Molecular Neurobiology

, Volume 55, Issue 5, pp 4098–4106 | Cite as

Hypoxanthine Induces Neuroenergetic Impairment and Cell Death in Striatum of Young Adult Wistar Rats

  • Helena Biasibetti-Brendler
  • Felipe Schmitz
  • Paula Pierozan
  • Bruna S. Zanotto
  • Caroline A. Prezzi
  • Rodrigo Binkowski de Andrade
  • Clovis M.D. Wannmacher
  • Angela T.S. Wyse


Hypoxanthine is the major purine involved in the salvage pathway of purines in the brain. High levels of hypoxanthine are characteristic of Lesch–Nyhan Disease. Since hypoxanthine is a purine closely related to ATP formation, the aim of this study was to investigate the effect of intrastriatal hypoxanthine administration on neuroenergetic parameters (pyruvate kinase, succinate dehydrogenase, complex II, cytochrome c oxidase, and ATP levels) and mitochondrial function (mitochondrial mass and membrane potential) in striatum of rats. We also evaluated the effect of cell death parameters (necrosis and apoptosis). Wistar rats of 60 days of life underwent stereotactic surgery and were divided into two groups: control (infusion of saline 0.9%) and hypoxanthine (10 μM). Intrastriatal hypoxanthine administration did not alter pyruvate kinase activity, but increased succinate dehydrogenase and complex II activities and diminished cytochrome c oxidase activity and immunocontent. Hypoxanthine injection decreased the percentage of cells with mitochondrial membrane label and increased mitochondrial membrane potential labeling. There was a decrease in the number of live cells and an increase in the number of apoptotic cells by caused hypoxanthine. Our findings show that intrastriatal hypoxanthine administration altered neuroenergetic parameters, and caused mitochondrial dysfunction and cell death by apoptosis, suggesting that these processes may be associated, at least in part, with neurological symptoms found in patients with Lesch–Nyhan Disease.


Hypoxanthine Neuroenergetic ATP Apoptosis 



This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-Brazil).

Compliance with Ethical Standards

Animal care followed the Guide for Care and Use of Laboratory Animals (NIH publication number 80-23 revised 1996) and the recommendations for animal care of the Brazilian Society for Neuroscience and Behavior. The project was approved by the local ethics committee (no. 25717).

Conflict of Interest

The authors declare that there are no conflicts of interest.


  1. 1.
    Jinnah H, Friedmann T (2001) Lesch-Nyhan disease and its variants. In: Scriver C, Beaudet A, Sly W, Valle D (eds) Metab. Mol. Bases Inherit. Dis. Mc Graw-Hill, New York, pp. 2537–2569Google Scholar
  2. 2.
    Nyhan WL (1978) Ataxia and disorders of purine metabolism: defects in hypoxanthine guanine phosphoribosyl transferase and clinical ataxia. Adv Neurol 21:279–287PubMedGoogle Scholar
  3. 3.
    Jinnah HA, De Gregorio L, Harris JC et al (2000) The spectrum of inherited mutations causing HPRT deficiency: 75 new cases and a review of 196 previously reported cases. Mutat Res Mutat Res 463:309–326. doi: 10.1016/S1383-5742(00)00052-1 CrossRefPubMedGoogle Scholar
  4. 4.
    Harkness RA (1988) Hypoxanthine, xanthine and uridine in body fluids, indicators of ATP depletion. J Chromatogr 429:255–278. doi: 10.1016/S0378-4347(00)83873-6 CrossRefPubMedGoogle Scholar
  5. 5.
    Rosenbloom FM, Kelley WN, Miller J et al (1967) Inherited disorder of purine metabolism. Correlation between central nervous system dysfunction and biochemical defects. JAMA 202:175–177. doi: 10.1001/jama.1967.03130160049007 CrossRefPubMedGoogle Scholar
  6. 6.
    Puig JG, Jimenez ML, Mateos FA, Fox IH (1989) Adenine nucleotide turnover in hypoxanthine-guanine phosphoribosyl-transferase deficiency: evidence for an increased contribution of purine biosynthesis de novo. Metabolism 38:410–418CrossRefPubMedGoogle Scholar
  7. 7.
    Visser JE, Bär PR, Jinnah HA (2000) Lesch-Nyhan disease and the basal ganglia. Brain Res Rev 32:449–475. doi: 10.1016/S0165-0173(99)00094-6 CrossRefPubMedGoogle Scholar
  8. 8.
    Göttle M, Prudente CN, Fu R et al (2014) Loss of dopamine phenotype among midbrain neurons in Lesch-Nyhan disease. Ann Neurol 76:95–107. doi: 10.1002/ana.24191 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Jinnah HA, Sabina RL, Van Den Berghe G (2013) Metabolic disorders of purine metabolism affecting the nervous system. Handb Clin Neurol 113:1827–1836. doi: 10.1016/B978-0-444-59565-2.00052-6 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Torres RJ, Puig JG (2008) The diagnosis of HPRT deficiency in the 21st century. Nucleosides Nucleotides Nucleic Acids 27:564–569. doi: 10.1080/15257770802135778 CrossRefPubMedGoogle Scholar
  11. 11.
    Gisbert de la Cuadra L, Torres RJ, Beltrán LM et al (2016) Development of new forms of self-injurious behavior following total dental extraction in Lesch–Nyhan disease. Nucleosides Nucleotides Nucleic Acids 35:524–528. doi: 10.1080/15257770.2016.1184276 CrossRefPubMedGoogle Scholar
  12. 12.
    Todd RD, Perlmutter JS (1998) Mutational and biochemical analysis of dopamine in dystonia. Mol Neurobiol 16:135–147. doi: 10.1007/BF02740641 CrossRefPubMedGoogle Scholar
  13. 13.
    Jinnah HA, Visser JE, Harris JC et al (2006) Delineation of the motor disorder of Lesch-Nyhan disease. Brain 129:1201–1217. doi: 10.1093/brain/awl056 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Torres RJ, Puig JG (2015) Hypoxanthine deregulates genes involved in early neuronal development. Implications in Lesch-Nyhan disease pathogenesis. J Inherit Metab Dis 38:1109–1118. doi: 10.1007/s10545-015-9854-4 CrossRefPubMedGoogle Scholar
  15. 15.
    Torres RJ, Prior C, Garcia MG, Puig JG (2016) A review of the implication of hypoxanthine excess in the physiopathology of Lesch–Nyhan disease. Nucleosides Nucleotides Nucleic Acids 35:507–516. doi: 10.1080/15257770.2016.1147579 CrossRefPubMedGoogle Scholar
  16. 16.
    Ceballos-Picot I, Mockel L, Potier M-C et al (2009) Hypoxanthine-guanine phosphoribosyl transferase regulates early developmental programming of dopamine neurons: implications for Lesch-Nyhan disease pathogenesis. Hum Mol Genet 18:2317–2327. doi: 10.1093/hmg/ddp164 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Bavaresco CS, Chiarani F, Wannmacher CMD et al (2007) Intrastriatal hypoxanthine reduces Na+, K+-ATPase activity and induces oxidative stress in the rats. Metab Brain Dis 22:1–11. doi: 10.1007/s11011-006-9037-y CrossRefPubMedGoogle Scholar
  18. 18.
    Bavaresco CS, Chiarani F, Kolling J et al (2008) Biochemical effects of pretreatment with vitamins E and C in rats submitted to intrastriatal hypoxanthine administration. Neurochem Int 52:1276–1283. doi: 10.1016/j.neuint.2008.01.008 CrossRefPubMedGoogle Scholar
  19. 19.
    Biasibetti H, Pierozan P, Rodrigues AF et al (2016) Hypoxanthine intrastriatal administration alters neuroinflammatory profile and redox status in striatum of infant and young adult rats. Mol Neurobiol:1–11. doi: 10.1007/s12035-016-9866-6
  20. 20.
    Ishii T, Miyazawa M, Onouchi H et al (2013) Model animals for the study of oxidative stress from complex II. Biochim Biophys Acta Bioenerg 1827:588–597. doi: 10.1016/j.bbabio.2012.10.016 CrossRefGoogle Scholar
  21. 21.
    Bélanger M, Allaman I, Magistretti PJ (2011) Brain energy metabolism: focus on astrocyte-neuron metabolic cooperation. Cell Metab 14:724–738. doi: 10.1016/j.cmet.2011.08.016 CrossRefPubMedGoogle Scholar
  22. 22.
    Magistretti PJ, Allaman I (2015) Neuron review. A cellular perspective on brain energy metabolism and functional imaging. Neuron 86:883–901. doi: 10.1016/j.neuron.2015.03.035 CrossRefPubMedGoogle Scholar
  23. 23.
    Bratic I, Trifunovic A (2010) Mitochondrial energy metabolism and ageing. Biochim Biophys Acta Bioenerg 1797:961–967. doi: 10.1016/j.bbabio.2010.01.004 CrossRefGoogle Scholar
  24. 24.
    Nelson DL, Cox MM (2013) Lehninger principles of biochemistry 6th ed. Book. doi: 10.1016/j.jse.2011.03.016
  25. 25.
    García-Bermúdez J, Cuezva JM (2016) The ATPase inhibitory factor 1 (IF1): a master regulator of energy metabolism and of cell survival. Biochim Biophys Acta Bioenerg 1857:1167–1182. doi: 10.1016/j.bbabio.2016.02.004 CrossRefGoogle Scholar
  26. 26.
    Johri A, Beal MF (2012) Mitochondrial dysfunction in neurodegenerative diseases. J Pharmacol Exp Ther 342:619–630. doi: 10.1124/jpet.112.192138 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Schurr A (2002) Energy metabolism, stress hormones and neural recovery from cerebral ischemia/hypoxia. Neurochem Int 41:1–8. doi: 10.1016/S0197-0186(01)00142-5 CrossRefPubMedGoogle Scholar
  28. 28.
    Beal MF (2005) Mitochondria take center stage in aging and neurodegeneration. Ann Neurol 58:495–505. doi: 10.1002/ana.20624 CrossRefPubMedGoogle Scholar
  29. 29.
    Petrozzi L, Ricci G, Giglioli NJ et al (2007) Mitochondria and neurodegeneration. Biosci Rep 27:87–104. doi: 10.1007/s10540-007-9038-z CrossRefPubMedGoogle Scholar
  30. 30.
    Paxinos G, Watson C (2006) The rat brain in stereotaxic coordinates. Sixth Edition by. Acad Press 170:547–612. doi: 10.1016/0143-4179(83)90049-5 Google Scholar
  31. 31.
    Leong SF, Lai JCK, Lim L, Clark JB (1981) Energy-metabolising enzymes in brain regions of adult and aging rats. J Neurochem 37:1548–1556. doi: 10.1111/j.1471-4159.1981.tb06326.x CrossRefPubMedGoogle Scholar
  32. 32.
    Fischer JC, Ruitenbeek W, Berden JA et al (1985) Differential investigation of the capacity of succinate oxidation in human skeletal muscle. Clin Chim Acta 153:23–36. doi: 10.1016/0009-8981(85)90135-4 CrossRefPubMedGoogle Scholar
  33. 33.
    Rustin P, Chretien D, Bourgeron T et al (1994) Biochemical and molecular investigations in respiratory chain deficiencies. Clin Chim Acta 228:35–51. doi: 10.1016/0009-8981(94)90055-8 CrossRefPubMedGoogle Scholar
  34. 34.
    Schmitz F, Pierozan P, Rodrigues AF et al (2016) Methylphenidate decreases ATP levels and impairs glutamate uptake and Na+, K+-ATPase activity in juvenile rat hippocampus. Mol Neurobiol. doi: 10.1007/s12035-016-0289-1
  35. 35.
    Hughes BP (1962) A method for the estimation of serum creatine kinase and its use in comparing creatine kinase and aldolase activity in normal and pathological sera. Clin Chim Acta 7:597–603. doi: 10.1016/0009-8981(62)90137-7 CrossRefPubMedGoogle Scholar
  36. 36.
    Agnello M, Morici G, Rinaldi AM (2008) A method for measuring mitochondrial mass and activity. Cytotechnology 56:145–149. doi: 10.1007/s10616-008-9143-2 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  38. 38.
    Eguchi Y, Shimizu S, Tsujimoto Y (1997) Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res 57:1835–1840PubMedGoogle Scholar
  39. 39.
    Micheli V, Camici M, Tozzi MG et al (2011) Neurological disorders of purine and pyrimidine metabolism. Curr Top Med Chem 11:923–947CrossRefPubMedGoogle Scholar
  40. 40.
    García MG, Puig JG, Torres RJ (2012) Adenosine, dopamine and serotonin receptors imbalance in lymphocytes of Lesch-Nyhan patients. J Inherit Metab Dis 35:1129–1135. doi: 10.1007/s10545-012-9470-5 CrossRefPubMedGoogle Scholar
  41. 41.
    Bavaresco CS, Chiarani F, Duringon E et al (2007) Intrastriatal injection of hypoxanthine reduces striatal serotonin content and impairs spatial memory performance in rats. Metab Brain Dis 22:67–76. doi: 10.1007/s11011-006-9038-x CrossRefPubMedGoogle Scholar
  42. 42.
    Willemoës M, Kilstrup M (2005) Nucleoside triphosphate synthesis catalysed by adenylate kinase is ADP dependent. Arch Biochem Biophys 444:195–199. doi: 10.1016/ CrossRefPubMedGoogle Scholar
  43. 43.
    Ferreira AGK, Lima DD, Delwing D et al (2010) Proline impairs energy metabolism in cerebral cortex of young rats. Metab Brain Dis 25:161–168. doi: 10.1007/s11011-010-9193-y CrossRefPubMedGoogle Scholar
  44. 44.
    de Andrade RB, Gemelli T, Rojas DB et al (2016) Evaluation of oxidative stress parameters and energy metabolism in cerebral cortex of rats subjected to sarcosine administration. Mol Neurobiol:1–11. doi: 10.1007/s12035-016-9984-1
  45. 45.
    Naseri NN, Bonica J, Xu H et al (2016) Novel metabolic abnormalities in the tricarboxylic acid cycle in peripheral cells from Huntington’s disease patients. PLoS One 11:1–17. doi: 10.1371/journal.pone.0160384 CrossRefGoogle Scholar
  46. 46.
    Bubber P, Haroutunian V, Fisch G et al (2005) Mitochondrial abnormalities in Alzheimer brain: mechanistic implications. Ann Neurol 57:695–703. doi: 10.1002/ana.20474 CrossRefPubMedGoogle Scholar
  47. 47.
    Lazzarino G, Amorini AM, Petzold A et al (2016) Serum compounds of energy metabolism impairment are related to disability, disease course and neuroimaging in multiple sclerosis. Mol Neurobiol:1–14. doi: 10.1007/s12035-016-0257-9
  48. 48.
    Stepanova A, Shurubor Y, Valsecchi F et al (2016) Differential susceptibility of mitochondrial complex II to inhibition by oxaloacetate in brain and heart. Biochim Biophys Acta Bioenerg 1857:1561–1568. doi: 10.1016/j.bbabio.2016.06.002 CrossRefGoogle Scholar
  49. 49.
    Caceda R, Gamboa JL, Boero JA et al (2001) Energetic metabolism in mouse cerebral cortex during chronic hypoxia. Neurosci Lett 301:171–174CrossRefPubMedGoogle Scholar
  50. 50.
    Weinberg JM, Venkatachalam MA, Roeser NF, Nissim I (2000) Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates. Proc Natl Acad Sci U S A 97:2826–2831. doi: 10.1073/pnas.97.6.2826 CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Milatovic D, Zivin M, Gupta RC, Dettbarn WD (2001) Alterations in cytochrome c oxidase activity and energy metabolites in response to kainic acid-induced status epilepticus. Brain Res 912:67–78. doi: 10.1016/s0006-8993(01)02657-9 CrossRefPubMedGoogle Scholar
  52. 52.
    Gupta RC, Milatovic D, Dettbarn W-D (2001) Depletion of energy metabolites following acetylcholinesterase inhibitor-induced status epilepticus: protection by antioxidants. Neurotoxicology 22:271–282. doi: 10.1016/S0161-813X(01)00013-4 CrossRefPubMedGoogle Scholar
  53. 53.
    Grimm S (2013) Respiratory chain complex II as general sensor for apoptosis. Biochim Biophys Acta Bioenerg 1827:565–572. doi: 10.1016/j.bbabio.2012.09.009 CrossRefGoogle Scholar
  54. 54.
    Owen L, Sunram-Lea SI (2011) Metabolic agents that enhance ATP can improve cognitive functioning: a review of the evidence for glucose, oxygen, pyruvate, creatine, and L-carnitine. Nutrients 3:735–755. doi: 10.3390/nu3080735 CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Dzeja PP, Terzic A (2003) Phosphotransfer networks and cellular energetics. J Exp Biol 206:2039–2047. doi: 10.1242/jeb.00426 CrossRefPubMedGoogle Scholar
  56. 56.
    Sas K, Robotka H, Toldi J, Vécsei L (2007) Mitochondria, metabolic disturbances, oxidative stress and the kynurenine system, with focus on neurodegenerative disorders. J Neurol Sci 257:221–239. doi: 10.1016/j.jns.2007.01.033 CrossRefPubMedGoogle Scholar
  57. 57.
    Reddy PH, Reddy TP (2011) Mitochondria as a therapeutic target for aging and neurodegenerative diseases. Curr Alzheimer Res 8:393–409. doi: 10.2174/156720511795745401 CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Jain IH, Zazzeron L, Goli R et al (2016) Hypoxia as a therapy for mitochondrial disease. Science 352:54–61. doi: 10.1126/science.aad9642 CrossRefPubMedPubMedCentralGoogle Scholar
  59. 59.
    Kolling J, Scherer EBS, Siebert C et al (2016) Severe hyperhomocysteinemia decreases respiratory Enzyme and Na+-K+ ATPase activities, and leads to mitochondrial alterations in rat amygdala. Neurotox Res 29:408–418. doi: 10.1007/s12640-015-9587-z CrossRefPubMedGoogle Scholar
  60. 60.
    Seminotti B, Amaral AU, Ribeiro RT et al (2016) Oxidative stress, disrupted energy metabolism, and altered signaling pathways in glutaryl-CoA dehydrogenase knockout mice: potential implications of quinolinic acid toxicity in the neuropathology of glutaric acidemia type I. Mol Neurobiol 53:6459–6475. doi: 10.1007/s12035-015-9548-9 CrossRefPubMedGoogle Scholar
  61. 61.
    Distelmaier F, Koopman WJH, Testa ER et al (2008) Life cell quantification of mitochondrial membrane potential at the single organelle level. Cytom Part A 73A:129–138. doi: 10.1002/cyto.a.20503 CrossRefGoogle Scholar
  62. 62.
    Iijima T, Mishima T, Akagawa K, Iwao Y (2006) Neuroprotective effect of propofol on necrosis and apoptosis following oxygen-glucose deprivation-relationship between mitochondrial membrane potential and mode of death. Brain Res 1099:25–32. doi: 10.1016/j.brainres.2006.04.117 CrossRefPubMedGoogle Scholar
  63. 63.
    Gottlieb RA, Carreira RS (2010) Autophagy in health and disease. 5. Mitophagy as a way of life. Am J Physiol—Cell Physiol 299:203–210. doi: 10.1152/ajpcell.00097.2010 CrossRefGoogle Scholar
  64. 64.
    Delmas D, Solary E, Latruffe N (2011) Resveratrol, a phytochemical inducer of multiple cell death pathways: apoptosis, autophagy and mitotic catastrophe. Curr Med Chem 18:1100–1121CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Helena Biasibetti-Brendler
    • 1
    • 2
  • Felipe Schmitz
    • 1
    • 2
  • Paula Pierozan
    • 2
  • Bruna S. Zanotto
    • 2
  • Caroline A. Prezzi
    • 2
  • Rodrigo Binkowski de Andrade
    • 1
  • Clovis M.D. Wannmacher
    • 1
    • 3
  • Angela T.S. Wyse
    • 1
    • 2
    • 3
  1. 1.Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, ICBSUFRGSPorto AlegreBrazil
  2. 2.Laboratório de Neuroproteção e Doenças Neurometabólicas, Departamento de Bioquímica, ICBSUFRGSPorto AlegreBrazil
  3. 3.Departamento de Bioquímica, ICBSUniversidade Federal do Rio Grande do SulPorto AlegreBrazil

Personalised recommendations