Neurotoxicity Research

, Volume 35, Issue 2, pp 475–483 | Cite as

Guanosine Protects Striatal Slices Against 6-OHDA-Induced Oxidative Damage, Mitochondrial Dysfunction, and ATP Depletion

  • Naiani Ferreira Marques
  • Caio Marcos Massari
  • Carla Inês TascaEmail author


Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by loss of dopaminergic neurons in substantia nigra pars compacta which induces severe motor symptoms. 6-OHDA is a neurotoxin widely used in PD animal models due to its high affinity by dopamine transporter, its rapid non-enzymatic auto-oxidation which generates reactive oxygen species (ROS), oxidative stress, and for induced mitochondrial dysfunction. We previously reported an in vitro protocol of 6-OHDA-induced toxicity in brain regions slices, as a simple and sensitive assay to screen for protective compounds related to PD. Guanosine (GUO), a guanine-based purine nucleoside, is a neuroprotective molecule that is showing promising effects as an antiparkinsonian agent. To investigate the mechanisms involved on GUO-induced neuroprotection, slices of cortex, striatum, and hippocampus were incubated with GUO in the presence of 6-OHDA (100 μM). 6-OHDA promoted a decrease in cellular viability and increased ROS generation in all brain regions. Disruption of mitochondrial potential, depletion in intracellular ATP levels, and increase in cell membrane permeabilization were evidenced in striatal slices. GUO prevented the increase in ROS generation, disruption in mitochondrial potential, and depletion of intracellular ATP induced by 6-OHDA in striatal slices. In conclusion, GUO was effective to prevent oxidative events before cell damage, such as mitochondrial disruption, intracellular ATP levels depletion, and ROS generation in striatal slices subjected to in vitro 6-OHDA-induced toxicity.


Parkinson’s disease 6-OHDA Guanosine In vitro 







Krebs–Ringer buffer


3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide


Dimethyl sulfoxide


Dichlorodihydrofluorescein diacetate






Carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone


Parkinson’s disease


Reactive oxygen species


Tetramethylrhodamine ethyl ester


Propidium iodide


Funding Information

Research supported by grants from the Brazilian funding agencies, CAPES (CAPES/PAJT), CNPq (INCT-EN for Excitotoxicity and Neuroprotection) and FAPESC (NENASC/PRONEX) to C.I.T. is recipient of CNPq productivity fellowship.

Compliance with Ethical Standards

Experiments followed the “The ARRIVE Guidelines” published in 2010 and were approved by the local Ethical Committee for Animal Research (CEUA/UFSC PP00955).


  1. Blandini F, Armentero MT, Martignoni E (2008) The 6-hydroxydopamine model: news from the past. Parkinsonism Relat Disord 14(Suppl 2):S124–S129. CrossRefPubMedGoogle Scholar
  2. Block ER, Nuttle J, Balcita-Pedicino JJ, Caltagarone J, Watkins SC, Sesack SR, Sorkin A (2015) Brain region-specific trafficking of the dopamine transporter. J Neurosci 35(37):12845–12858. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bose A, Beal MF (2016) Mitochondrial dysfunction in Parkinson’s disease. J Neurochem 139(Suppl 1):216–231. CrossRefPubMedGoogle Scholar
  4. Braak H, Ghebremedhin E, Rüb U, Bratzke H, Del Tredici K (2004) Stages in the development of Parkinson’s disease-related pathology. Cell Tissue Res 318(1):121–134. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Cunha MP, Martín-de-Saavedra MD, Romero A, Egea J, Ludka FK, Tasca CI, Farina M, Rodrigues ALS, López MG (2014) Both creatine and its product phosphocreatine reduce oxidative stress and afford neuroprotection in an in vitro Parkinson’s model. ASN Neuro 6(6):175909141455494. CrossRefGoogle Scholar
  6. Dal-Cim T, Ludka FK, Martins WC, Reginato C, Parada E, Egea J, López MG, Tasca CI (2013a) Guanosine controls inflammatory pathways to afford neuroprotection of hippocampal slices under oxygen and glucose deprivation conditions (research support, non-U.S. Gov’t). J Neurochem 126(4):437–450. CrossRefPubMedGoogle Scholar
  7. Dal-Cim T, Ludka FK, Martins WC, Reginato C, Parada E, Egea J, López MG, Tasca CI (2013b) Guanosine controls inflammatory pathways to afford neuroprotection of hippocampal slices under oxygen and glucose deprivation conditions. J Neurochem 126(4):437–450. CrossRefPubMedGoogle Scholar
  8. Dal-Cim T, Martins WC, Santos AR, Tasca CI (2011) Guanosine is neuroprotective against oxygen/glucose deprivation in hippocampal slices via large conductance Ca2+-activated K+ channels, phosphatidilinositol-3 kinase/protein kinase B pathway activation and glutamate uptake. Neuroscience 183:212–220. CrossRefPubMedGoogle Scholar
  9. Dal-Cim T, Molz S, Egea J, Parada E, Romero A, Budni J, Martín de Saavedra MD, Barrio L, Tasca CI, López MG (2012) Guanosine protects human neuroblastoma SH-SY5Y cells against mitochondrial oxidative stress by inducing heme oxigenase-1 via PI3K/Akt/GSK-3β pathway. Neurochem Int 61(3):397–404. CrossRefPubMedGoogle Scholar
  10. de Lau LM, Breteler MM (2006) Epidemiology of Parkinson’s disease. Lancet Neurol 5(6):525–535. CrossRefPubMedGoogle Scholar
  11. Dias V, Junn E, Mouradian MM (2013) The role of oxidative stress in Parkinson’s disease. J Parkinsons Dis 3(4):461–491. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Dobrachinski F, da Rosa Gerbatin R, Sartori G, Ferreira Marques N, Zemolin AP, Almeida Silva LF, Franco JL, Freire Royes LF, Rechia Fighera M, Antunes Soares FA (2017) Regulation of mitochondrial function and glutamatergic system are the target of guanosine effect in traumatic brain injury. J Neurotrauma 34(7):1318–1328. CrossRefPubMedGoogle Scholar
  13. Egea J, Rosa AO, Sobrado M, Gandía L, López MG, García AG (2007) Neuroprotection afforded by nicotine against oxygen and glucose deprivation in hippocampal slices is lost in alpha7 nicotinic receptor knockout mice. Neuroscience 145(3):866–872. CrossRefPubMedGoogle Scholar
  14. Garver DL, Cedarbaum J, Maas JW (1975) Blood-brain barrier to 6-hydroxydopamine: uptake by heart and brain. Life Sci 17(7):1081–1084CrossRefGoogle Scholar
  15. Glinka YY, Youdim MB (1995) Inhibition of mitochondrial complexes I and IV by 6-hydroxydopamine. Eur J Pharmacol 292(3–4):329–332PubMedGoogle Scholar
  16. Goedert M, Spillantini MG, Del Tredici K, Braak H (2013) 100 years of Lewy pathology. Nat Rev Neurol 9(1):13–24. CrossRefPubMedGoogle Scholar
  17. Herraiz T, Galisteo J (2015) Hydroxyl radical reactions and the radical scavenging activity of β-carboline alkaloids. Food Chem 172:640–649. CrossRefPubMedGoogle Scholar
  18. Hong SJ, Zhang D, Zhang LH, Yang P, Wan J, Yu Y, Wang TH, Feng ZT, Li LH, Yew DTW (2015) Expression of dopamine transporter in the different cerebral regions of methamphetamine-dependent rats. Hum Exp Toxicol 34(7):707–717. CrossRefPubMedGoogle Scholar
  19. Kostrzewa RM, Jacobowitz DM (1974) Pharmacological actions of 6-hydroxydopamine. Pharmacol Rev 26(3):199–288PubMedGoogle Scholar
  20. Lanznaster D, Dal-Cim T, Piermartiri TC, Tasca CI (2016) Guanosine: a neuromodulator with therapeutic potential in brain disorders. Aging Dis 7(5):657–679. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Liu Y, Peterson DA, Kimura H, Schubert D (1997) Mechanism of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. J Neurochem 69(2):581–593CrossRefGoogle Scholar
  22. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275PubMedGoogle Scholar
  23. Magalingam KB, Radhakrishnan A, Haleagrahara N (2014) Protective effects of flavonol isoquercitrin, against 6-hydroxy dopamine (6-OHDA)-induced toxicity in PC12 cells. BMC Res Notes 7:49. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Massari CM, Castro AA, Dal-Cim T, Lanznaster D, Tasca CI (2016) In vitro 6-hydroxydopamine-induced toxicity in striatal, cerebrocortical and hippocampal slices is attenuated by atorvastatin and MK-801. Toxicol in Vitro 37:162–168. CrossRefPubMedGoogle Scholar
  25. Massari CM, López-Cano M, Núñez F, Fernández-Dueñas V, Tasca CI, Ciruela F (2017) Antiparkinsonian efficacy of guanosine in rodent models of movement disorder. Front Pharmacol 8:700. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Mizuno Y, Ohta S, Tanaka M, Takamiya S, Suzuki K, Sato T, Oya H, Ozawa T, Kagawa Y (1989) Deficiencies in complex I subunits of the respiratory chain in Parkinson’s disease. Biochem Biophys Res Commun 163(3):1450–1455CrossRefGoogle Scholar
  27. Molz S, Dal-Cim T, Budni J, Martín-de-Saavedra MD, Egea J, Romero A, del Barrio L, Rodrigues ALS, López MG, Tasca CI (2011) Neuroprotective effect of guanosine against glutamate-induced cell death in rat hippocampal slices is mediated by the phosphatidylinositol-3 kinase/Akt/ glycogen synthase kinase 3β pathway activation and inducible nitric oxide synthase inhibition. J Neurosci Res 89(9):1400–1408. CrossRefPubMedGoogle Scholar
  28. Molz S, Decker H, Dal-Cim T, Cremonez C, Cordova FM, Leal RB, Tasca CI (2008) Glutamate-induced toxicity in hippocampal slices involves apoptotic features and p38 MAPK signaling. Neurochem Res 33(1):27–36. CrossRefPubMedGoogle Scholar
  29. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65(1–2):55–63CrossRefGoogle Scholar
  30. Navarro A, Boveris A, Bández MJ, Sánchez-Pino MJ, Gómez C, Muntané G, Ferrer I (2009) Human brain cortex: mitochondrial oxidative damage and adaptive response in Parkinson disease and in dementia with Lewy bodies. Free Radic Biol Med 46(12):1574–1580. CrossRefPubMedGoogle Scholar
  31. Parker WD, Boyson SJ, Parks JK (1989) Abnormalities of the electron transport chain in idiopathic Parkinson’s disease. Ann Neurol 26(6):719–723. CrossRefPubMedGoogle Scholar
  32. Perier C, Vila M (2012) Mitochondrial biology and Parkinson’s disease. Cold Spring Harb Perspect Med 2(2):a009332. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Piermartiri TC, Vandresen-Filho S, de Araújo Herculano B, Martins WC, Dal’agnolo D, Stroeh E et al (2009) Atorvastatin prevents hippocampal cell death due to quinolinic acid-induced seizures in mice by increasing Akt phosphorylation and glutamate uptake. Neurotox Res 16(2):106–115. CrossRefPubMedGoogle Scholar
  34. Poewe W, Seppi K, Tanner CM, Halliday GM, Brundin P, Volkmann J, Schrag AE, Lang AE (2017) Parkinson disease. Nat Rev Dis Primers 3:17013. CrossRefPubMedGoogle Scholar
  35. Przedborski S (2017) The two-century journey of Parkinson disease research. Nat Rev Neurosci 18(4):251–259. CrossRefPubMedGoogle Scholar
  36. Schober A (2004) Classic toxin-induced animal models of Parkinson’s disease: 6-OHDA and MPTP. Cell Tissue Res 318(1):215–224. CrossRefPubMedGoogle Scholar
  37. Segura-Aguilar J, Kostrzewa RM (2015) Neurotoxin mechanisms and processes relevant to Parkinson’s disease: an update. Neurotox Res 27(3):328–354. CrossRefPubMedGoogle Scholar
  38. Su C, Elfeki N, Ballerini P, D’Alimonte I, Bau C, Ciccarelli R, Caciagli F, Gabriele J, Jiang S (2009) Guanosine improves motor behavior, reduces apoptosis, and stimulates neurogenesis in rats with parkinsonism. J Neurosci Res 87(3):617–625. CrossRefPubMedGoogle Scholar
  39. Thomaz DT, Dal-Cim TA, Martins WC, Cunha MP, Lanznaster D, de Bem AF, Tasca CI (2016) Guanosine prevents nitroxidative stress and recovers mitochondrial membrane potential disruption in hippocampal slices subjected to oxygen/glucose deprivation. Purinergic Signal 12(4):707–718. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Programa de Pós-Graduação em Bioquímica, Centro de Ciências BiológicasUniversidade Federal de Santa CatarinaFlorianópolisBrazil
  2. 2.Departamento de Bioquímica, CCBUFSCFlorianópolisBrazil

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