref PubMed Scopus (1283) Google Scholar), and recently, endogenous 15d-PGJ2 has been shown to protect PC12 cells from nitrosative-induced cell death and increase PPARγ expression (15Lim S.Y. Jang J.H. Na H.K. Lu S.C. Rahman I. Surh Y.J. J. Biol. Chem. 2004; 279: 46263-46270Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). To determine whether PPARγ could be involved in neural cell fate, we studied its expression and transcriptional activity in cells exposed to the differentiating effect of nerve growth factor (NGF) using both a PPARγ agonist-dependent and non-dependent approach. NGF signaling is one of the best characterized neurotrophic and differentiation-inducing pathways in nerve cells (for recent reviews, see Refs. 16Teng K.K. Hempstead B.L. Cell. Mol. Life Sci. 2004; 61: 35-48Crossref PubMed Scopus (195) Google Scholar and 17Segal R.A. Annu Rev. Neurosci. 2003; 26: 299-330Crossref PubMed Scopus (372) Google Scholar)). Neurotrophins, such as NGF, exert their effects by interacting with two structurally unrelated membrane receptors, the tropomyosin-related tyrosine kinase (Trk) receptors and the neurotrophin receptor (p75). Whereas p75 binds all neurotrophins, Trk family members exhibit ligand selectivity, NGF being the preferred ligand of TrkA (18Ip N.Y. Stitt T.N. Tapley P. Klein R. Glass D.J. Fandl J. Greene L.A. Barbacid M. Yancopoulos G.D. Neuron. 1993; 10: 137-149Abstract Full Text PDF PubMed Scopus (481) Google Scholar). NGF induces an array of biological responses mainly elucidated using the rat pheochromocytoma cell line PC12 (19Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4861) Google Scholar), which expresses both NGF receptors (20Kaplan D.R. Hempstead B.L. Martin-Zanca D. Chao M.V. Parada L.F. Science. 1991; 252: 554-558Crossref PubMed Scopus (1140) Google Scholar, 21Radeke M.J. Misko T.P. Hsu C. Herzenberg L.A. Shooter E.M. Nature. 1987; 325: 593-597Crossref PubMed Scopus (726) Google Scholar) and differentiates into sympathetic-like neurons when exposed to NGF (19Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4861) Google Scholar). A sustained activation of extracellular-regulated MAP kinases extracellular signal-regulated kinases 1 and 2 is involved in NGF-induced differentiation of PC12 cells, whereas epidermal growth factor (EGF) induces an opposite proliferative effect, paradoxically also via extracellular signal-regulated kinase MAP kinase but through a transient activation of this pathway (22Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4235) Google Scholar). We found that NGF, but not EGF, induces increased PPARγ transcriptional activity and modulates its expression in PC12 neural cells. Rescue experiments using a PC12 cell mutant lacking the TrkA receptor but expressing p75 show that NGF activates PPARγ through the TrkA receptor. Our results suggest that PPARγ is a novel target gene of the NGF-signaling pathway, opening new avenues of research on its potential participation in neuronal differentiation and/or survival pathways. Chemicals—Chemicals, drugs, culture media, and sera were obtained from Sigma and Invitrogen, and rosiglitazone and carbaprostacylin were from Cayman Chemical (Ann Arbor, MI). Stock solutions of drugs were prepared in dimethyl sulfoxide (Me2SO) and added to culture medium (0.01% final Me2SO concentration). Plasmids—The reporter plasmid containing three tandem repeats of the peroxisomal proliferator response element (PPRE) from the acyl-CoA oxidase gene fused to the herpes virus thymidine kinase promoter upstream of the coding sequence for luciferase was a kind gift of Dr. R. M. Evans, Howard Hughes Medical Institute, Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, CA. The expression vector containing only the coding sequence for luciferase under the control of the herpes virus thymidine kinase promoter (Clontech, Palo Alto, CA) was used as the control. Co-transfections for reporter gene assays were carried out using 0.3 μg of reporter plasmid and 40 ng of a CMV-β-Gal vector (Clontech) for normalization. An expression vector containing full-length murine PPARγ1 cloned in the pCMX vector was also a kind gift of Dr. R. M. Evans. A PPARγ1 double mutant of Leu468 and Glu471 to alanine was generated by site-directed mutagenesis using QuikChange XL (Stratagene, La Jolla, CA), verified by sequencing, and inserted into the pCMX expression vector. Mutation to alanine of these two highly conserved amino acids in helix 12 of the ligand binding domain of human PPARγ has been shown to result in a strong constitutive repression of PPARγ (23Gurnell M. Wentworth J.M. Agostini M. Adams M. Collingwood T.N. Provenzano C. Browne P.O. Rajanayagam O. Burris T.P. Schwabe J.W. Lazar M.A. Chatterjee V.K. J. Biol. Chem. 2000; 275: 5754-5759Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). An expression vector encoding the full-length rat TrkA cDNA under the control of the cytomegalovirus promoter was obtained from Dr. Philip A. Barker, Montreal Neurological Institute, McGill University, Montreal, Canada. Cell Culture and Transfections—PC12 and nnr5 cells were maintained and in medium containing and on rat of PC12 cells were carried out using to the PC12 cells were in and with a of μg of the pCMX expression vector the wild type the dominant or with μg of In all cells were also with the CMV-β-Gal (Clontech) vector for normalization. NGF treatment PC12 cells were in medium containing cells were in medium for in the and of were prepared as in and Res. 1991; PubMed Scopus Google Scholar). In the cells were with and the were in of containing and and for The were in of a containing for the containing the nuclear was in nuclear was using a of the assays were as μg of nuclear was in of binding 1 for by with of containing a binding A of PPARγ was by CA) to the binding for the or of of the binding were used as 2 μg of PPARγ was added the of the and was from The binding was on a in and for were using and has been Jung P. L. M. J. Neurosci. Res. 2003; PubMed Scopus Google Scholar, Jung P. K.M. A. M. J. Neurochem. 2003; PubMed Scopus Google Scholar). PC12 were by and to Life were by and in 2 were using cDNA for PPARγ and were using in the of was for The were by of The were PPARγ, rat we used the from Science. The used for were The was from Science. were as for PPARγ, with for by of for for 1 for 12 for with for by of for for 1 for 12 The expression of PPARγ with to was for all cells were in high as Jung P. L. M. J. Neurosci. Res. 2003; PubMed Scopus Google Scholar, Jung P. K.M. A. M. J. Neurochem. 2003; PubMed Scopus Google Scholar). The of was using a and μg of was by and to a membrane and PPARγ were from The PPARγ an from amino acids to A PPARγ to a in the of PPARγ CA) was also used to the with the and a of in PC12 cells and rat used as the control. The was not by a results using the are was from PC12 cells on were using PPARs with a was using the were and using a NGF PPARγ a gene reporter with a plasmid containing the PPRE, we found that NGF induces PPAR transcriptional activity activity is and and the point which a in the luciferase is To the PPAR for this effect, PC12 cells were with the reporter gene and for in the of PPAR agonists and In the of NGF, only the PPARγ agonist rosiglitazone a TZD, a increase in transcriptional which was by a PPARγ and agonists of and were NGF was a increase in PPAR transcriptional activity was as from and agonists were effect, whereas a in transcriptional activity was in the of that was by results were obtained with The NGF effect was highly and in PC12 cells obtained from two suggest that PPARγ is for the NGF-induced PPAR transcriptional activity in PC12 cells. To this we used PC12 cells with the PPAR reporter gene and coding for a PPARγ DN, a wild type PPARγ or the vector see and To the of we the effect of concentrations in PPAR transcriptional activity in cells cells increased transcriptional activity in a manner when with cells. The effect was and control in cells, whereas with both and in inhibition of the transcriptional shown in in the of NGF cells with the vector show the transcriptional PPAR activity when with in the of activity is and increased by NGF in PC12 cells, whereas in cells the effect is with both and the effect of and is when with suggest that NGF induces increased transcriptional activity of To this we used to determine whether PPARγ indeed binds to in nuclear from control PC12 cells and cells for in the and of NGF of PPAR to was in nuclear from control cells and was in the of of NGF treatment in an increase of the PPAR binding activity that was but it was difficult to because are for nuclear The binding was with that the is result shows that PPARγ binds to in both control and cells when transcriptional activity NGF PPARγ we whether NGF induces in PPARγ Because it has been reported that PC12 cells have PPARγ by (9Jung K.M. Park K.S. Oh J.H. Jung S.Y. Yang K.H. Song Y.S. Son D.J. Park Y.H. Yun Y.P. Lee M.K. Oh K.W. Hong J.T. Mol. Pharmacol. 2003; 63: 607-616Crossref PubMed Scopus (58) Google Scholar), we used two of the PPARγ and both a in PC12 cells and in used as control be to the culture or to or of PPARγ is that PPARγ has been by in PC12 cells (15Lim S.Y. Jang J.H. Na H.K. Lu S.C. Rahman I. Surh Y.J. J. Biol. Chem. 2004; 279: 46263-46270Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). A transient in PPARγ and NGF was in which is with the reported of this receptor activation J.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google PPARγ expression not of NGF treatment but to a of in and of PC12 cells, as by or The NGF-induced PPARγ expression of PPARγ and transcriptional activation. were in or expression NGF treatment To determine whether these are in PPARγ we used PPARγ In and PC12 cells, PPARγ a whereas in cells, the PPARγ from the of NGF treatment was PPARγ in the whereas in and cells, PPARγ was in the cell and its in the was when with cells. The that the luciferase in the reporter gene shown in of NGF treatment be in of a and experiments with the of PPARγ as by and with the increased PPARγ expression that PPARγ was using a of the PPARγ and In to PPARγ, were for both and which a nuclear in cells as as in cells for the treatment a these results suggest a the and PC12 cell differentiation PPARγ PC12 cells were for and in the of NGF and for with PPARγ with a and by were with as under and The is also of PC12 cells in the of NGF the are In control cells PPARγ is in both the and and in the of NGF most cells only or whereas in and cells PPARγ is in the cell and its in the is when with or cells. results were obtained with a PPARγ to a of the PPARγ not NGF-induced PPARγ on the TrkA whether TrkA or p75 was involved in PPARγ, we used nnr5 cells, a PC12 mutant in TrkA that not in response to NGF but that expresses p75 J. Martin-Zanca D. Chao M.V. Parada L.F. Greene L.A. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). In with J. Martin-Zanca D. Chao M.V. Parada L.F. Greene L.A. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar), nnr5 cells with a plasmid containing the full-length TrkA receptor NGF-induced neurite PC12 and nnr5 cells PPARγ shown in in nnr5 cells but not NGF induces PPAR transcriptional which is by a PPARγ that TrkA is for NGF-induced PPARγ transcriptional TrkA of nnr5 cells results in increased PPAR transcriptional activation and the induces an of luciferase which is by a PPARγ as in PC12 cells. results show that TrkA activation induces PPARγ transcriptional In this study we show that NGF increases PPARγ transcriptional activity in PC12 cells, that this nuclear transcription factor is a novel target of the NGF-signaling using PPARγ agonists and or PPARγ or repression and also this of PPARγ from the and NGF treatment is also with this with the of PPARγ as by and with the reported of this receptor activation J.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). rescue experiments with nnr5 cells lacking the TrkA receptor show that NGF-induced TrkA activation is for PPARγ transcriptional from the by which NGF induces increased PPARγ transcriptional activity and the of the The increase in PPARγ by NGF, which that NGF induces the transcriptional activity of The of nuclear PPARγ of NGF treatment this as is to that TrkA activation results in the of endogenous PPARγ In PC12 cells, TrkA activation induces a and of through the pathway G. J. Neurochem. PubMed Scopus Google Scholar), which is for neurite S. Y. T. M. A. S. K. M. J. 2003; PubMed Scopus Google Scholar). of have been as PPARγ L. P. Chen R.M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar), a by the of this in PPARγ responses in J.T. Welch J.S. Ricote M. C.J. C. D. Glass C.K. Nature. PubMed Scopus Google Scholar). it has been shown that nitrosative-induced in PC12 cells results in the of endogenous 15d-PGJ2 and increased PPARγ expression (15Lim S.Y. Jang J.H. Na H.K. Lu S.C. Rahman I. Surh Y.J. J. Biol. Chem. 2004; 279: 46263-46270Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). could also a role in the NGF-induced PPARγ transcriptional PPARγ is by extracellular signal-regulated kinase 2 and MAP kinases M. M.J. D. Lazar M.A. Chatterjee V.K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), in inhibition or activation of PPARγ transcriptional activity B. J. G. A. S. S. D. D.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In human cells, PPARγ cell differentiation is under in which the receptor signaling pathway is and results from of PPARγ and its to the J. Lee J. C. J. J. Cell Sci. 2004; PubMed Scopus Google Scholar). this it is to that in PC12 cells, the pathway is and activation of PPARγ by be and the of activation of extracellular signal-regulated kinase MAP kinases is for PC12 cell differentiation and survival by NGF but also for cell proliferation (22Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4235) Google Scholar). The of growth biological through has considerable interest in controversial even though it is that in PC12 cells responses are by the of extracellular signal-regulated kinase activation (22Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4235) Google Scholar, Y. J. K. M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, S. W. Landreth G.E. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). NGF induces sustained activation of this signaling pathway, in cell differentiation and survival, whereas only a activation, cell Because we show that NGF but not induces increased PPARγ transcriptional results a new the signaling pathways activated by these two growth and suggest that PPARγ signaling could be to cell differentiation and/or cell survival in In with this we have found that activation of PPARγ by three rat neurons and from the in PPARγ activation results in inhibition of a target gene of the NGF survival pathway R.A. C. M. Exp. Cell Res. Google Scholar). research interest in PPARγ function in brain cells is mainly in PPARγ activation in glial cells, in of has been involved in the effect of and anti-inflammatory drugs in neurodegenerative diseases (1Combs C.K. Johnson D.E. Karlo J.C. Cannady S.B. Landreth G.E. J. Neurosci. 2000; 20: 558-567Crossref PubMed Google Scholar, D.L. Diabetes Technol. Ther. 2003; 5: 67-73Crossref PubMed Scopus (105) Google Scholar, M.T. T. D.L. J. Neurosci. 2000; 20: PubMed Google Scholar). is sustained by the that PPARγ agonists responses and (1Combs C.K. Johnson D.E. Karlo J.C. Cannady S.B. Landreth G.E. J. Neurosci. 2000; 20: 558-567Crossref PubMed Google Scholar). the Alzheimer's disease for type 2 with rosiglitazone of treatment S. 2003; PubMed Scopus Google Scholar), and a TZD, D.L. L. Landreth G.E. G. Heneka M.T. PubMed Scopus Google Scholar). Our suggest the of a direct effect of neuronal PPARγ activation in survival and/or Because NGF is also a target in the treatment of neural J.M. M.A. J. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar), the the the and pathways in neurons could have in the pharmacological approach to neurodegenerative

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.