ISPITIVANJE NEUROPROTEKTIVNOG EFEKTA INHIBICIJE RO-KINAZE U ĆELIJSKOM MODELU NEURODEGENERACIJE IZAZVANOM 1-METIL-4-FENILPIRIDINIJUMOM (MPP+)
Ključne reči:
Parkinsonova bolest, Ro-kinaza, fasudil, MPP , Akt, AMPK
Sažetak
Uvod: Model neurodegeneracije indukovan primenom 1-metil 4-fenil 1,2,3,6-tetrahidropiridijuma (MPTP) jedan je od najčešće korišćenih modela ove bolesti u ekperimentalnim uslovima. MPTP, tj. njegov metabolit MPP+, inhibira kompleks I u mitohondrijama, povećava proizvodnju slobodnih radikala što vodi ka depleciji ATP-a i ćelijskoj smrti. Inhibicija Ro-kinaze, enzima uključenog u regulaciju važnih procesa u ćeliji, kao što su organizacija citoskeleta, aksonogeneza, regulacija vezikularnog transporta, apoptoza, dovodi do njihovog poboljšanog preživljavanja.
Cilj: Cilj ovog rada je bio ispitivanje efekta inhibicije Ro-kinaze u ćelijskom modelu neurodegeneracije izazvane MPP+-om, kao i uloga regulatonih kinaza i to protein kinaze B/Akt i adenozin monofosfatom aktivirane kinaze (AMPK) u tom procesu.
Materijal i metode: Eksperimenti su rađeni na ćelijskoj liniji humanog neuroblastoma SH-SY5Y. MTT test je korišćen za određivanje vijabiliteta ćelija nakon tretmana MPP+-om inhibitorom Ro-kinaze, fasudilom. Za određivanje nivoa aktivacije proteina uključenih u kontrolu metaboličkih puteva, AMPK i Akt, korišćena je imunoblot metoda, a kvantifikacija je urađena denzitometrijom.
Rezultati: Pokazali smo da tretman MPP+-om dovodi do pada vijabiliteta ćelija neuroblastoma u odnosu na kontrolne (netretirane) ćelije, dok se u uslovima pretretmana fasudilom uočava dozno-zavisni porast vijabiliteta u odnosu na tretman samo MPP+-om. Ispitivanje ciljnih proteina je pokazalo da u uslovima tretmana fasudilom i MPP+-om dolazi do značajanog porasta u aktivaciji Akt-a, dok nema značajnih promena u aktivaciji AMPK.
Zaključak: Inhibicija Ro-kinaze, upotrebom fasudila, poboljšava preživljavanje ćelija neuroblastoma u uslovima citotoksičnog dejstva MPP+ neurotoksina i dovodi do aktivacije Akt/PI3K signalnog puta.
Reference
1. Bose A, Beal MF. Mitochondrial dysfunction in Parkinson’s disease. J Neurochem. 2016 Oct;139(S1):216–31.
2. Apostolski Slobodan, Bulat Petar, Bumbaširević Ljiljana, Cerovac Nataša DN. Neurologija za studente. drugo. Kostić Vladimir, vojodić Nikola PI, editor. Beograd; 2018.
3. Kumar V, Abbas AA, Fausto N, Mitchell RN. Robinsove osnove patologije. 8. Boričić I, Đuričić S, editors. Beograd: Data Status; 2010. 893,894.
4. Keane H, Ryan BJ, Jackson B, Whitmore A, Wade-Martins R. Protein-protein interaction networks identify targets which rescue the MPP+ cellular model of Parkinson’s disease. Sci Rep. 2015 Dec 26;5(1):17004.
5. Langston J, Ballard P, Tetrud J, Irwin I. Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis. Science (80- ). 1983 Feb 25;219(4587):979–80.
6. Stanley Burns R, Markey SP, Phillips JM, Chiueh CC. The Neurotoxicity of 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine in the Monkey and Man. Can J Neurol Sci / J Can des Sci Neurol. 1984 Feb 18;11(S1):166–8.
7. Heikkila RE, Cabbat FS, Manzino L, Duvoisin RC. Effects of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine on neostriatal dopamine in mice. Neuropharmacology. 1984 Jun;23(6):711–3.
8. Ransom BR, Kunis DM, Irwin I, Langston JW. Astrocytes convert the parkinsonism inducing neurotoxin, MPTP, to its active metabolite, MPP+. Neurosci Lett. 1987 Apr;75(3):323–8.
9. Brooks WJ, Jarvis MF, Wagner GC. Astrocytes as a primary locus for the conversion MPTP into MPP+. J Neural Transm. 1989 Feb;76(1):1–12.
10. Langston JW, Irwin I, Langston EB, Forno LS. 1-Methyl-4-phenylpyridinium ion (MPP+): Identification of a metabolite of MPTP, a toxin selective to the substantia nigra. Neurosci Lett. 1984 Jul 13;48(1):87–92.
11. Markey SP, Johannessen JN, Chiueh CC, Burns RS, Herkenham MA. Intraneuronal generation of a pyridinium metabolite may cause drug-induced parkinsonism. Nature. 1984 Oct;311(5985):464–7.
12. Ramsay RR, Singer TP. Energy-dependent uptake of N-methyl-4-phenylpyridinium, the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, by mitochondria. J Biol Chem. 1986 Jun 15;261(17):7585–7.
13. Jackson-Lewis V, Jay Smeyne R. From Man to Mouse: The MPTP Model of Parkinson Disease. Mov Disord. 2005 Jan 1;149–60.
14. Ramsay RR, Salach JI, Singer TP. Uptake of the neurotoxin 1-methyl-4-phenylpyridine (MPP+) by mitochondria and its relation to the inhibition of the mitochondrial oxidation of NAD+-linked substrates by MPP+. Biochem Biophys Res Commun. 1986 Jan;134(2):743–8.
15. Ito K, Eguchi Y, Imagawa Y, Akai S, Mochizuki H, Tsujimoto Y. MPP+ induces necrostatin-1- and ferrostatin-1-sensitive necrotic death of neuronal SH-SY5Y cells. Cell Death Discov. 2017 Feb 27;3(December 2016):17013.
16. Choi SJ, Panhelainen A, Schmitz Y, Larsen KE, Kanter E, Wu M, et al. Changes in Neuronal Dopamine Homeostasis following 1-Methyl-4-phenylpyridinium (MPP+) Exposure. J Biol Chem. 2015 Mar 13;290(11):6799.
17. Ara J, Przedborski S, Naini AB, Jackson-Lewis V, Trifiletti RR, Horwitz J, et al. Inactivation of tyrosine hydroxylase by nitration following exposure to peroxynitrite and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Proc Natl Acad Sci. 1998 Jun 23;95(13):7659–63.
18. Jenner P. Oxidative stress and Parkinson’s disease. Handb Clin Neurol. 2007;83:507-20.
19. Labandeira-Garcia JL, Rodríguez-Perez AI, Villar-Cheda B, Borrajo A, Dominguez-Meijide A, Guerra MJ. Rho Kinase and Dopaminergic Degeneration. Neurosci. 2015 Dec 16;21(6):616–29.
20. Villar-Cheda B, Dominguez-Meijide A, Joglar B, Rodriguez-Perez AI, Guerra MJ, Labandeira-Garcia JL. Involvement of microglial RhoA/Rho-Kinase pathway activation in the dopaminergic neuron death. Role of angiotensin via angiotensin type 1 receptors. Neurobiol Dis. 2012 Aug 1;47(2):268–79.
21. Hashimoto R, Nakamura Y, Kosako H, Amano M, Kaibuchi K, Inagaki M, et al. Distribution of Rho-kinase in the bovine brain. Biochem Biophys Res Commun. 1999 Sep 24;263(2):575–9.
22. Komagome R, Kimura K, Saito M. Postnatal changes in Rho and Rho-related proteins in the mouse brain. Jpn J Vet Res. 2000 Feb;47(3–4):127–33.
23. Zhao Y, Zhang Q, Xi J, Li Y, Ma C, Xiao B. Multitarget intervention of Fasudil in the neuroprotection of dopaminergic neurons in MPTP-mouse model of Parkinson’s disease. J Neurol Sci. 2015 Jun;353(1–2):28–37.
24. Koch JC, Tönges L, Barski E, Michel U, Bähr M, Lingor P. ROCK2 is a major regulator of axonal degeneration, neuronal death and axonal regeneration in the CNS. Cell Death Dis. 2014 May 15;5(5):e1225–e1225.
25. Tonges L, Frank T, Tatenhorst L, Saal KA, Koch JC, Szego EM, et al. Inhibition of rho kinase enhances survival of dopaminergic neurons and attenuates axonal loss in a mouse model of Parkinson’s disease. Brain. 2012 Nov 1;135(11):3355–70.
26. Wu J, Li J, Hu H, Liu P, Fang Y, Wu D. Rho-Kinase Inhibitor, Fasudil, Prevents Neuronal Apoptosis via the Akt Activation and PTEN Inactivation in the Ischemic Penumbra of Rat Brain. Cell Mol Neurobiol. 2012 Oct 3;32(7):1187–97.
27. Sako K, Tsuchiya M, Yonemasu Y, Asano T. HA1077, a novel calcium antagonistic antivasospasm drug, increases both cerebral blood flow and glucose metabolism in conscious rats. Eur J Pharmacol. 1991 Dec;209(1–2):39–43.
28. Noda K, Nakajima S, Godo S, Saito H, Ikeda S, Shimizu T, et al. Rho-Kinase Inhibition Ameliorates Metabolic Disorders through Activation of AMPK Pathway in Mice. Claret M, editor. PLoS One. 2014 Nov 3;9(11):e110446.
29. Carling D. AMPK signalling in health and disease. Curr Opin Cell Biol. 2017 Apr;45:31-37.
30. Keeney PM, Xie J, Capaldi RA, Bennett JP. Parkinson’s Disease Brain Mitochondrial Complex I Has Oxidatively Damaged Subunits and Is Functionally Impaired and Misassembled. J Neurosci. 2006 May 10;26(19):5256–64.
31. Manning BD, Cantley LC. AKT/PKB Signaling: Navigating Downstream. Cell. 2007 Jun 29;129(7):1261–74.
32. Song G, Ouyang G, Bao S. The activation of Akt/PKB signaling pathway and cell survival. J Cell Mol Med. 2005 Jan-Mar;9(1):59-71.
33. Malagelada C, Jin ZH, Greene LA. RTP801 Is Induced in Parkinson’s Disease and Mediates Neuron Death by Inhibiting Akt Phosphorylation/Activation. J Neurosci. 2008 Dec 31;28(53):14363–71.
34. Jovanovic-Tucovic M, Harhaji-Trajkovic L, Dulovic M, Tovilovic-Kovacevic G, Zogovic N, Jeremic M, et al. AMP-activated protein kinase inhibits MPP+-induced oxidative stress and apoptotic death of SH-SY5Y cells through sequential stimulation of Akt and autophagy. Eur J Pharmacol. 2019 Nov 15;863:172677.
35. Aleyasin H, Rousseaux MWC, Marcogliese PC, Hewitt SJ, Irrcher I, Joselin AP, et al. DJ-1 protects the nigrostriatal axis from the neurotoxin MPTP by modulation of the AKT pathway. Proc Natl Acad Sci U S A. 2010 Feb;107(7):3186–91.
36. Kim C, Park S. IGF-1 protects SH-SY5Y cells against MPP+-induced apoptosis via PI3K/PDK-1/Akt pathway. Endocr Connect. 2018 Mar 1;7(3):443–55.
37. Zhao Y, Hu X, Liu Y, Dong S, Wen Z, He W, et al. ROS signaling under metabolic stress: cross-talk between AMPK and AKT pathway. Mol Cancer. 2017 Dec 13;16(1):79.
38. Kruger NJ. The Bradford method for protein quantitation. Methods Mol Biol. 1994;32:9-15.
39. Oyarce AM, Fleming PJ. Multiple forms of human dopamine β-hydroxylase in SH-SY5Y neuroblastoma cells. Arch Biochem Biophys. 1991 Nov 1 ;290(2):503–10.
40. Xicoy H, Wieringa B, Martens GJM. The SH-SY5Y cell line in Parkinson’s disease research: a systematic review. Mol Neurodegener 2017 121. 2017 Jan 24;12(1):1–11.
41. Zhao Y, Zhang Q, Xi J, Xiao B, Li Y, Ma C. Neuroprotective effect of fasudil on inflammation through PI3K/Akt and Wnt/β-catenin dependent pathways in a mice model of Parkinson’s disease. Int J Clin Exp Pathol. 2015 Mar;8(3):2354–64.
42. Dulovic M, Jovanovic M, Xilouri M, Stefanis L, Harhaji-Trajkovic L, Kravic-Stevovic T, et al. The protective role of AMP-activated protein kinase in alpha-synuclein neurotoxicity in vitro. Neurobiol Dis. 2014 Mar;63:1–11.
43. Leslie NR, Downes CP. PTEN: The down side of PI 3-kinase signalling. Cell Signal. 2002 Apr;14(4):285–95.
2. Apostolski Slobodan, Bulat Petar, Bumbaširević Ljiljana, Cerovac Nataša DN. Neurologija za studente. drugo. Kostić Vladimir, vojodić Nikola PI, editor. Beograd; 2018.
3. Kumar V, Abbas AA, Fausto N, Mitchell RN. Robinsove osnove patologije. 8. Boričić I, Đuričić S, editors. Beograd: Data Status; 2010. 893,894.
4. Keane H, Ryan BJ, Jackson B, Whitmore A, Wade-Martins R. Protein-protein interaction networks identify targets which rescue the MPP+ cellular model of Parkinson’s disease. Sci Rep. 2015 Dec 26;5(1):17004.
5. Langston J, Ballard P, Tetrud J, Irwin I. Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis. Science (80- ). 1983 Feb 25;219(4587):979–80.
6. Stanley Burns R, Markey SP, Phillips JM, Chiueh CC. The Neurotoxicity of 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine in the Monkey and Man. Can J Neurol Sci / J Can des Sci Neurol. 1984 Feb 18;11(S1):166–8.
7. Heikkila RE, Cabbat FS, Manzino L, Duvoisin RC. Effects of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine on neostriatal dopamine in mice. Neuropharmacology. 1984 Jun;23(6):711–3.
8. Ransom BR, Kunis DM, Irwin I, Langston JW. Astrocytes convert the parkinsonism inducing neurotoxin, MPTP, to its active metabolite, MPP+. Neurosci Lett. 1987 Apr;75(3):323–8.
9. Brooks WJ, Jarvis MF, Wagner GC. Astrocytes as a primary locus for the conversion MPTP into MPP+. J Neural Transm. 1989 Feb;76(1):1–12.
10. Langston JW, Irwin I, Langston EB, Forno LS. 1-Methyl-4-phenylpyridinium ion (MPP+): Identification of a metabolite of MPTP, a toxin selective to the substantia nigra. Neurosci Lett. 1984 Jul 13;48(1):87–92.
11. Markey SP, Johannessen JN, Chiueh CC, Burns RS, Herkenham MA. Intraneuronal generation of a pyridinium metabolite may cause drug-induced parkinsonism. Nature. 1984 Oct;311(5985):464–7.
12. Ramsay RR, Singer TP. Energy-dependent uptake of N-methyl-4-phenylpyridinium, the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, by mitochondria. J Biol Chem. 1986 Jun 15;261(17):7585–7.
13. Jackson-Lewis V, Jay Smeyne R. From Man to Mouse: The MPTP Model of Parkinson Disease. Mov Disord. 2005 Jan 1;149–60.
14. Ramsay RR, Salach JI, Singer TP. Uptake of the neurotoxin 1-methyl-4-phenylpyridine (MPP+) by mitochondria and its relation to the inhibition of the mitochondrial oxidation of NAD+-linked substrates by MPP+. Biochem Biophys Res Commun. 1986 Jan;134(2):743–8.
15. Ito K, Eguchi Y, Imagawa Y, Akai S, Mochizuki H, Tsujimoto Y. MPP+ induces necrostatin-1- and ferrostatin-1-sensitive necrotic death of neuronal SH-SY5Y cells. Cell Death Discov. 2017 Feb 27;3(December 2016):17013.
16. Choi SJ, Panhelainen A, Schmitz Y, Larsen KE, Kanter E, Wu M, et al. Changes in Neuronal Dopamine Homeostasis following 1-Methyl-4-phenylpyridinium (MPP+) Exposure. J Biol Chem. 2015 Mar 13;290(11):6799.
17. Ara J, Przedborski S, Naini AB, Jackson-Lewis V, Trifiletti RR, Horwitz J, et al. Inactivation of tyrosine hydroxylase by nitration following exposure to peroxynitrite and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Proc Natl Acad Sci. 1998 Jun 23;95(13):7659–63.
18. Jenner P. Oxidative stress and Parkinson’s disease. Handb Clin Neurol. 2007;83:507-20.
19. Labandeira-Garcia JL, Rodríguez-Perez AI, Villar-Cheda B, Borrajo A, Dominguez-Meijide A, Guerra MJ. Rho Kinase and Dopaminergic Degeneration. Neurosci. 2015 Dec 16;21(6):616–29.
20. Villar-Cheda B, Dominguez-Meijide A, Joglar B, Rodriguez-Perez AI, Guerra MJ, Labandeira-Garcia JL. Involvement of microglial RhoA/Rho-Kinase pathway activation in the dopaminergic neuron death. Role of angiotensin via angiotensin type 1 receptors. Neurobiol Dis. 2012 Aug 1;47(2):268–79.
21. Hashimoto R, Nakamura Y, Kosako H, Amano M, Kaibuchi K, Inagaki M, et al. Distribution of Rho-kinase in the bovine brain. Biochem Biophys Res Commun. 1999 Sep 24;263(2):575–9.
22. Komagome R, Kimura K, Saito M. Postnatal changes in Rho and Rho-related proteins in the mouse brain. Jpn J Vet Res. 2000 Feb;47(3–4):127–33.
23. Zhao Y, Zhang Q, Xi J, Li Y, Ma C, Xiao B. Multitarget intervention of Fasudil in the neuroprotection of dopaminergic neurons in MPTP-mouse model of Parkinson’s disease. J Neurol Sci. 2015 Jun;353(1–2):28–37.
24. Koch JC, Tönges L, Barski E, Michel U, Bähr M, Lingor P. ROCK2 is a major regulator of axonal degeneration, neuronal death and axonal regeneration in the CNS. Cell Death Dis. 2014 May 15;5(5):e1225–e1225.
25. Tonges L, Frank T, Tatenhorst L, Saal KA, Koch JC, Szego EM, et al. Inhibition of rho kinase enhances survival of dopaminergic neurons and attenuates axonal loss in a mouse model of Parkinson’s disease. Brain. 2012 Nov 1;135(11):3355–70.
26. Wu J, Li J, Hu H, Liu P, Fang Y, Wu D. Rho-Kinase Inhibitor, Fasudil, Prevents Neuronal Apoptosis via the Akt Activation and PTEN Inactivation in the Ischemic Penumbra of Rat Brain. Cell Mol Neurobiol. 2012 Oct 3;32(7):1187–97.
27. Sako K, Tsuchiya M, Yonemasu Y, Asano T. HA1077, a novel calcium antagonistic antivasospasm drug, increases both cerebral blood flow and glucose metabolism in conscious rats. Eur J Pharmacol. 1991 Dec;209(1–2):39–43.
28. Noda K, Nakajima S, Godo S, Saito H, Ikeda S, Shimizu T, et al. Rho-Kinase Inhibition Ameliorates Metabolic Disorders through Activation of AMPK Pathway in Mice. Claret M, editor. PLoS One. 2014 Nov 3;9(11):e110446.
29. Carling D. AMPK signalling in health and disease. Curr Opin Cell Biol. 2017 Apr;45:31-37.
30. Keeney PM, Xie J, Capaldi RA, Bennett JP. Parkinson’s Disease Brain Mitochondrial Complex I Has Oxidatively Damaged Subunits and Is Functionally Impaired and Misassembled. J Neurosci. 2006 May 10;26(19):5256–64.
31. Manning BD, Cantley LC. AKT/PKB Signaling: Navigating Downstream. Cell. 2007 Jun 29;129(7):1261–74.
32. Song G, Ouyang G, Bao S. The activation of Akt/PKB signaling pathway and cell survival. J Cell Mol Med. 2005 Jan-Mar;9(1):59-71.
33. Malagelada C, Jin ZH, Greene LA. RTP801 Is Induced in Parkinson’s Disease and Mediates Neuron Death by Inhibiting Akt Phosphorylation/Activation. J Neurosci. 2008 Dec 31;28(53):14363–71.
34. Jovanovic-Tucovic M, Harhaji-Trajkovic L, Dulovic M, Tovilovic-Kovacevic G, Zogovic N, Jeremic M, et al. AMP-activated protein kinase inhibits MPP+-induced oxidative stress and apoptotic death of SH-SY5Y cells through sequential stimulation of Akt and autophagy. Eur J Pharmacol. 2019 Nov 15;863:172677.
35. Aleyasin H, Rousseaux MWC, Marcogliese PC, Hewitt SJ, Irrcher I, Joselin AP, et al. DJ-1 protects the nigrostriatal axis from the neurotoxin MPTP by modulation of the AKT pathway. Proc Natl Acad Sci U S A. 2010 Feb;107(7):3186–91.
36. Kim C, Park S. IGF-1 protects SH-SY5Y cells against MPP+-induced apoptosis via PI3K/PDK-1/Akt pathway. Endocr Connect. 2018 Mar 1;7(3):443–55.
37. Zhao Y, Hu X, Liu Y, Dong S, Wen Z, He W, et al. ROS signaling under metabolic stress: cross-talk between AMPK and AKT pathway. Mol Cancer. 2017 Dec 13;16(1):79.
38. Kruger NJ. The Bradford method for protein quantitation. Methods Mol Biol. 1994;32:9-15.
39. Oyarce AM, Fleming PJ. Multiple forms of human dopamine β-hydroxylase in SH-SY5Y neuroblastoma cells. Arch Biochem Biophys. 1991 Nov 1 ;290(2):503–10.
40. Xicoy H, Wieringa B, Martens GJM. The SH-SY5Y cell line in Parkinson’s disease research: a systematic review. Mol Neurodegener 2017 121. 2017 Jan 24;12(1):1–11.
41. Zhao Y, Zhang Q, Xi J, Xiao B, Li Y, Ma C. Neuroprotective effect of fasudil on inflammation through PI3K/Akt and Wnt/β-catenin dependent pathways in a mice model of Parkinson’s disease. Int J Clin Exp Pathol. 2015 Mar;8(3):2354–64.
42. Dulovic M, Jovanovic M, Xilouri M, Stefanis L, Harhaji-Trajkovic L, Kravic-Stevovic T, et al. The protective role of AMP-activated protein kinase in alpha-synuclein neurotoxicity in vitro. Neurobiol Dis. 2014 Mar;63:1–11.
43. Leslie NR, Downes CP. PTEN: The down side of PI 3-kinase signalling. Cell Signal. 2002 Apr;14(4):285–95.
Objavljeno
2022/01/11
Broj časopisa
Rubrika
Originalni naučni članak