THE NEUROPROTECTIVE EFFECT OF Rho-KINASE INHIBITION IN 1-METHYL-4-PHENYLPYRIDINIUM (MPP+)-INDUCED CELLULAR MODEL OF NEURODEGENERATION
Keywords:
Parkinson’s disease, Rho-kinase, fasudil, MPP , Akt, AMPK
Abstract
Introduction: The 1-methyl 4-phenyl 1,2,3,6-tetrahydropiridium (MPTP) induced model of neurodegeneration in Parkinson’s disease (PD) is one of the most commonly used experimental models. MPTP, or rather its metabolite MPP+, leads to inhibition of mitochondrial complex I, an increase in free radicals’ production and ATP depletion, all resulting in cellular demise and death. Rho-kinase is an enzyme involved with numerous cell regulatory mechanisms, such as cytoskeleton organization, axonogenesis, vesicular transport regulation and apoptosis regulation, which are important for cell survival.
Aim: Our aim was to investigate the effects of Rho-kinase inhibition on the MPP+ induced model of neurodegeneration and the role of Akt and adenosine monophosphate-activated protein kinase (AMPK) signaling pathways in this process.
Materials and methods: The experiments were performed on the human neuroblastoma SH-SY5Y cell line. The MTT test was used to measure the viability of the cells after the MPP+ and/or Rho-kinase inhibitor, fasudil, treatments. Changes in activation levels, or rather expression of pAMPK, pAkt, AMPK and Akt, were measured with the immunoblotting method, and the protein levels were quantified by densitometry.
Results: MPP+ treatment caused a dose-depedent decrease in cellular viability, compared to the control group (untreated cells), while fasudil treatment prior to MPP+ exposure, improved cell viability a dose-dependent increase, compared to MPP+ treatment. Analysis of activation status of target proteins showed an increase in Akt activation after the fasudil treatment, while the AMPK activation was not significantly changed.
Conclusion: Inhibition of Rho-kinase using fasudil causes a decrease in MPP+ induced cell death, which is possibly mediated by an activation of the Akt/PI3K signaling pathway.
References
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18. Jenner P. Oxidative stress and Parkinson’s disease. Handb Clin Neurol. 2007;83:507-20.
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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.
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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.
Published
2022/01/11
Section
Original Scientific Paper