Abstract
Nitric oxide (NO) has chemical properties that make it uniquely suitable as an intracellular and intercellular messenger. NO is produced by the activity of the enzyme nitric oxide synthases (NOS). There is substantial and mounting evidence that slight abnormalities of NO may underlie a wide range of neurodegenerative disorders. NO participates of the oxidative stress and inflammatory processes that contribute to the progressive dopaminergic loss in Parkinson’s disease (PD). The present study aimed to evaluate in vitro and in vivo the effects of neuronal NOS-targeted siRNAs on the injury caused in dopaminergic neurons by the toxin 6-hidroxydopamine (6-OHDA). First, we confirmed (immunohistochemistry and Western blotting) that SH-SY5Y cell lineage expresses the dopaminergic marker tyrosine hydroxylase (TH) and the protein under analysis, neuronal NOS (nNOS). We designed four siRNAs by using the BIOPREDsi algorithm choosing the one providing the highest knockdown of nNOS mRNA in SH-SY5Y cells, as determined by qPCR. siRNA 4400 carried by liposomes was internalized into cells, caused a concentration-dependent knockdown on nNOS, and reduced the toxicity induced by 6-OHDA (p < 0.05). Regarding in vivo action in the dopamine-depleted animals, intra-striatal injection of siRNA 4400 at 4 days prior 6-OHDA produced a decrease in the rotational behavior induced by apomorphine. Finally, siRNA 4400 mitigated the loss of TH(+) cells in substantia nigra dorsal and ventral part. In conclusion, the suppression of nNOS enzyme by targeted siRNAs modified the progressive death of dopaminergic cells induced by 6-OHDA and merits further pre-clinical investigations as a neuroprotective approach for PD.
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Adams D, Gonzalez-Duarte A, O’Riordan WD, Yang CC, Ueda M, Kristen AV, Tournev I, Schmidt HH, Coelho T, Berk JL, Lin KP, Vita G, Attarian S, Planté-Bordeneuve V, Mezei MM, Campistol JM, Buades J, Brannagan TH III, Kim BJ, Oh J, Parman Y, Sekijima Y, Hawkins PN, Solomon SD, Polydefkis M, Dyck PJ, Gandhi PJ, Goyal S, Chen J, Strahs AL, Nochur SV, Sweetser MT, Garg PP, Vaishnaw AK, Gollob JA, Suhr OB (2018) Patisiran, an RNAi therapeutic, for hereditary transthyretin amyloidosis. N Engl J Med 379:11–21. https://doi.org/10.1056/NEJMoa1716153
Agholme L, Lindstrom T, Kagedal K, Marcusson J, Hallbeck M (2010) An in vitro model for neuroscience: differentiation of SH-SY5Y cells into cells with morphological and biochemical characteristics of mature neurons. J Alzheimers Dis 20:1069–1082. https://doi.org/10.3233/JAD-2010-091363
Alberio T, Lopiano L, Fasano M (2012) Cellular models to investigate biochemical pathways in Parkinson's disease. FEBS J 279:1146–1155. https://doi.org/10.1111/j.1742-4658.2012.08516.x
Alderton WK, Cooper CE, Knowles RG (2001) Nitric oxide synthases: structure, function and inhibition. Biochem J 357:593–615
Antonelli MC, Guillemin GJ, Raisman-Vozari R, del-Bel EA, Aschner M, Collins MA, Tizabi Y, Moratalla R, West AK (2012) New strategies in neuroprotection and neurorepair. Neurotox Res 21:49–56. https://doi.org/10.1007/s12640-011-9265-8
Birmingham A, Anderson E, Sullivan K, Reynolds A, Boese Q, Leake D, Karpilow J, Khvorova A (2007) A protocol for designing siRNAs with high functionality and specificity. Nat Protoc 2:2068–2078. https://doi.org/10.1038/nprot.2007.278
Bobbin ML, Rossi JJ (2016) RNA interference (RNAi)-based therapeutics: delivering on the promise? Annu Rev Pharmacol Toxicol 56:103–122. https://doi.org/10.1146/annurev-pharmtox-010715-103633
Bortolanza M, Bariotto-Dos-Santos KD, Dos-Santos-Pereira M, da-Silva CA, Del-Bel E (2016) Antidyskinetic effect of 7-Nitroindazole and sodium nitroprusside associated with amantadine in a rat model of Parkinson's disease. Neurotox Res 30:88–100. https://doi.org/10.1007/s12640-016-9618-4
Brown GC (2010) Nitric oxide and neuronal death. Nitric Oxide 23:153–165. https://doi.org/10.1016/j.niox.2010.06.001
Calabrese V, Mancuso C, Calvani M, Rizzarelli E, Butterfield DA, Stella AM (2007) Nitric oxide in the central nervous system: neuroprotection versus neurotoxicity. Nat Rev Neurosci 8:766–775. https://doi.org/10.1038/nrn2214
Castania V, Issy AC, Silveira JW, Ferreira FR, Titze-de-Almeida SS, Resende FFB, Ferreira NR, Titze-de-Almeida R, Defino HLA, del Bel E (2017) The presence of the neuronal nitric oxide synthase isoform in the intervertebral disk. Neurotox Res 31:148–161. https://doi.org/10.1007/s12640-016-9676-7
Cheng B, Martinez AA, Morado J, Scofield V, Roberts JL, Maffi SK (2013) Retinoic acid protects against proteasome inhibition associated cell death in SH-SY5Y cells via the AKT pathway. Neurochem Int 62:31–42. https://doi.org/10.1016/j.neuint.2012.10.014
Cheung YT, Lau WK, Yu MS, Lai CS, Yeung SC, So KF, Chang RC (2009) Effects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitro model in neurotoxicity research. Neurotoxicology 30:127–135. https://doi.org/10.1016/j.neuro.2008.11.001
Cohen ZR, Ramishetti S, Peshes-Yaloz N, Goldsmith M, Wohl A, Zibly Z, Peer D (2015) Localized RNAi therapeutics of chemoresistant grade IV glioma using hyaluronan-grafted lipid-based nanoparticles. ACS Nano 9:1581–1591. https://doi.org/10.1021/nn506248s
Cunha LC, Del Bel E, Pardo L, Stuhmer W, Titze DEAR (2013) RNA interference with EAG1 enhances interferon gamma injury to glioma cells in vitro. Anticancer Res 33:865–870
de Boer AG, Gaillard PJ (2007) Drug targeting to the brain. Annu Rev Pharmacol Toxicol 47:323–355. https://doi.org/10.1146/annurev.pharmtox.47.120505.105237
Del Bel EA et al (2005) Role of nitric oxide on motor behavior. Cell Mol Neurobiol 25:371–392
Del-Bel E, Padovan-Neto FE, Raisman-Vozari R, Lazzarini M (2011) Role of nitric oxide in motor control: implications for Parkinson's disease pathophysiology and treatment. Curr Pharm Des 17:471–488
Del-Bel E et al (2014) Counteraction by nitric oxide synthase inhibitor of neurochemical alterations of dopaminergic system in 6-OHDA-lesioned rats under L-DOPA treatment. Neurotox Res 25:33–44. https://doi.org/10.1007/s12640-013-9406-3
Dexter DT, Carter CJ, Wells FR, Javoy-Agid F, Agid Y, Lees A, Jenner P, Marsden CD (1989) Basal lipid peroxidation in substantia nigra is increased in Parkinson's disease. J Neurochem 52:381–389
Dotsch J, Harmjanz A, Christiansen H, Hanze J, Lampert F, Rascher W (2000) Gene expression of neuronal nitric oxide synthase and adrenomedullin in human neuroblastoma using real-time PCR. Int J Cancer 88:172–175
Douhou A et al (2002) Effect of chronic treatment with riluzole on the nigrostriatal dopaminergic system in weaver mutant mice. Exp Neurol 176:247–253
Dunkel P, Chai CL, Sperlagh B, Huleatt PB, Matyus P (2012) Clinical utility of neuroprotective agents in neurodegenerative diseases: current status of drug development for Alzheimer's, Parkinson's and Huntington's diseases, and amyotrophic lateral sclerosis. Expert Opin Investig Drugs 21:1267–1308. https://doi.org/10.1517/13543784.2012.703178
Ebadi M, Sharma SK (2003) Peroxynitrite and mitochondrial dysfunction in the pathogenesis of Parkinson's disease. Antioxid Redox Signal 5:319–335. https://doi.org/10.1089/152308603322110896
Elmen J et al (2005) Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality. Nucleic Acids Res 33:439–447. https://doi.org/10.1093/nar/gki193
Fahn S (2018) The 200-year journey of Parkinson disease: reflecting on the past and looking towards the future. Parkinsonism Relat Disord 46(Suppl 1):S1–S5. https://doi.org/10.1016/j.parkreldis.2017.07.020
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811. https://doi.org/10.1038/35888
Gaki GS, Papavassiliou AG (2014) Oxidative stress-induced signaling pathways implicated in the pathogenesis of Parkinson's disease. NeuroMolecular Med 16:217–230. https://doi.org/10.1007/s12017-014-8294-x
Gomes MZ, Del Bel EA (2003) Effects of electrolytic and 6-hydroxydopamine lesions of rat nigrostriatal pathway on nitric oxide synthase and nicotinamide adenine dinucleotide phosphate diaphorase. Brain Res Bull 62:107–115
Gomes MZ, Raisman-Vozari R, Del Bel EA (2008) A nitric oxide synthase inhibitor decreases 6-hydroxydopamine effects on tyrosine hydroxylase and neuronal nitric oxide synthase in the rat nigrostriatal pathway. Brain Res 1203:160–169. https://doi.org/10.1016/j.brainres.2008.01.088
Grant MK, Cuadra AE, El-Fakahany EE (2002) Endogenous expression of nNOS protein in several neuronal cell lines. Life Sci 71:813–817
Haik KL, Shear DA, Hargrove C, Patton J, Mazei-Robison M, Sandstrom MI, Dunbar GL (2008) 7-nitroindazole attenuates 6-hydroxydopamine-induced spatial learning deficits and dopamine neuron loss in a presymptomatic animal model of Parkinson's disease. Exp Clin Psychopharmacol 16:178–189. https://doi.org/10.1037/1064-1297.16.2.178
Hajeri PB, Singh SK (2009) siRNAs: their potential as therapeutic agents--part I. Designing of siRNAs. Drug Discov Today 14:851–858. https://doi.org/10.1016/j.drudis.2009.06.001
Hara MR, Snyder SH (2007) Cell signaling and neuronal death. Annu Rev Pharmacol Toxicol 47:117–141. https://doi.org/10.1146/annurev.pharmtox.47.120505.105311
Helmschrodt C, Höbel S, Schöniger S, Bauer A, Bonicelli J, Gringmuth M, Fietz SA, Aigner A, Richter A, Richter F (2017) Polyethylenimine nanoparticle-mediated siRNA delivery to reduce alpha-Synuclein expression in a model of Parkinson's disease. Mol Ther Nucleic Acids 9:57–68. https://doi.org/10.1016/j.omtn.2017.08.013
Herbison AE, Simonian SX, Norris PJ, Emson PC (1996) Relationship of neuronal nitric oxide synthase immunoreactivity to GnRH neurons in the ovariectomized and intact female rat. J Neuroendocrinol 8:73–82
Huesken D, Lange J, Mickanin C, Weiler J, Asselbergs F, Warner J, Meloon B, Engel S, Rosenberg A, Cohen D, Labow M, Reinhardt M, Natt F, Hall J (2005) Design of a genome-wide siRNA library using an artificial neural network. Nat Biotechnol 23:995–1001. https://doi.org/10.1038/nbt1118
Hunot S, Boissiere F, Faucheux B, Brugg B, Mouatt-Prigent A, Agid Y, Hirsch EC (1996) Nitric oxide synthase and neuronal vulnerability in Parkinson's disease. Neuroscience 72:355–363
Jimenez-Jimenez FJ, Alonso-Navarro H, Herrero MT, Garcia-Martin E, Agundez JA (2016) An update on the role of nitric oxide in the neurodegenerative processes of Parkinson's disease. Curr Med Chem 23:2666–2679
Kalia LV, Lang AE (2015) Parkinson's disease. Lancet 386:896–912. https://doi.org/10.1016/S0140-6736(14)61393-3
Kavya R, Saluja R, Singh S, Dikshit M (2006) Nitric oxide synthase regulation and diversity: implications in Parkinson's disease. Nitric Oxide 15:280–294. https://doi.org/10.1016/j.niox.2006.07.003
Kempster PA, O'Sullivan SS, Holton JL, Revesz T, Lees AJ (2010) Relationships between age and late progression of Parkinson's disease: a clinico-pathological study. Brain 133:1755–1762. https://doi.org/10.1093/brain/awq059
Khvorova A, Watts JK (2017) The chemical evolution of oligonucleotide therapies of clinical utility. Nat Biotechnol 35:238–248. https://doi.org/10.1038/nbt.3765
Kiss JP, Vizi ES (2001) Nitric oxide: a novel link between synaptic and nonsynaptic transmission. Trends Neurosci 24:211–215
Ku SH, Jo SD, Lee YK, Kim K, Kim SH (2016) Chemical and structural modifications of RNAi therapeutics. Adv Drug Deliv Rev 104:16–28. https://doi.org/10.1016/j.addr.2015.10.015
Kumari R, Kumar JB, Luthra PM (2015) Post-lesion administration of 7-NI attenuated motor and non-motor deficits in 6-OHDA induced bilaterally lesioned female rat model of Parkinson's disease. Neurosci Lett 589:191–195. https://doi.org/10.1016/j.neulet.2014.12.030
Kwon MJ, Oh E, Lee S, Roh MR, Kim SE, Lee Y, Choi YL, in YH, Park T, Koh SS, Shin YK (2009) Identification of novel reference genes using multiplatform expression data and their validation for quantitative gene expression analysis. PLoS One 4:e6162. https://doi.org/10.1371/journal.pone.0006162
Ledford H (2018) Gene-silencing technology gets first drug approval after 20-year wait. Nature 560:291–292. https://doi.org/10.1038/d41586-018-05867-7
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Method 25:402–408. https://doi.org/10.1006/meth.2001.1262
Lonser RR, Sarntinoranont M, Morrison PF, Oldfield EH (2015) Convection-enhanced delivery to the central nervous system. J Neurosurg 122:697–706. https://doi.org/10.3171/2014.10.JNS14229
Lopes FM, Schröder R, Júnior MLCF, Zanotto-Filho A, Müller CB, Pires AS, Meurer RT, Colpo GD, Gelain DP, Kapczinski F, Moreira JCF, Fernandes MC, Klamt F (2010) Comparison between proliferative and neuron-like SH-SY5Y cells as an in vitro model for Parkinson disease studies. Brain Res 1337:85–94. https://doi.org/10.1016/j.brainres.2010.03.102
Mathupala SP (2009) Delivery of small-interfering RNA (siRNA) to the brain. Expert Opin Ther Pat 19:137–140. https://doi.org/10.1517/13543770802680195
Matveeva O, Nechipurenko Y, Rossi L, Moore B, Saetrom P, Ogurtsov AY, Atkins JF, Shabalina SA (2007) Comparison of approaches for rational siRNA design leading to a new efficient and transparent method. Nucleic Acids Res 35:e63. https://doi.org/10.1093/nar/gkm088
Meister G, Tuschl T (2004) Mechanisms of gene silencing by double-stranded RNA. Nature 431:343–349. https://doi.org/10.1038/nature02873
Moncada S, Bolanos JP (2006) Nitric oxide, cell bioenergetics and neurodegeneration. J Neurochem 97:1676–1689. https://doi.org/10.1111/j.1471-4159.2006.03988.x
Mukherjee P, Cinelli MA, Kang S, Silverman RB (2014) Development of nitric oxide synthase inhibitors for neurodegeneration and neuropathic pain. Chem Soc Rev 43:6814–6838. https://doi.org/10.1039/c3cs60467e
Niu S, Zhang LK, Zhang L, Zhuang S, Zhan X, Chen WY, du S, Yin L, You R, Li CH, Guan YQ (2017) Inhibition by multifunctional magnetic nanoparticles loaded with alpha-Synuclein RNAi plasmid in a Parkinson's disease model. Theranostics 7:344–356. https://doi.org/10.7150/thno.16562
Padovan-Neto FE, Cavalcanti-Kiwiatkoviski R, Carolino RO, Anselmo-Franci J, Del Bel E (2015) Effects of prolonged neuronal nitric oxide synthase inhibition on the development and expression of L-DOPA-induced dyskinesia in 6-OHDA-lesioned rats. Neuropharmacology 89:87–99. https://doi.org/10.1016/j.neuropharm.2014.08.019
Padovan-Neto FE, Echeverry MB, Chiavegatto S, Del-Bel E (2011) Nitric oxide synthase inhibitor improves De novo and long-term l-DOPA-induced dyskinesia in Hemiparkinsonian rats. Front Syst Neurosci 5:40. https://doi.org/10.3389/fnsys.2011.00040
Padovan-Neto FE, Echeverry MB, Tumas V, Del-Bel EA (2009) Nitric oxide synthase inhibition attenuates L-DOPA-induced dyskinesias in a rodent model of Parkinson's disease. Neuroscience 159:927–935. https://doi.org/10.1016/j.neuroscience.2009.01.034
Padovan-Neto FE, Ferreira NR, de Oliveira-Tavares D, de Aguiar D, da Silva CA, Raisman-Vozari R, Del Bel E (2013) Anti-dyskinetic effect of the neuronal nitric oxide synthase inhibitor is linked to decrease of FosB/deltaFosB expression. Neurosci Lett 541:126–131. https://doi.org/10.1016/j.neulet.2013.02.015
Pardridge WM (2007) Blood-brain barrier delivery. Drug Discov Today 12:54–61. https://doi.org/10.1016/j.drudis.2006.10.013
Paxinos G, Watson C (2005) The Rat Brain in Stereotaxic Coordinates. 5th edn. Elsevier Academic Press, San Diego
Presgraves SP, Ahmed T, Borwege S, Joyce JN (2004) Terminally differentiated SH-SY5Y cells provide a model system for studying neuroprotective effects of dopamine agonists. Neurotox Res 5:579–598
Przedborski S (2005) Pathogenesis of nigral cell death in Parkinson's disease. Parkinsonism Relat Disord 11(Suppl 1):S3–S7. https://doi.org/10.1016/j.parkreldis.2004.10.012
Ramot Y, Rotkopf S, Gabai RM, Zorde Khvalevsky E, Muravnik S, Marzoli GA, Domb AJ, Shemi A, Nyska A (2016) Preclinical safety evaluation in rats of a polymeric matrix containing an siRNA drug used as a local and prolonged delivery system for pancreatic Cancer therapy. Toxicol Pathol 44:856–865. https://doi.org/10.1177/0192623316645860
Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A (2004) Rational siRNA design for RNA interference. Nat Biotechnol 22:326–330. https://doi.org/10.1038/nbt936
Sales TT, Resende FF, Chaves NL, Titze-De-Almeida SS, Bao SN, Brettas ML, Titze-De-Almeida R (2016) Suppression of the Eag1 potassium channel sensitizes glioblastoma cells to injury caused by temozolomide. Oncol Lett 12:2581–2589. https://doi.org/10.3892/ol.2016.4992
Smith CIE, Zain R (2019) Therapeutic oligonucleotides: state of the art. Annu Rev Pharmacol Toxicol 59:605–630. https://doi.org/10.1146/annurev-pharmtox-010818-021050
Solis O, Espadas I, Del-Bel EA, Moratalla R (2015) Nitric oxide synthase inhibition decreases l-DOPA-induced dyskinesia and the expression of striatal molecular markers in Pitx3(−/−) aphakia mice. Neurobiol Dis 73:49–59. https://doi.org/10.1016/j.nbd.2014.09.010
Soto-Sanchez C, Martinez-Navarrete G, Humphreys L, Puras G, Zarate J, Pedraz JL, Fernandez E (2015) Enduring high-efficiency in vivo transfection of neurons with non-viral magnetoparticles in the rat visual cortex for optogenetic applications. Nanomedicine 11:835–843. https://doi.org/10.1016/j.nano.2015.01.012
Titze de Almeida SS, Horst CH, Soto-Sanchez C, Fernandez E, Titze de Almeida R (2018) Delivery of miRNA-targeted oligonucleotides in the rat striatum by Magnetofection with Neuromag((R)) Molecules 23 https://doi.org/10.3390/molecules23071825
Titze-de-Almeida R, David C, Titze-de-Almeida SS (2017) The race of 10 synthetic RNAi-based drugs to the pharmaceutical market. Pharm Res 34:1339–1363. https://doi.org/10.1007/s11095-017-2134-2
Titze-de-Almeida SS, Lustosa CF, Horst CH, Bel ED, Titze-de-Almeida R (2014) Interferon gamma potentiates the injury caused by MPP(+) on SH-SY5Y cells, which is attenuated by the nitric oxide synthases inhibition. Neurochem Res 39:2452–2464. https://doi.org/10.1007/s11064-014-1449-1
Valera E, Masliah E (2016) Therapeutic approaches in Parkinson's disease and related disorders. J Neurochem 139 Suppl 1:346–352. https://doi.org/10.1111/jnc.13529
Vert JP, Foveau N, Lajaunie C, Vandenbrouck Y (2006) An accurate and interpretable model for siRNA efficacy prediction. BMC Bioinformatics 7:520. https://doi.org/10.1186/1471-2105-7-520
Vistica DT, Skehan P, Scudiero D, Monks A, Pittman A, Boyd MR (1991) Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production. Cancer Res 51:2515–2520
Weber C, Mello de Queiroz F, Downie BR, Suckow A, Stuhmer W, Pardo LA (2006) Silencing the activity and proliferative properties of the human EagI Potassium Channel by RNA interference. J Biol Chem 281:13030–13037. https://doi.org/10.1074/jbc.M600883200
Xicoy H, Wieringa B, Martens GJ (2017) The SH-SY5Y cell line in Parkinson's disease research: a systematic review. Mol Neurodegener 12:10. https://doi.org/10.1186/s13024-017-0149-0
Xie HR, Hu LS, Li GY (2010) SH-SY5Y human neuroblastoma cell line: in vitro cell model of dopaminergic neurons in Parkinson's disease. Chin Med J 123:1086–1092
Zhou L, Zhu DY (2009) Neuronal nitric oxide synthase: structure, subcellular localization, regulation, and clinical implications. Nitric Oxide 20:223–230. https://doi.org/10.1016/j.niox.2009.03.001
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This publication was funded by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; award number 467467/2014-5); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; award number PNPD 0299081/2010 and PNPD 3731-37/2010); Fundação de Apoio a Pesquisa do Distrito Federal (FAPDF; award number 0193.001009/2015) and Instituto Nacional de Ciência e Tecnologia (INCT).
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Titze-de-Almeida, R., Titze-de-Almeida, S.S., Ferreira, N.R. et al. Suppressing nNOS Enzyme by Small-Interfering RNAs Protects SH-SY5Y Cells and Nigral Dopaminergic Neurons from 6-OHDA Injury. Neurotox Res 36, 117–131 (2019). https://doi.org/10.1007/s12640-019-00043-9
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DOI: https://doi.org/10.1007/s12640-019-00043-9