Neurochemical Research

, Volume 39, Issue 1, pp 37–45 | Cite as

Effects of Amide Creatine Derivatives in Brain Hippocampal Slices, and Their Possible Usefulness for Curing Creatine Transporter Deficiency

  • Patrizia Garbati
  • Enrico Adriano
  • Annalisa Salis
  • Silvia Ravera
  • Gianluca Damonte
  • Enrico Millo
  • Maurizio Balestrino
Original Paper


The creatine/phosphocreatine system carries ATP from production to consumption sites and buffers the intracellular content of ATP at times of energy deprivation. The creatine transporter deficiency syndrome is an X-linked disease caused by a defective creatine transporter into the central nervous system. This disease is presently untreatable because creatine lacking its carrier cannot cross neither the blood–brain barrier nor the cell plasma membranes. Possible strategies to cure this condition are to couple creatine to molecules which have their own carrier, to exploit the latter to cross biological membranes or to modify the creatine molecule to make it more lipophilic, in such a way that it may more easily cross lipid-rich biological membranes. Such molecules could moreover be useful for treatment of stroke or other ischemic brain syndromes of normal (transporter working) tissue. In this paper we tested four molecules in in vitro hippocampal slices experiments to investigate whether or not they had a neuroprotective effect similar to that of creatine. On two of them we also performed biochemical measurements to investigate whether or not they were able to increase the creatine and phosphocreatine content of the hippocampal slices with and without block of the transporter. We found that these molecules increase levels of creatine after block of the transporter, and significantly increased the levels of phosphocreatine. Both significantly increased the total creatine content in both conditions of active and blocked transporter. This shows that these molecules are capable of entering cells through biological membranes without using the creatine transporter. By contrast, neither of them was able to delay synaptic block during anoxia of normal (transporter functioning) tissue. We conclude that these compounds might possibly be useful for therapy of creatine transporter deficiency, but further research is needed to understand their possible role in anoxia/ischemia of normal tissue.


Creatine Creatine transporter deficiency Creatine derivatives Neuroprotection Glycine damage Hippocampal slices 


  1. 1.
    Wyss M, Kaddurah-Daouk R (2000) Creatine and creatinine metabolism. Physiol Rev 80(3):1107–1213PubMedGoogle Scholar
  2. 2.
    Greenhaff PL (2001) The creatine–phosphocreatine system: there’s more than one song in its repertoire. J Physiol 537(3):657. doi: 10.1113/jphysiol.2001.013478 PubMedCrossRefGoogle Scholar
  3. 3.
    Balestrino M, Lensman M, Parodi M, Perasso L, Rebaudo R, Melani R, Polenov S, Cupello A (2002) Role of creatine and in neuronal protection from anoxic and ischemic damage. Amino Acids 23(1–3):221–229. doi: 10.1007/s00726-001-0133-3 PubMedCrossRefGoogle Scholar
  4. 4.
    Perasso L, Spallarossa P, Gandolfo C, Ruggeri P, Balestrino M (2013) Therapeutic use of creatine in brain or heart ischemia: available data and future perspectives. Med Res Rev 33(2):336–363. doi: 10.1002/med.20255 PubMedCrossRefGoogle Scholar
  5. 5.
    Klivenyi P, Ferrante RJ, Matthews RT, Bogdanov MB, Klein AM, Andreassen OA, Mueller G, Wermer M, Kaddurah-Daouk R, Beal MF (1999) Neuroprotective effects of creatine in a transgenic animal model of amyotrophic lateral sclerosis. Nat Med 5:347–350. doi: 10.1038/6568 PubMedCrossRefGoogle Scholar
  6. 6.
    Adhihetty PJ, Beal MF (2008) Creatine and its potential therapeutic value for targeting cellular energy impairment in neurodegenerative diseases. Neuromol Med 10(4):275–290. doi: 10.1007/s12017-008-8053-y CrossRefGoogle Scholar
  7. 7.
    Beal MF (2011) Neuroprotective effects of creatine. Amino Acids 40(5):1305–1313. doi: 10.1007/s00726-011-0851-0 PubMedCrossRefGoogle Scholar
  8. 8.
    Wang J, Xiao Y, Chen S, Zhong C, Luo M, Luo H (2012) Creatine for Parkinson’s disease. Cochrane Database Syst Rev Issue 2. doi: 10.1002/14651858.CD009646
  9. 9.
    Perasso L, Cupello A, Lunardi GL, Principato C, Gandolfo C, Balestrino M (2003) Kinetics of creatine in blood and brain after intraperitoneal injection in the rat. Brain Res 974(1–2):37–42. doi: 10.1016/S0006-8993(03)02547-2 PubMedCrossRefGoogle Scholar
  10. 10.
    Lunardi G, Parodi A, Perasso L, Pohvozcheva AV, Scarrone S, Adriano E, Florio T, Gandolfo C, Cupello A, Burov SV, Balestrino M (2006) The creatine transporter mediates the uptake of creatine by brain tissue, but not the uptake of two creatine-derived compounds. Neuroscience 142(4):991–997. doi: 10.1016/j.neuroscience.2006.06.058 PubMedCrossRefGoogle Scholar
  11. 11.
    Snow RJ, Murphy RM (2001) Creatine and the creatine transporter: a review. Mol Cell Biochem 224(1–2):169–181. doi: 10.1023/A:1011908606819 PubMedCrossRefGoogle Scholar
  12. 12.
    deGrauw TJ, Cecil KM, Byars AW, Salomons GS, Ball WS, Jakobs C (2003) The clinical syndrome of creatine transporter deficiency. Mol Cell Biochem 244(1–2):45–48. doi: 10.1023/A:1022487218904 PubMedCrossRefGoogle Scholar
  13. 13.
    Salomons GS, van Dooren SJ, Verhoeven NM, Marsden D, Schwartz C, Cecil KM, DeGrauw TJ, Jakobs C (2003) X-linked creatine transporter defect: an overview. J Inherit Metab Dis 26(2–3):309–318PubMedCrossRefGoogle Scholar
  14. 14.
    Rosenberg EH, Almeida LS, Kleefstra T, deGrauw RS, Yntema HG, Bahi N, Moraine C, Ropers HH, Fryns JP, deGrauw TJ, Jakobs C, Salomons GS (2004) High prevalence of SLC6A8 deficiency in X-linked mental retardation. Am J Hum Genet 75(1):97–105. doi: 10.1086/422102 PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Newmeyer A, Cecil KM, Schapiro M, Clark JF, Degrauw TJ (2005) Incidence of brain creatine transporter deficiency in males with developmental delay referred for brain magnetic resonance imaging. J Dev Behav Pediatr 26(4):276–282. doi: 10.1097/00004703-200508000-00003 PubMedCrossRefGoogle Scholar
  16. 16.
    Clark AJ, Rosenberg EH, Almeida LS, Wood TC, Jakobs C, Stevenson RE, Schwartz CE, Salomons GS (2006) X-linked creatine transporter (SLC6A8) mutations in about 1% of males with mental retardation of unknown etiology. Hum Genet 119(6):604–610. doi: 10.1007/s00439-006-0162-9 PubMedCrossRefGoogle Scholar
  17. 17.
    Lion-François L, Cheillan D, Pitelet G, Acquaviva-Bourdain C, Bussy G, Cotton F, Guibaud L, Gérard D, Rivier C, Vianey-Saban C, Jakobs C, Salomons GS, des Portes V (2006) High frequency of creatine deficiency syndromes in patients with unexplained mental retardation. Neurology 67(9):1713–1714. doi: 10.1212/01.wnl.0000239153.39710.81 PubMedCrossRefGoogle Scholar
  18. 18.
    Arias A, Corbella M, Fons C, Sempere A, García-Villoria J, Ormazabal A, Poo P, Pineda M, Vilaseca MA, Campistol J, Briones P, Pàmpols T, Salomons GS, Ribes A, Artuch R (2007) Creatine transporter deficiency: prevalence among patients with mental retardation and pitfalls in metabolite screening. Clin Biochem 40(16–17):1328–1331. doi: 10.1016/j.clinbiochem.2007.07.010 PubMedCrossRefGoogle Scholar
  19. 19.
    Burov S, Leko M, Dorosh M, Dobrodumov A, Veselkina O (2011) Creatinyl amino acids—new hybrid compounds with neuroprotective activity. J Pept Sci 17(9):620–626. doi: 10.1002/psc.1379 PubMedCrossRefGoogle Scholar
  20. 20.
    Perasso L, Lunardi G, Risso F, Pohvozcheva A, Leko M, Gandolfo C, Florio T, Cupello A, Burov S, Balestrino M (2008) Protective effects of some creatine derivatives in brain tissue anoxia. Neurochem Res 33:765–775. doi: 10.1007/s11064-007-9492-9 PubMedCrossRefGoogle Scholar
  21. 21.
    Balestrino M, Rebaudo R, Lunardi G (1999) Exogenous creatine delays anoxic depolarization and protects from hypoxic damage: dose-effect relationship. Brain Res 816:124–130. doi: 10.1016/S0006-8993(98)01131-7 PubMedCrossRefGoogle Scholar
  22. 22.
    Millo E, Balestrino M, Damonte G, Garbati P, Adriano E, Salis A (2012) Procedimento per sintetizzare derivati della creatina. Patent number TO2012 A001098Google Scholar
  23. 23.
    Garbati P, Salis A, Adriano E, Galatini A, Damonte G, Balestrino M, Millo E (2013) A new method to synthesize creatine derivatives. Amino Acids 44(6):1–13. doi: 10.1007/s00726-013-1525-x Google Scholar
  24. 24.
    Adriano E, Garbati P, Damonte G, Salis A, Armirotti A, Balestrino M (2011) Searching for a therapy of creatine transporter deficiency: some effects of creatine ethyl ester in brain slices in vitro. Neuroscience 199:386–393. doi: 10.1016/j.neuroscience.2011.09.018 PubMedCrossRefGoogle Scholar
  25. 25.
    Andersen P, Bliss TV, Skrede KK (1971) Unit analysis of hippocampal population spikes. Exp Brain Res 13:208–221. doi: 10.1007/BF00234086 PubMedGoogle Scholar
  26. 26.
    Ballanyi K (1999) In vitro preparations. In: Windhorst U, Johansson H (eds) Modern techniques in neuroscience research. Springer, Heidelberg, pp 307–326CrossRefGoogle Scholar
  27. 27.
    Whittingham TS, Lipton P (1981) Cerebral synaptic transmission during anoxia is protected by creatine. J Neurochem 37:1618–1621PubMedCrossRefGoogle Scholar
  28. 28.
    Kass IS, Lipton P (1989) Protection of hippocampal slices from young rats against anoxic transmission damage is due to better maintenance of ATP. J Physiol 413:1–11PubMedGoogle Scholar
  29. 29.
    Schedel JM, Tanaka H, Kiyonaga A, Shindo M, Schutz Y (1999) Acute creatine ingestion in human: consequences on serum creatine and creatinine concentrations. Life Sci 65(23):2463–2470. doi: 10.1016/S0024-3205(99)00512-3 PubMedCrossRefGoogle Scholar
  30. 30.
    Dai W, Vinnakota S, Qian X, Kunze DL, Sarkar HK (1999) Molecular characterization of the human CRT-1 creatine transporter expressed in Xenopus oocytes. Arch Biochem Biophys 361:75–84. doi: 10.1006/abbi.1998.0959 PubMedCrossRefGoogle Scholar
  31. 31.
    Smith PK, Krohon RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150(1):76–85PubMedCrossRefGoogle Scholar
  32. 32.
    Bergmeyer HU (1974) In: Bergmeyer HV (ed) Methods of enzymatic analysis, vol 1. Verlag Chemie/Academic Press, New York/London, pp 425–426Google Scholar
  33. 33.
    Song W, Chattipakorn SC, McMahon LL (2006) Glycine-gated chloride channels depress synaptic transmission in rat hippocampus. J Neurophysiol 95(4):2366–2379. doi: 10.1152/jn.00386.2005 PubMedCrossRefGoogle Scholar
  34. 34.
    Betz H (1992) Structure and function of inhibitory glycine receptors. Q Rev Biophys 25:381–394PubMedCrossRefGoogle Scholar
  35. 35.
    Kirsch J (2006) Glycinergic transmission. Cell Tissue Res 326(2):535–540. doi: 10.1007/s00441-006-0261-x PubMedCrossRefGoogle Scholar
  36. 36.
    Barth A, Nguyen LB, Barth L, Newell DW (2004) Glycine-induced neurotoxicity in organotypic hippocampal slice cultures. Exp Brain Res 161(3):351–357. doi: 10.1007/s00221-004-2079-7 PubMedCrossRefGoogle Scholar
  37. 37.
    Saransaari P, Oja SS (2001) Characteristics of hippocampal glycine release in cell-damaging conditions in the adult and developing mouse. Neurochem Res 26(7):845–852. doi: 10.1023/A:1011624421505 PubMedCrossRefGoogle Scholar
  38. 38.
    Prass K, Royl G, Lindauer U, Freyer D, Megow D, Dirnagl U, Stöckler-Ipsiroglu G, Wallimann T, Priller J (2007) Improved reperfusion and neuroprotection by creatine in a mouse model of stroke. J Cereb Blood Flow Metab 27(3):452–459. doi: 10.1038/sj.jcbfm.9600351 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Patrizia Garbati
    • 1
  • Enrico Adriano
    • 1
  • Annalisa Salis
    • 2
    • 5
  • Silvia Ravera
    • 3
  • Gianluca Damonte
    • 4
    • 5
  • Enrico Millo
    • 4
    • 5
  • Maurizio Balestrino
    • 1
  1. 1.Department of Neuroscience, Ophthalmology, Genetics, Maternal-Infantile SciencesUniversity of GenovaGenoaItaly
  2. 2.Department of Hearth Environmental and Life Science (DISTAV)University of GenovaGenoaItaly
  3. 3.Biochemistry Lab, Department of Pharmacy (DIFAR)University of GenovaGenoaItaly
  4. 4.Section of Biochemistry, Department of Experimental MedicineUniversity of GenovaGenoaItaly
  5. 5.Center of Excellence for Biomedical ResearchUniversity of GenovaGenoaItaly

Personalised recommendations