Abstract
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.
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Abbreviations
- 6-OHDA:
-
6-Hydroxydopamine
- GUO:
-
Guanosine
- KRB:
-
Krebs–Ringer buffer
- MTT:
-
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- DMSO:
-
Dimethyl sulfoxide
- DCFH-DA:
-
Dichlorodihydrofluorescein diacetate
- DCFH:
-
Dichlorodihydrofluorescein
- DCF:
-
Dichlorofluorescein
- FCCP:
-
Carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone
- PD:
-
Parkinson’s disease
- ROS:
-
Reactive oxygen species
- TMRE:
-
Tetramethylrhodamine ethyl ester
- PI:
-
Propidium iodide
References
Blandini F, Armentero MT, Martignoni E (2008) The 6-hydroxydopamine model: news from the past. Parkinsonism Relat Disord 14(Suppl 2):S124–S129. https://doi.org/10.1016/j.parkreldis.2008.04.015
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. https://doi.org/10.1523/JNEUROSCI.1391-15.2015
Bose A, Beal MF (2016) Mitochondrial dysfunction in Parkinson’s disease. J Neurochem 139(Suppl 1):216–231. https://doi.org/10.1111/jnc.13731
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. https://doi.org/10.1007/s00441-004-0956-9
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. https://doi.org/10.1177/1759091414554945
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. https://doi.org/10.1111/jnc.12324
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. https://doi.org/10.1111/jnc.12324
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. https://doi.org/10.1016/j.neuroscience.2011.03.022
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. https://doi.org/10.1016/j.neuint.2012.05.021
de Lau LM, Breteler MM (2006) Epidemiology of Parkinson’s disease. Lancet Neurol 5(6):525–535. https://doi.org/10.1016/S1474-4422(06)70471-9
Dias V, Junn E, Mouradian MM (2013) The role of oxidative stress in Parkinson’s disease. J Parkinsons Dis 3(4):461–491. https://doi.org/10.3233/JPD-130230
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. https://doi.org/10.1089/neu.2016.4563
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. https://doi.org/10.1016/j.neuroscience.2006.12.036
Garver DL, Cedarbaum J, Maas JW (1975) Blood-brain barrier to 6-hydroxydopamine: uptake by heart and brain. Life Sci 17(7):1081–1084
Glinka YY, Youdim MB (1995) Inhibition of mitochondrial complexes I and IV by 6-hydroxydopamine. Eur J Pharmacol 292(3–4):329–332
Goedert M, Spillantini MG, Del Tredici K, Braak H (2013) 100 years of Lewy pathology. Nat Rev Neurol 9(1):13–24. https://doi.org/10.1038/nrneurol.2012.242
Herraiz T, Galisteo J (2015) Hydroxyl radical reactions and the radical scavenging activity of β-carboline alkaloids. Food Chem 172:640–649. https://doi.org/10.1016/j.foodchem.2014.09.091
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. https://doi.org/10.1177/0960327114555929
Kostrzewa RM, Jacobowitz DM (1974) Pharmacological actions of 6-hydroxydopamine. Pharmacol Rev 26(3):199–288
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. https://doi.org/10.14336/AD.2016.0208
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):581–593
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275
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. https://doi.org/10.1186/1756-0500-7-49
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. https://doi.org/10.1016/j.tiv.2016.09.015
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. https://doi.org/10.3389/fphar.2017.00700
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):1450–1455
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. https://doi.org/10.1002/jnr.22681
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. https://doi.org/10.1007/s11064-007-9402-1
Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65(1–2):55–63
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. https://doi.org/10.1016/j.freeradbiomed.2009.03.007
Parker WD, Boyson SJ, Parks JK (1989) Abnormalities of the electron transport chain in idiopathic Parkinson’s disease. Ann Neurol 26(6):719–723. https://doi.org/10.1002/ana.410260606
Perier C, Vila M (2012) Mitochondrial biology and Parkinson’s disease. Cold Spring Harb Perspect Med 2(2):a009332. https://doi.org/10.1101/cshperspect.a009332
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. https://doi.org/10.1007/s12640-009-9057-6
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. https://doi.org/10.1038/nrdp.2017.13
Przedborski S (2017) The two-century journey of Parkinson disease research. Nat Rev Neurosci 18(4):251–259. https://doi.org/10.1038/nrn.2017.25
Schober A (2004) Classic toxin-induced animal models of Parkinson’s disease: 6-OHDA and MPTP. Cell Tissue Res 318(1):215–224. https://doi.org/10.1007/s00441-004-0938-y
Segura-Aguilar J, Kostrzewa RM (2015) Neurotoxin mechanisms and processes relevant to Parkinson’s disease: an update. Neurotox Res 27(3):328–354. https://doi.org/10.1007/s12640-015-9519-y
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. https://doi.org/10.1002/jnr.21883
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. https://doi.org/10.1007/s11302-016-9534-3
Funding
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.
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Experiments followed the “The ARRIVE Guidelines” published in 2010 and were approved by the local Ethical Committee for Animal Research (CEUA/UFSC PP00955).
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Marques, N.F., Massari, C.M. & Tasca, C.I. Guanosine Protects Striatal Slices Against 6-OHDA-Induced Oxidative Damage, Mitochondrial Dysfunction, and ATP Depletion. Neurotox Res 35, 475–483 (2019). https://doi.org/10.1007/s12640-018-9976-1
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DOI: https://doi.org/10.1007/s12640-018-9976-1