Amino Acids

pp 1–11 | Cite as

Epileptic seizures and oxidative stress in a mouse model over-expressing spermine oxidase

  • Alessia Leonetti
  • Giulia Baroli
  • Emiliano Fratini
  • Stefano Pietropaoli
  • Manuela Marcoli
  • Paolo Mariottini
  • Manuela CervelliEmail author
Original Article
Part of the following topical collections:
  1. Polyamines: Biochemical and Pathophysiological Properties


Several studies have demonstrated high polyamine levels in brain diseases such as epilepsy. Epilepsy is the fourth most common neurological disorder and affects people of all ages. Excitotoxic stress has been associated with epilepsy and it is considered one of the main causes of neuronal degeneration and death. The transgenic mouse line Dach-SMOX, with CD1 background, specifically overexpressing spermine oxidase in brain cortex, has been proven to be highly susceptible to epileptic seizures and excitotoxic stress induced by kainic acid. In this study, we analysed the effect of spermine oxidase over-expression in a different epileptic model, pentylenetetrazole. Behavioural evaluations of transgenic mice compared to controls showed a higher susceptibility towards pentylentetrazole. High-performance liquid chromatography analysis of transgenic brain from treated mice revealed altered polyamine content. Immunoistochemical analysis indicated a rise of 8-oxo-7,8-dihydro-2′-deoxyguanosine, demonstrating an increase in oxidative damage, and an augmentation of system x c as a defence mechanism. This cascade of events can be initially linked to an increase in protein kinase C alpha, as shown by Western blot. This research points out the role of spermine oxidase, as a hydrogen peroxide producer, in the oxidative stress during epilepsy. Moreover, Dach-SMOX susceptibility demonstrated by two different epileptic models strongly indicates this transgenic mouse line as a potential animal model to study epilepsy.


Excitotoxicity Glutamate toxicity Pentylentetrazole treatment Polyamines Spermine oxidase 



This work was supported by the Roma Tre University contribution to the laboratories (CAL/2017 and CAL/2018) to M.C. and P.M and by the Ph.D. School (Department of Science) contribution 2017 to A.L. and S.P and 2018 to G.B. The authors wish to thank Mrs Rosetta Ponzo for the revision of the English text and Prof. G. Maura (University of Genova, Italy) for useful discussion.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The experiments were carried out in accordance with the ethical guidelines for the conduction of animal research of the European Community’s Council Directive 2010/63/EU. Formal approval of these experiments was obtained from the Italian Ministry of Health with the approved protocol N°964/2015-PR.


  1. Amendola R, Cervelli M, Fratini E, Polticelli F, Sallustio DE, Mariottini P (2009) Spermine metabolism and anticancer therapy. Curr Cancer Drug Targets 9:118–130CrossRefGoogle Scholar
  2. Amendola R, Cervelli M, Fratini E, Sallustio DE, Tempera G, Ueshima T, Mariottini P, Agostinelli E (2013) Reactive oxygen species spermine metabolites generated from amine oxidases and radiation represent a therapeutic gain in cancer treatments. Int J Oncol 43:813–820CrossRefGoogle Scholar
  3. Amendola R, Cervelli M, Tempera G, Fratini E, Varesio L, Mariottini P, Agostinelli E (2014) Spermine metabolism and radiation-derived reactive oxygen species for future therapeutic implications in cancer: an additive or adaptive response. Amino Acids 46:487–498CrossRefGoogle Scholar
  4. Bannai S (1986) Exchange of cystine and glutamate across plasma membrane of human fibroblasts. J Biol Chem 26:2256–2263Google Scholar
  5. Binder DK, Oshio K, Ma T, Verkman AS, Manley GT (2004) Increased seizure threshold in mice lacking aquaporin-4 water channels. Neuro Rep 15:259–262Google Scholar
  6. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  7. Bridges RJ, Natale NR, Patel SA (2012) System xc cystine/glutamate antiporter: an update on molecular pharmacology and roles within the CNS. Br J Pharmacol 165:20–34CrossRefGoogle Scholar
  8. Buckingham SC, Robel S (2013) Glutamate and tumor-associated epilepsy: glial cell dysfunction in the peritumoral environment. Neurochem Int 63:696–701CrossRefGoogle Scholar
  9. Buckingham SC, Campbell SL, Haas BR, Montana V, Robel S, Ogunrinu T, Sontheimer H (2011) Glutamate release by primary brain tumors induces epileptic activity. Nat Med 17:1269–1274CrossRefGoogle Scholar
  10. Capone C, Cervelli M, Angelucci E, Colasanti M, Macone A, Mariottini P, Persichini T (2013) A role for spermine oxidase as a mediator of reactive oxygen species production in HIV-Tat-induced neuronal toxicity. Free Radic Biol Med 63:99–107CrossRefGoogle Scholar
  11. Carmona-Aparicio L, Pérez-Cruz C, Zavala-Tecuapetla C, Granados-Rojas L, Rivera-Espinosa L, Montesinos-Correa H, Hernández-Damián J, Pedraza-Chaverri J, Sampieri Aristides III, Coballase-Urrutia E, Cárdenas-Rodríguez N (2015) Overview of Nrf2 as therapeutic Target in epilepsy. Int J Mol 16:18348–18367CrossRefGoogle Scholar
  12. Casero RA, Pegg AE (2009) Polyamine catabolism and disease. Biochem J 421:323–338CrossRefGoogle Scholar
  13. Ceci R, Duranti G, Leonetti A, Pietropaoli S, Spinozzi F, Marcocci L, Amendola R, Cecconi F, Sabatini S, Mariottini P, Cervelli M (2017) Adaptive responses of heart and skeletal muscle to spermine oxidase overexpression: evaluation of a new transgenic mouse model. Free Radic Biol Med 103:216–225CrossRefGoogle Scholar
  14. Cervelli M, Fratini E, Amendola R, Bianchi M, Signori E, Ferraro E, Lisi A, Federico R, Marcocci L, Mariottini P (2009) Increased spermine oxidase (SMO) activity as a novel differentiation marker of myogenic C2C12 cells. Int J Biochem Cell Biol 41:934–944CrossRefGoogle Scholar
  15. Cervelli M, Bellavia G, Fratini E, Amendola R, Polticelli F, Barba M, Federico R, Signore F, Gucciardo G, Grillo R, Woster PM, Casero RA Jr, Mariottini P (2010) Spermine oxidase (SMO) activity in breast tumor tissues and biochemical analysis of the anticancer spermine analogues BENSpm and CPENSpm. BMC Cancer 10:555CrossRefGoogle Scholar
  16. Cervelli M, Amendola R, Polticelli F, Mariottini P (2012) Spermine oxidase: ten years after. Amino Acids 42:441–450CrossRefGoogle Scholar
  17. Cervelli M, Salvi D, Polticelli F, Amendola R, Mariottini P (2013a) Structure-function relationships in the evolutionary framework of spermine oxidase. J Mol Evol 76:365–370CrossRefGoogle Scholar
  18. Cervelli M, Bellavia G, D’Amelio M, Cavallucci V, Moreno S, Berger J, Nardacci R, Marcoli M, Maura G, Piacentini M, Amendola R, Cecconi F, Mariottini P (2013b) A new transgenic mouse model for studying the neurotoxicity of spermine oxidase dosage in the response to excitotoxic injury. PLoS One 8:e64810CrossRefGoogle Scholar
  19. Cervelli M, Pietropaoli S, Signore F, Amendola R, Mariottini P (2014a) Polyamines metabolism and breast cancer: state of the art and perspectives. Breast Cancer Res Treat 148:233–248CrossRefGoogle Scholar
  20. Cervelli M, Angelucci E, Germani F, Amendola R, Mariottini P (2014b) Inflammation, carcinogenesis and neurodegeneration studies in transgenic animal models for polyamine research. Amino Acids 46:521–530CrossRefGoogle Scholar
  21. Cervelli M, Leonetti A, Cervoni L, Ohkubo S, Xhani M, Stano P, Federico R, Polticelli F, Mariottini P, Agostinelli E (2016) Stability of spermine oxidase to thermal and chemical denaturation: comparison with bovine serum amine oxidase. Amino Acids 48:2283–2291CrossRefGoogle Scholar
  22. Cervetto C, Vergani L, Passalacqua M, Ragazzoni M, Venturini A, Cecconi F, Berretta N, Mercuri N, D’Amelio M, Maura G, Mariottini P, Voci A, Marcoli M, Cervelli M (2016) Astrocyte-dependent vulnerability to excitotoxicity in spermine oxidase-overexpressing mouse. Neuro Mol Med 18:50–68CrossRefGoogle Scholar
  23. Chaturvedi R, Asim M, Romero-Gallo J, Barry DP, Hoge S, de Sablet T, Delgado AG, Wroblewski LE, Piazuelo MB, Yan F, Israel DA, Casero RA Jr, Correa P, Gobert AP, Polk DB, Peek RM Jr, Wilson KT (2011) Spermine oxidase mediates the gastric cancer risk associated with Helicobacter pylori CagA. Gastroenterology 141:1696–1708CrossRefGoogle Scholar
  24. Choi DW (1994) Calcium and excitotoxic neuronal injury. Ann N Y Acad Sci 747:162–171CrossRefGoogle Scholar
  25. Claycomb RJ, Hewett SJ, Hewett JA (2011) Prophylactic, prandial rofecoxib treatment lacks efficacy against acute PTZ-induced seizure generation and kindling acquisition. Epilepsia 52:273–283Google Scholar
  26. de Groot J, Sontheimer H (2011) Glutamate and the biology of gliomas. Glia 59:1181–1189CrossRefGoogle Scholar
  27. Doble A (1999) The role of excitotoxicity in neurodegenerative disease: implications for therapy. Pharmacol Ther 81:163–221CrossRefGoogle Scholar
  28. Doi T, Ueda Y, Nagatomo K, Willmore LJ (2009) Role of glutamate and GABA transporters in development of pentylenetetrazol-kindling. Neurochem Res 34:1324–1331CrossRefGoogle Scholar
  29. Elger CE, Schmidt D (2008) Modern management of epilepsy: a practical approach. Epilepsy Behav 12:501–539CrossRefGoogle Scholar
  30. Erdoğan F, Gölgeli A, Arman F, Ersoy AO (2004) The effects of pentylenetetrazole-induced status epilepticus on behavior, emotional memory, and learning in rats. Epilepsy Behav 5:388–393CrossRefGoogle Scholar
  31. Giorgi O, Carboni G, Frau V, Orlandi M, Valentini V, Feldman A, Corda MG (1996) Anticonvulsant effect of felbamate in the pentylenetetrazole kindling model of epilepsy in the rat. Naunyn Schmiedebergs Arch Pharmacol 354:173–178CrossRefGoogle Scholar
  32. Goodwin AC, Jadallah S, Toubaji A, Lecksell K, Hicks JL, Kowalski J, Bova GS, De Marzo AM, Netto GJ, Casero RA Jr (2008) Increased spermine oxidase expression in human prostate cancer and prostatic intraepithelial neoplasia tissues. Prostate 68:766–772CrossRefGoogle Scholar
  33. Huang HC, Nguyen T, Pickett CB (2000) Regulation of the antioxidant response element by protein kinase C-mediated phosphorylation of NF-E2-related factor 2. PNAS 97:12475–12480CrossRefGoogle Scholar
  34. Huang RQ, Bell-Horner CL, Dibas MI, Covey DF, Drewe JA, Dillon GH (2001) Pentylenetetrazol-induced inhibition of recombinant gamma-aminobutyric acid type A (GABA(A)) receptors: mechanism and site of action. J Pharmacol Exp Ther 298:986–995Google Scholar
  35. Kauppinen RA, Alhonen LI (1995) Transgenic animals as models in the study of the neurobiological role of polyamines. Progr Neurobiol 47:545–563CrossRefGoogle Scholar
  36. Lewerenz J, Hewett SJ, Huang Y, Lambros M, Gout PW, Massie KA, Smolders I, Methner A, Pergande M, Smith SB, Ganapathy V, Maher P (2013) The cystine/glutamate antiporter system xc- in health and disease: from molecular mechanisms to novel therapeutic opportunities. Antioxid Redox Signal 18:522–555CrossRefGoogle Scholar
  37. Lipton SA, Rosenberg PA (1994) Excitatory amino acids as a final common pathway for neurologic disorders. N Engl J Med 330:613–622CrossRefGoogle Scholar
  38. Lüttjohann A, Fabene PF, van Luijtelaar G (2009) A revised Racine’s scale for PTZ-induced seizures in rats. Physiol Behav 98:579–586CrossRefGoogle Scholar
  39. Mastrantonio R, Cervelli M, Pietropaoli S, Mariottini P, Colasanti M, Persichini T (2016) HIV-Tat induces the Nrf2/ARE pathway through NMDA receptor-elicited spermine oxidase activation in human neuroblastoma cells. PLoS One 11:e0149802CrossRefGoogle Scholar
  40. Naseer MI, Ullah I, Al-Qahtani MH, Karim S, Ullah N, Ansari SA, Kim MO, Bibi F (2013) Decreased GABABR expression and increased neuronal cell death in developing rat brain after PTZ-induced seizure. Neurol Sci 34:497–503CrossRefGoogle Scholar
  41. Patel M (2004) Mitochondrial dysfunction and oxidative stress: cause and consequence of epileptic seizures. Free Radical Biol Med 37:1951–1962CrossRefGoogle Scholar
  42. Pietropaoli S, Leonetti A, Cervetto C, Venturini A, Mastrantonio R, Baroli G, Persichini T, Colasanti M, Maura G, Marcoli M, Mariottini P, Cervelli M (2018) Glutamate excitotoxicity linked to spermine oxidase overexpression. Mol Neurobiol 55:7259–7270CrossRefGoogle Scholar
  43. Polticelli F, Salvi D, Mariottini P, Amendola R, Cervelli M (2012) Molecular evolution of the polyamine oxidase gene family in Metazoa. BMC Evol Biol 12:90CrossRefGoogle Scholar
  44. Psarropoulou C, Matsokis N, Angelatou F, Kostopoulos G (1994) Pentylenetetrazol-induced seizures decrease gamma-aminobutyric acid-mediated recurrent inhibition and enhance adenosine-mediated depression. Epilepsia 35:12–19CrossRefGoogle Scholar
  45. Puttachary S, Sharma S, Stark S, Thippeswamy T (2015) Seizure-induced oxidative stress in temporal lobe epilepsy. Biomed Res Int 2015:745613CrossRefGoogle Scholar
  46. Qaisiya M, Zabetta CDC, Bellarosa C, Tiribelli C (2014) Bilirubin mediated oxidative stress involves antioxidant response activation via Nrf2 pathway. Cell Signal 26:512–520CrossRefGoogle Scholar
  47. Racine RJ (1972) Modification of seizure activity by electrical stimulation: II motor seizure. Electroencephalogr Clin Neurophysiol 32:281–294CrossRefGoogle Scholar
  48. Rauca C, Zerbe R, Jantze H (1999) Formation of free hydroxyl radicals after pentylenetetrazol-induced seizure and kindling. Brain Res 847:347–351CrossRefGoogle Scholar
  49. Rea G, Bocedi A, Cervelli M (2004) Question: what is the biological function of the polyamines? IUBMB Life 56:167–169CrossRefGoogle Scholar
  50. Sajadian A, Esteghamat S, Karimzadeh F, Eshaghabadi A, Sieg F, Speckmann EJ, Meuth S, Seidenbecher T, Budde T, Gorji A (2015) Anticonvulsant effect of neural regeneration peptide 2945 on pentylenetetrazol-induced seizures in rats. Neuropeptides 49:15–23CrossRefGoogle Scholar
  51. Sato H, Tamba M, Ishii T, Bannai S (1999) Cloning and expression of a plasma membrane cystine/glutamate exchange transporter composed of two distinct proteins. J Biol Chem 274:11455–11458CrossRefGoogle Scholar
  52. Tavladoraki P, Cervelli M, Antonangeli F, Minervini G, Stano P, Federico R, Mariottini P, Polticelli F (2011) Probing mammalian spermine oxidase enzyme-substrate complex through molecular modeling, site-directed mutagenesis and biochemical characterization. Amino Acids 40:1115–1126CrossRefGoogle Scholar
  53. Tremblay R, Hewitt K, Lesiuk H, Mealing G, Morley P, Durkin JP (1999) Evidence that brain-derived neurotrophic factor neuroprotection is linked to its ability to reverse the NMDA-induced inactivation of protein kinase C in cortical neurons. J Neurochem 72:102–111CrossRefGoogle Scholar
  54. Volterra A, Trotti D, Tromba C, Floridi S, Racagni G (1994) Glutamate uptake inhibition by oxygen free radicals in rat cortical astrocytes. J Neurosci 5:2924–2932CrossRefGoogle Scholar
  55. Williams K (1997) Interactions of polyamines with ion channels. Biochem J 325:289–297CrossRefGoogle Scholar
  56. World Health Organization (WHO) (2019) Epilepsy Fact sheet.
  57. Zahedi K, Huttinger F, Morrison R, Murray-Stewart T, Casero RA, Strauss KI (2010) Polyamine catabolism is enhanced after traumatic brain injury. J Neurotrauma 27:515–525CrossRefGoogle Scholar
  58. Zahedi K, Barone S, Destefano-Shields C, Brooks M, Murray-Stewart T, Dunworth M, Li W, Doherty JR, Hall MA, Smith RD, Cleveland JL, Casero RA Jr, Soleimani M (2017) Activation of endoplasmic reticulum stress response by enhanced polyamine catabolism is important in the mediation of cisplatin-induced acute kidney injury. PLoS One 12:e0184570CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of ScienceUniversity of Rome “Roma Tre”RomeItaly
  2. 2.Interuniversity Consortium of Structural and Systems BiologyRomeItaly
  3. 3.Section of Pharmacology and Toxicology, Department of PharmacyUniversity of GenovaGenoaItaly
  4. 4.Center of Excellence for Biomedical Research, University of GenovaGenoaItaly

Personalised recommendations