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Impact of atypical mitochondrial cyclic-AMP level in nephropathic cystinosis

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Abstract

Nephropathic cystinosis (NC) is a rare disease caused by mutations in the CTNS gene encoding for cystinosin, a lysosomal transmembrane cystine/H+ symporter, which promotes the efflux of cystine from lysosomes to cytosol. NC is the most frequent cause of Fanconi syndrome (FS) in young children, the molecular basis of which is not well established. Proximal tubular cells have very high metabolic rate due to the active transport of many solutes. Not surprisingly, mitochondrial disorders are often characterized by FS. A similar mechanism may also apply to NC. Because cAMP has regulatory properties on mitochondrial function, we have analyzed cAMP levels and mitochondrial targets in CTNS−/− conditionally immortalized proximal tubular epithelial cells (ciPTEC) carrying the classical homozygous 57-kb deletion (delCTNS−/−) or with compound heterozygous loss-of-function mutations (mutCTNS/). Compared to wild-type cells, cystinotic cells had significantly lower mitochondrial cAMP levels (delCTNS/ ciPTEC by 56% ± 10.5, P < 0.0001; mutCTNS/ by 26% ± 4.3, P < 0.001), complex I and V activities, mitochondrial membrane potential, and SIRT3 protein levels, which were associated with increased mitochondrial fragmentation. Reduction of complex I and V activities was associated with lower expression of part of their subunits. Treatment with the non-hydrolysable cAMP analog 8-Br-cAMP restored mitochondrial potential and corrected mitochondria morphology. Treatment with cysteamine, which reduces the intra-lysosomal cystine, was able to restore mitochondrial cAMP levels, as well as most other abnormal mitochondrial findings. These observations were validated in CTNS-silenced HK-2 cells, indicating a pivotal role of mitochondrial cAMP in the proximal tubular dysfunction observed in NC.

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References

  1. Acin-Perez R, Salazar E, Kamenetsky M, Buck J, Levin LR, Manfredi G (2009) Cyclic AMP produced inside mitochondria regulates oxidative phosphorylation. Cell Metab 9:265–276

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Ahn BH, Kim HS, Song S, Lee IH, Liu J, Vassilopoulos A, Deng CX, Finkel T (2008) A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis. Proc Natl Acad Sci USA 105:14447–14452

    Article  PubMed  Google Scholar 

  3. Arnold I, Pfeiffer K, Neupert W, Stuart RA, Schagger H (1998) Yeast mitochondrial F1F0-ATP synthase exists as a dimer: identification of three dimer-specific subunits. EMBO J 17:7170–7178

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Bartsocas CS, Bernstein J, Orloff S, Chandra R, Schulman JD (1986) A familial syndrome of growth retardation, severe Fanconi-type renal disease and glomerular changes–a new entity? Int J Pediatr Nephrol 7:101–106

    PubMed  CAS  Google Scholar 

  5. Bell EL, Guarente L (2011) The SirT3 divining rod points to oxidative stress. Mol Cell 42:561–568

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Bellomo F, Piccoli C, Cocco T, Scacco S, Papa F, Gaballo A, Boffoli D, Signorile A, D’Aprile A, Scrima R, Sardanelli AM, Capitanio N, Papa S (2006) Regulation by the cAMP cascade of oxygen free radical balance in mammalian cells. Antioxid Redox Signal 8:495–502

    Article  PubMed  CAS  Google Scholar 

  7. Besouw M, Masereeuw R, van den Heuvel L, Levtchenko E (2013) Cysteamine: an old drug with new potential. Drug Discov Today 18:785–792

    Article  PubMed  CAS  Google Scholar 

  8. Bornhovd C, Vogel F, Neupert W, Reichert AS (2006) Mitochondrial membrane potential is dependent on the oligomeric state of F1F0-ATP synthase supracomplexes. J Biol Chem 281:13990–13998

    Article  PubMed  CAS  Google Scholar 

  9. Cherqui S, Courtoy PJ (2017) The renal Fanconi syndrome in cystinosis: pathogenic insights and therapeutic perspectives. Nat Rev Nephrol 13:115–131

    Article  PubMed  CAS  Google Scholar 

  10. Cherqui S, Sevin C, Hamard G, Kalatzis V, Sich M, Pequignot MO, Gogat K, Abitbol M, Broyer M, Gubler MC, Antignac C (2002) Intralysosomal cystine accumulation in mice lacking cystinosin, the protein defective in cystinosis. Mol Cell Biol 22:7622–7632

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. De Rasmo D, Micelli L, Santeramo A, Signorile A, Lattanzio P, Papa S (2016) cAMP regulates the functional activity, coupling efficiency and structural organization of mammalian FOF1 ATP synthase. Biochim Biophys Acta 1857:350–358

    Article  PubMed  CAS  Google Scholar 

  12. De Rasmo D, Signorile A, Larizza M, Pacelli C, Cocco T, Papa S (2012) Activation of the cAMP cascade in human fibroblast cultures rescues the activity of oxidatively damaged complex I. Free Radic Biol Med 52:757–764

    Article  PubMed  CAS  Google Scholar 

  13. De Rasmo D, Signorile A, Papa F, Roca E, Papa S (2010) cAMP/Ca2+ response element-binding protein plays a central role in the biogenesis of respiratory chain proteins in mammalian cells. IUBMB Life 62:447–452

    PubMed  Google Scholar 

  14. De Rasmo D, Signorile A, Santeramo A, Larizza M, Lattanzio P, Capitanio G, Papa S (2015) Intramitochondrial adenylyl cyclase controls the turnover of nuclear-encoded subunits and activity of mammalian complex I of the respiratory chain. Biochim Biophys Acta 1853:183–191

    Article  PubMed  CAS  Google Scholar 

  15. Di Benedetto G, Scalzotto E, Mongillo M, Pozzan T (2013) Mitochondrial Ca(2)(+) uptake induces cyclic AMP generation in the matrix and modulates organelle ATP levels. Cell Metab 17:965–975

    Article  PubMed  CAS  Google Scholar 

  16. Emma F, Montini G, Parikh SM, Salviati L (2016) Mitochondrial dysfunction in inherited renal disease and acute kidney injury. Nat Rev Nephrol 12:267–280

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Emma F, Nesterova G, Langman C, Labbe A, Cherqui S, Goodyer P, Janssen MC, Greco M, Topaloglu R, Elenberg E, Dohil R, Trauner D, Antignac C, Cochat P, Kaskel F, Servais A, Wuhl E, Niaudet P, Vant Hoff W, Gahl W, Levtchenko E (2014) Nephropathic cystinosis: an international consensus document. Nephrol Dial Transplant 29(Suppl 4):iv87–iv94

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Francis SH, Busch JL, Corbin JD, Sibley D (2010) cGMP-dependent protein kinases and cGMP phosphodiesterases in nitric oxide and cGMP action. Pharmacol Rev 62:525–563

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Gahl WA, Bashan N, Tietze F, Bernardini I, Schulman JD (1982) Cystine transport is defective in isolated leukocyte lysosomes from patients with cystinosis. Science 217:1263–1265

    Article  PubMed  CAS  Google Scholar 

  20. Habersetzer J, Ziani W, Larrieu I, Stines-Chaumeil C, Giraud MF, Brethes D, Dautant A, Paumard P (2013) ATP synthase oligomerization: from the enzyme models to the mitochondrial morphology. Int J Biochem Cell Biol 45:99–105

    Article  PubMed  CAS  Google Scholar 

  21. Ivanova EA, van den Heuvel LP, Elmonem MA, De Smedt H, Missiaen L, Pastore A, Mekahli D, Bultynck G, Levtchenko EN (2016) Altered mTOR signalling in nephropathic cystinosis. J Inherit Metab Dis 39:457–464

    Article  PubMed  CAS  Google Scholar 

  22. Koentges C, Bode C, Bugger H (2016) SIRT3 in cardiac physiology and disease. Front Cardiovasc Med 3:38

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Kumar A, Bachhawat AK (2010) A futile cycle, formed between two ATP-dependant gamma-glutamyl cycle enzymes, gamma-glutamyl cysteine synthetase and 5-oxoprolinase: the cause of cellular ATP depletion in nephrotic cystinosis? J Biosci 35:21–25

    Article  PubMed  CAS  Google Scholar 

  24. Lazarou M, McKenzie M, Ohtake A, Thorburn DR, Ryan MT (2007) Analysis of the assembly profiles for mitochondrial- and nuclear-DNA-encoded subunits into complex I. Mol Cell Biol 27:4228–4237

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Lee CH, MacKinnon R (2017) Structures of the human HCN1 hyperpolarization-activated channel. Cell 168(111–120):e11

    Google Scholar 

  26. Levtchenko EN, Wilmer MJ, Janssen AJ, Koenderink JB, Visch HJ, Willems PH, de Graaf-Hess A, Blom HJ, van den Heuvel LP, Monnens LA (2006) Decreased intracellular ATP content and intact mitochondrial energy generating capacity in human cystinotic fibroblasts. Pediatr Res 59:287–292

    Article  PubMed  CAS  Google Scholar 

  27. Litvin TN, Kamenetsky M, Zarifyan A, Buck J, Levin LR (2003) Kinetic properties of “soluble” adenylyl cyclase. Synergism between calcium and bicarbonate. J Biol Chem 278:15922–15926

    Article  PubMed  CAS  Google Scholar 

  28. Mannucci L, Pastore A, Rizzo C, Piemonte F, Rizzoni G, Emma F (2006) Impaired activity of the gamma-glutamyl cycle in nephropathic cystinosis fibroblasts. Pediatr Res 59:332–335

    Article  PubMed  CAS  Google Scholar 

  29. Mao Z, Choo YS, Lesort M (2006) Cystamine and cysteamine prevent 3-NP-induced mitochondrial depolarization of Huntington’s disease knock-in striatal cells. Eur J Neurosci 23:1701–1710

    Article  PubMed  Google Scholar 

  30. McEvoy B, Sumayao R, Slattery C, McMorrow T, Newsholme P (2015) Cystine accumulation attenuates insulin release from the pancreatic beta-cell due to elevated oxidative stress and decreased ATP levels. J Physiol 593:5167–5182

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Mello T, Materozzi M, Galli A (2016) PPARs and Mitochondrial Metabolism: from NAFLD to HCC. PPAR Res 2016:7403230

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Mitznegg P (1973) On the mechanism of radioprotection by cysteamine. II. The significance of cyclic 3′,5′-AMP for the cysteamine-induced radioprotective effects in white mice. Int J Radiat Biol Relat Stud Phys Chem Med 24:339–344

    Article  PubMed  CAS  Google Scholar 

  33. Morigi M, Perico L, Rota C, Longaretti L, Conti S, Rottoli D, Novelli R, Remuzzi G, Benigni A (2015) Sirtuin 3-dependent mitochondrial dynamic improvements protect against acute kidney injury. J Clin Investig 125:715–726

    Article  PubMed  Google Scholar 

  34. Nakamura A, Johns EJ, Imaizumi A, Yanagawa Y, Kohsaka T (2001) Activation of beta(2)-adrenoceptor prevents shiga toxin 2-induced TNF-alpha gene transcription. J Am Soc Nephrol 12:2288–2299

    PubMed  CAS  Google Scholar 

  35. Napolitano G, Johnson JL, He J, Rocca CJ, Monfregola J, Pestonjamasp K, Cherqui S, Catz SD (2015) Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis. EMBO Mol Med 7:158–174

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Papa S, De Rasmo D (2013) Complex I deficiencies in neurological disorders. Trends Mol Med 19:61–69

    Article  PubMed  CAS  Google Scholar 

  37. Paumard P, Vaillier J, Coulary B, Schaeffer J, Soubannier V, Mueller DM, Brethes D, di Rago JP, Velours J (2002) The ATP synthase is involved in generating mitochondrial cristae morphology. EMBO J 21:221–230

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Plotegher N, Duchen MR (2017) Mitochondrial dysfunction and neurodegeneration in lysosomal storage disorders. Trends Mol Med 23:116–134

    Article  PubMed  CAS  Google Scholar 

  39. Raggi C, Luciani A, Nevo N, Antignac C, Terryn S, Devuyst O (2014) Dedifferentiation and aberrations of the endolysosomal compartment characterize the early stage of nephropathic cystinosis. Hum Mol Genet 23:2266–2278

    Article  PubMed  CAS  Google Scholar 

  40. Ravnskjaer K, Madiraju A, Montminy M (2016) Role of the cAMP pathway in glucose and lipid metabolism. Handb Exp Pharmacol 233:29–49

    Article  PubMed  CAS  Google Scholar 

  41. Rodighiero S, Bazzini C, Ritter M, Furst J, Botta G, Meyer G, Paulmichl M (2008) Fixation, mounting and sealing with nail polish of cell specimens lead to incorrect FRET measurements using acceptor photobleaching. Cell Physiol Biochem 21:489–498

    Article  PubMed  CAS  Google Scholar 

  42. Sansanwal P, Yen B, Gahl WA, Ma Y, Ying L, Wong LJ, Sarwal MM (2010) Mitochondrial autophagy promotes cellular injury in nephropathic cystinosis. J Am Soc Nephrol 21:272–283

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Schapira AH, Olanow CW, Greenamyre JT, Bezard E (2014) Slowing of neurodegeneration in Parkinson’s disease and Huntington’s disease: future therapeutic perspectives. Lancet 384:545–555

    Article  PubMed  CAS  Google Scholar 

  44. Signorile A, Santeramo A, Tamma G, Pellegrino T, D’Oria S, Lattanzio P, De Rasmo D (2017) Mitochondrial cAMP prevents apoptosis modulating Sirt3 protein level and OPA1 processing in cardiac myoblast cells. Biochim Biophys Acta 1864:355–366

    Article  PubMed  CAS  Google Scholar 

  45. Stork PJ, Schmitt JM (2002) Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation. Trends Cell Biol 12:258–266

    Article  PubMed  CAS  Google Scholar 

  46. Sumayao R, McEvoy B, Newsholme P, McMorrow T (2016) Lysosomal cystine accumulation promotes mitochondrial depolarization and induction of redox-sensitive genes in human kidney proximal tubular cells. J Physiol 594:3353–3370

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Taub M, Cutuli F (2012) Activation of AMP kinase plays a role in the increased apoptosis in the renal proximal tubule in cystinosis. Biochem Biophys Res Commun 426:516–521

    Article  PubMed  CAS  Google Scholar 

  48. Town M, Jean G, Cherqui S, Attard M, Forestier L, Whitmore SA, Callen DF, Gribouval O, Broyer M, Bates GP, van’t Hoff W, Antignac C (1998) A novel gene encoding an integral membrane protein is mutated in nephropathic cystinosis. Nat Genet 18:319–324

    Article  PubMed  CAS  Google Scholar 

  49. Valsecchi F, Ramos-Espiritu LS, Buck J, Levin LR, Manfredi G (2013) cAMP and mitochondria. Physiology (Bethesda) 28:199–209

    CAS  Google Scholar 

  50. Vinogradov AD, Grivennikova VG (2016) Oxidation of NADH and ROS production by respiratory complex I. Biochim Biophys Acta 1857:863–871

    Article  PubMed  CAS  Google Scholar 

  51. Vowinckel J, Hartl J, Butler R, Ralser M (2015) MitoLoc: a method for the simultaneous quantification of mitochondrial network morphology and membrane potential in single cells. Mitochondrion 24:77–86

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. Walker JE (2013) The ATP synthase: the understood, the uncertain and the unknown. Biochem Soc Trans 41:1–16

    Article  PubMed  CAS  Google Scholar 

  53. Wang Z, Zhang L, Liang Y, Zhang C, Xu Z, Zhang L, Fuji R, Mu W, Li L, Jiang J, Ju Y, Wang Z (2015) Cyclic AMP mimics the anti-ageing effects of calorie restriction by up-regulating sirtuin. Sci Rep 5:12012

    Article  PubMed  PubMed Central  Google Scholar 

  54. Wilmer MJ, de Graaf-Hess A, Blom HJ, Dijkman HB, Monnens LA, van den Heuvel LP, Levtchenko EN (2005) Elevated oxidized glutathione in cystinotic proximal tubular epithelial cells. Biochem Biophys Res Commun 337:610–614

    Article  PubMed  CAS  Google Scholar 

  55. Wilmer MJ, Emma F, Levtchenko EN (2010) The pathogenesis of cystinosis: mechanisms beyond cystine accumulation. Am J Physiol Renal Physiol 299:F905–F916

    Article  PubMed  CAS  Google Scholar 

  56. Wilmer MJ, Kluijtmans LA, van der Velden TJ, Willems PH, Scheffer PG, Masereeuw R, Monnens LA, van den Heuvel LP, Levtchenko EN (2011) Cysteamine restores glutathione redox status in cultured cystinotic proximal tubular epithelial cells. Biochim Biophys Acta 1812:643–651

    Article  PubMed  CAS  Google Scholar 

  57. Wilmer MJ, Saleem MA, Masereeuw R, Ni L, van der Velden TJ, Russel FG, Mathieson PW, Monnens LA, van den Heuvel LP, Levtchenko EN (2010) Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters. Cell Tissue Res 339:449–457

    Article  PubMed  Google Scholar 

  58. Wilmer MJ, van den Heuvel LP, Rodenburg RJ, Vogel RO, Nijtmans LG, Monnens LA, Levtchenko EN (2008) Mitochondrial complex V expression and activity in cystinotic fibroblasts. Pediatr Res 64:495–497

    Article  PubMed  CAS  Google Scholar 

  59. Wittig I, Schagger H (2009) Supramolecular organization of ATP synthase and respiratory chain in mitochondrial membranes. Biochim Biophys Acta 1787:672–680

    Article  PubMed  CAS  Google Scholar 

  60. Zippin JH, Chen Y, Straub SG, Hess KC, Diaz A, Lee D, Tso P, Holz GG, Sharp GW, Levin LR, Buck J (2013) CO2/HCO3(−)- and calcium-regulated soluble adenylyl cyclase as a physiological ATP sensor. J Biol Chem 288:33283–33291

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Acknowledgements

FB, FE, and DDR are supported by a Research Grant from Cystinosis Research Foundation. DDR and AS are supported by a Research Grant from University of Bari “Aldo Moro”. We thank Mr. Paolo Lattanzio for technical assistance.

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Correspondence to Francesco Bellomo or Domenico De Rasmo.

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Supplemental Figure S1. The sAC-dependent cAMP level is lower in

CTNS/ ciPTEC and increased by cysteamine treatment. ciPTEC were treated in the absence or in the presence of 50 μM KH7 or 100 μM cysteamine (MEA), for 4 and 24 h respectively. After the treatment, the medium was aspirated and 1 ml of 0.1 M HCl was added to the cell layer followed by 10 min incubation at 37 °C. The lysed cells were centrifuged at 1,300×g for 10 min at 4 °C. The supernatants were used to determine cAMP concentration using a direct immunoassay kit (Assay Designs) as described by the manufacturer. Total protein concentration was determined by Bio Rad protein assay. The cAMP levels in the samples were normalized to the protein concentration and expressed as pmol/mg protein. The histograms represent the mean values of duplicate determinations of cAMP level in three separate experiments. Error bars indicate standard deviations. Significant differences were calculated with Student’s t test (JPEG 257 kb)

Supplemental Figure S2.

HK-2 cells silenced for CTNS gene show a significative reduction of CTNS relative gene expression. Human kidney-2 cells (HK-2) were transfected with siGENOME human CTNS gene (siCTNS) or non-targeting siRNA (Mock). Where indicated the cells were treated for 24 h with DMSO (vehicle) or 100 µM cysteamine (MEA). Analysis of CTNS gene expression, normalized to the expression of GAPDH, show a reduction in siCTNS of 68% (P < 0.0001) and 64% (P < 0.02) compared to control and mock respectively. Significant differences were calculated with Student’s t test (JPEG 124 kb)

Supplemental Figure S3.

Cysteamine or 8Br-cAMP treatment ameliorate mitochondrial morphology in HK-2 cells double silenced for CTNS and SIRT3 genes. In panel A, HK-2 were co-transfected with human CTNS gene and SIRT3 gene (Santa Cruz, Biotechnology, Dallas, Texas) (siRNA) or non-targeting siRNA (Mock). Where indicated the cells were treated for 24 h with DMSO (vehicle) or 100 µM cysteamine (MEA) or 100 µM 8Br-cAMP. Analysis of CTNS gene expression, normalized to the expression of GAPDH, shows 80% reduction of CTNS gene expression (P < 0.0001) and 40% reduction of SIRT3 gene expression (P < 0.04) compared to control. Panel B, quantification of mitochondrial fragmentation index (f index) in HK-2 cells not transfected (Control), transfected with non-targeting siRNA (Mock) or co-transfected with human CTNS gene and SIRT3 gene (siRNA) for 72 h and treated with 100 µM cysteamine (MEA) or 100 µM 8Br-cAMP during last 24 h of incubation. Data are the mean values (± SEM) of the analyses performed in cell numbers of n = 30 for HK-2 cells. Significant differences were calculated with Student’s t test (JPEG 395 kb)

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Bellomo, F., Signorile, A., Tamma, G. et al. Impact of atypical mitochondrial cyclic-AMP level in nephropathic cystinosis. Cell. Mol. Life Sci. 75, 3411–3422 (2018). https://doi.org/10.1007/s00018-018-2800-5

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