Ginkgolide B ameliorates MPTP-induced neuroinflammation and neurodegeneration by improving mitochondrial electron transport chain complex I

Abstract

Although the pathology and clinical symptoms of Parkinson's disease (PD) are well-defined, the cellular and molecular mechanisms underlying the selective degeneration of dopaminergic neurons remain unclear. Mitochondrial dysfunction and neuroinflammation are increasingly recognized as central contributors to the pathogenesis of PD. The leaf extract of Ginkgolide, Ginkgo biloba, is known for its neuroprotective properties in several neurodegenerative diseases. In the present study, we sought to investigate the neuroprotective mechanism of Ginkgolide B (BN52021), a terpene lactone derived from the leaf of Ginkgo biloba, in an animal model of PD. Adult C57BL/6 mice treated with MPTP (30 mg/ kg b.wt.) exhibited significant motor deficits, ameliorated by cotreatment with BN52021 (20 mg/ Kg b.wt.), as evidenced by improved motor behaviors. MPTP administration resulted in a marked reduction in the mitochondrial complex I activity and antioxidant enzymes, specifically in the substantia nigra, whereas the striatum remained unaffected. Notably, BN52021 cotreatment restored the complex I function and antioxidant enzymes in the substantia nigra, highlighting its region-specific neuroprotective properties. Additionally, MPTP exposure significantly increased myeloperoxidase activity, a marker of oxidative stress and inflammation mitigated by BN52021. Moreover, the inflammatory markers NLRP3, MCP-1, and IL-1β were significantly upregulated following MPTP administration, indicating the activation of the inflammasome pathway. However, coadministration of MPTP with BN52021 effectively suppressed the upregulation of these inflammatory markers, suggesting a strong anti-inflammatory effect. These findings underscore the therapeutic potential of Ginkgolide in PD, primarily through its ability to enhance mitochondrial electron transport complex I activity, restore antioxidant defense, and suppress neuroinflammation.

Keywords

• Ginkgolide B
• Inflammasome
• MPTP
• Neurodegeneration
• Neuroprotection
• Parkinson's disease

Supporting Institution

Annamalai University

Project Number

1

Ethical Statement

Approval of the Research Protocol: N/A Informed Consent: N/A Registry and Registration No. of the Study: The present animal studies were approved by the Institutional Animal Ethics Committee, Rajah Muthiah Medical College, Annamalai University (Approval ID: AU-IAEC/PR/1283/10/20). Animal Studies: All animal studies were carried out following the national and international guidelines and the relevant national laws on the protection of animals. Patient Consent for Publication: N/A

Thanks

The authors acknowledge DST-FIST for sponsoring the Department of Biotechnology, School of Integrative Biology, Central University of Tamil Nadu.

References

1. Barone FC, Hillegass LM, Price WJ, White RF, Lee EV, Feuerstein GZ, Sarau HM, Clark RK, and Griswold DE. (1991). Polymorphonuclear leukocyte infiltration into cerebral focal ischemic tissue: myeloperoxidase activity assay and histologic verification. J Neurosci Res. 29 (3): 336-45. https://doi.org/10.1002/jnr.490290309.
2. Beal MF. (2003). Mitochondria, oxidative damage, and inflammation in Parkinson's disease. Ann N Y Acad Sci. 991: 120-31. https://doi.org/10.1111/j.1749-6632.2003.tb07470.x.
3. Bellizzi MJ, Geathers JS, Allan KC, and Gelbard HA. (2016). Platelet-Activating Factor Receptors Mediate Excitatory Postsynaptic Hippocampal Injury in Experimental Autoimmune Encephalomyelitis. J Neurosci. 36 (4): 1336-46. https://doi.org/10.1523/JNEUROSCI.1171-15.2016.
4. Berman SB, and Hastings TG. (1999). Dopamine oxidation alters mitochondrial respiration and induces permeability transition in brain mitochondria: implications for Parkinson's disease. J Neurochem. 73 (3): 1127-37. https://doi.org/10.1046/j.1471-4159.1999.0731127.x.
5. Block ML, and Hong JS. (2007). Chronic microglial activation and progressive dopaminergic neurotoxicity. Biochem Soc Trans. 35 (Pt 5): 1127-32. https://doi.org/10.1042/BST0351127.
6. Brahadeeswaran S, Sivagurunathan N, and Calivarathan L. (2022). Inflammasome Signaling in the Aging Brain and Age-Related Neurodegenerative Diseases. Mol Neurobiol. 59 (4): 2288-304. https://doi.org/10.1007/s12035-021-02683-5.
7. Carlberg I, and Mannervik B. (1975). Purification and characterization of the flavoenzyme glutathione reductase from rat liver. J Biol Chem. 250 (14): 5475-80doi: http://www.ncbi.nlm.nih.gov/pubmed/237922.
8. Cassarino DS, Parks JK, Parker WD, Jr., and Bennett JP, Jr. (1999). The parkinsonian neurotoxin MPP+ opens the mitochondrial permeability transition pore and releases cytochrome c in isolated mitochondria via an oxidative mechanism. Biochim Biophys Acta. 1453 (1): 49-62. https://doi.org/10.1016/s0925-4439(98)00083-0.

9. Chen Y, Zhang Y, Li L, and Holscher C. (2015). Neuroprotective effects of geniposide in the MPTP mouse model of Parkinson's disease. Eur J Pharmacol. 768: 21-7. https://doi.org/10.1016/j.ejphar.2015.09.029.
10. Claiborne A. 1985. Catalase activity (CRC Press: Boca Raton). Dauer W, and Przedborski S. (2003). Parkinson's disease: mechanisms and models. Neuron. 39 (6): 889-909. https://doi.org/10.1016/s0896-6273(03)00568-3.
11. Gordon D, Dafinca R, Scaber J, Alegre-Abarrategui J, Farrimond L, Scott C, Biggs D, Kent L, Oliver PL, Davies B, Ansorge O, Wade-Martins R, and Talbot K. (2019). Single-copy expression of an amyotrophic lateral sclerosis-linked TDP-43 mutation (M337V) in BAC transgenic mice leads to altered stress granule dynamics and progressive motor dysfunction. Neurobiol Dis. 121: 148-62. https://doi.org/10.1016/j.nbd.2018.09.024.
12. Harms AS, Ferreira SA, and Romero-Ramos M. (2021). Periphery and brain, innate and adaptive immunity in Parkinson's disease. Acta Neuropathol. 141 (4): 527-45. https://doi.org/10.1007/s00401-021-02268-5.
13. Holbrook JA, Jarosz-Griffiths HH, Caseley E, Lara-Reyna S, Poulter JA, Williams-Gray CH, Peckham D, and McDermott MF. (2021). Neurodegenerative Disease and the NLRP3 Inflammasome. Front Pharmacol. 12: 643254. https://doi.org/10.3389/fphar.2021.643254.
14. Jackson-Lewis V, and Przedborski S. (2007). Protocol for the MPTP mouse model of Parkinson's disease. Nat Protoc. 2 (1): 141-51. https://doi.org/10.1038/nprot.2006.342.
15. Jankovic J. (2008). Parkinson's disease: clinical features and diagnosis. J Neurol Neurosurg Psychiatry. 79 (4): 368-76. https://doi.org/10.1136/jnnp.2007.131045.
16. Joers V, Tansey MG, Mulas G, and Carta AR. (2017). Microglial phenotypes in Parkinson's disease and animal models of the disease. Prog Neurobiol. 155: 57-75. https://doi.org/10.1016/j.pneurobio.2016.04.006.
17. Kelley AE. (2001). Measurement of rodent stereotyped behavior. Curr Protoc Neurosci. Chapter 8: Unit 8 8. https://doi.org/10.1002/0471142301.ns0808s04.
18. Liu Y, Liu W, Xiong S, Luo J, Li Y, Zhao Y, Wang Q, Zhang Z, Chen X, and Chen T. (2020). Highly stabilized nanocrystals delivering Ginkgolide B in protecting against the Parkinson's disease. Int J Pharm. 577: 119053. https://doi.org/10.1016/j.ijpharm.2020.119053.
19. Long-Smith CM, Sullivan AM, and Nolan YM. (2009). The influence of microglia on the pathogenesis of Parkinson's disease. Prog Neurobiol. 89 (3): 277-87. https://doi.org/10.1016/j.pneurobio.2009.08.001.
20. Maniradhan M, Sivagurunathan N, Unnikrishnan AK, Anbiah VS, and Calivarathan L. (2024). Selenium ameliorates oxidized phospholipid-mediated testicular dysfunction and epididymal sperm abnormalities following Bisphenol A exposure in adult Wistar rats. Reprod Toxicol. 130: 108751. https://doi.org/10.1016/j.reprotox.2024.108751.
21. Marklund S, and Marklund G. (1974). Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem. 47 (3): 469-74. https://doi.org/10.1111/j.1432-1033.1974.tb03714.x.
22. Matsuura K, Kabuto H, Makino H, and Ogawa N. (1997). Pole test is a useful method for evaluating the mouse movement disorder caused by striatal dopamine depletion. J Neurosci Methods. 73 (1): 45-8. https://doi.org/10.1016/s0165-0270(96)02211-x.
23. McGeer PL, Itagaki S, Boyes BE, and McGeer EG. (1988). Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains. Neurology. 38 (8): 1285-91. https://doi.org/10.1212/wnl.38.8.1285.
24. Meireles J, and Massano J. (2012). Cognitive impairment and dementia in Parkinson's disease: clinical features, diagnosis, and management. Front Neurol. 3: 88. https://doi.org/10.3389/fneur.2012.00088.
25. Mizuno Y, Suzuki K, and Sone N. (1990). Inhibition of ATP synthesis by 1-methyl-4-phenylpyridinium ion (MPP+) in mouse brain in vitro and in vivo. Adv Neurol. 53: 197-200doi: http://www.ncbi.nlm.nih.gov/pubmed/2122644.
26. Mohandas J, Marshall JJ, Duggin GG, Horvath JS, and Tiller DJ. (1984). Differential distribution of glutathione and glutathione-related enzymes in rabbit kidney. Possible implications in analgesic nephropathy. Biochem Pharmacol. 33 (11): 1801-7. https://doi.org/10.1016/0006-2952(84)90353-8.
27. Nagatsu T, Mogi M, Ichinose H, and Togari A. (2000a). Changes in cytokines and neurotrophins in Parkinson's disease. J Neural Transm Suppl. no. 10.1007/978-3-7091-6301-6_19(60): 277-90. https://doi.org/10.1007/978-3-7091-6301-6_19.
28. Nagatsu T, Mogi M, Ichinose H, and Togari A. (2000b). Cytokines in Parkinson's disease. J Neural Transm Suppl. doi: http://www.ncbi.nlm.nih.gov/pubmed/11128604(58): 143-51.
29. Oberpichler H, Sauer D, Rossberg C, Mennel HD, and Krieglstein J. (1990). PAF antagonist ginkgolide B reduces postischemic neuronal damage in rat brain hippocampus. J Cereb Blood Flow Metab. 10 (1): 133-5. https://doi.org/10.1038/jcbfm.1990.17.
30. Ogawa N, Hirose Y, Ohara S, Ono T, and Watanabe Y. (1985). A simple quantitative bradykinesia test in MPTP-treated mice. Res Commun Chem Pathol Pharmacol. 50 (3): 435-41doi: http://www.ncbi.nlm.nih.gov/pubmed/3878557.
31. Pike AF, Szabo I, Veerhuis R, and Bubacco L. (2022). The potential convergence of NLRP3 inflammasome, potassium, and dopamine mechanisms in Parkinson's disease. NPJ Parkinsons Dis. 8 (1): 32. https://doi.org/10.1038/s41531-022-00293-z.
32. Pirunkaset E, Boonyarat C, Maneenet J, Khamphukdee C, Daodee S, Monthakantirat O, Awale S, Kijjoa A, and Chulikhit Y. (2024). Effect of Diacetylcurcumin Manganese Complex on Rotenone-Induced Oxidative Stress, Mitochondria Dysfunction, and Inflammation in the SH-SY5Y Parkinson's Disease Cell Model. Molecules. 29 (5). https://doi.org/10.3390/molecules29050957.
33. Ramsay RR, Salach JI, Dadgar J, and Singer TP. (1986). Inhibition of mitochondrial NADH dehydrogenase by pyridine derivatives and its possible relation to experimental and idiopathic parkinsonism. Biochem Biophys Res Commun. 135 (1): 269-75. https://doi.org/10.1016/0006-291x(86)90972-1.
34. Russell SM, Davey J, and Mayer RJ. (1979). The vectorial orientation of human monoamine oxidase in the mitochondrial outer membrane. Biochem J. 181 (1): 7-14. https://doi.org/10.1042/bj1810007.
35. Sanchez-Guajardo V, Barnum CJ, Tansey MG, and Romero-Ramos M. (2013). Neuroimmunological processes in Parkinson's disease and their relation to alpha-synuclein: microglia as the referee between neuronal processes and peripheral immunity. ASN Neuro. 5 (2): 113-39. https://doi.org/10.1042/AN20120066.
36. Scheiblich H, Bousset L, Schwartz S, Griep A, Latz E, Melki R, and Heneka MT. (2021). Microglial NLRP3 Inflammasome Activation upon TLR2 and TLR5 Ligation by Distinct alpha-Synuclein Assemblies. J Immunol. 207 (8): 2143-54. https://doi.org/10.4049/jimmunol.2100035.
37. Schmittgen TD, and Livak KJ. (2008). Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 3 (6): 1101-8. https://doi.org/10.1038/nprot.2008.73.
38. Shao L, Dong C, Geng D, He Q, and Shi Y. (2021). Ginkgolide B protects against cognitive impairment in senescence-accelerated P8 mice by mitigating oxidative stress, inflammation and ferroptosis. Biochem Biophys Res Commun. 572: 7-14. https://doi.org/10.1016/j.bbrc.2021.07.081.
39. Sivagurunathan N, Rahamathulla MP, Al-Dossary H, and Calivarathan L. (2024). Emerging Role of Long Noncoding RNAs in Regulating Inflammasome-Mediated Neurodegeneration in Parkinson's Disease. Mol Neurobiol. 61 (7): 4619-32. https://doi.org/10.1007/s12035-023-03809-7.
40. Spinazzi M, Casarin A, Pertegato V, Salviati L, and Angelini C. (2012). Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells. Nat Protoc. 7 (6): 1235-46. https://doi.org/10.1038/nprot.2012.058.
41. Sun J, Li H, Jin Y, Yu J, Mao S, Su KP, Ling Z, and Liu J. (2021). Probiotic Clostridium butyricum ameliorated motor deficits in a mouse model of Parkinson's disease via gut microbiota-GLP-1 pathway. Brain Behav Immun. 91: 703-15. https://doi.org/10.1016/j.bbi.2020.10.014.
42. Suzuki K, Mizuno Y, and Yoshida M. (1990). Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-like compounds on mitochondrial respiration. Adv Neurol. 53: 215-8doi: http://www.ncbi.nlm.nih.gov/pubmed/2122645.
43. Tillerson JL, Caudle WM, Reveron ME, and Miller GW. (2002). Detection of behavioral impairments correlated to neurochemical deficits in mice treated with moderate doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Exp Neurol. 178 (1): 80-90. https://doi.org/10.1006/exnr.2002.8021.
44. Wu T, Fang X, Xu J, Jiang Y, Cao F, and Zhao L. (2020). Synergistic Effects of Ginkgolide B and Protocatechuic Acid on the Treatment of Parkinson's Disease. Molecules. 25 (17). https://doi.org/10.3390/molecules25173976.
45. Yildizhan K, Cinar R, and Naziroglu M. (2022). The involvement of TRPM2 on the MPP(+)-induced oxidative neurotoxicity and apoptosis in hippocampal neurons from neonatal mice: protective role of resveratrol. Neurol Res. 44 (7): 636-44. https://doi.org/10.1080/01616412.2022.2027644.
46. Yildizhan K, and Naziroglu M. (2020). Glutathione Depletion and Parkinsonian Neurotoxin MPP(+)-Induced TRPM2 Channel Activation Play Central Roles in Oxidative Cytotoxicity and Inflammation in Microglia. Mol Neurobiol. 57 (8): 3508-25. https://doi.org/10.1007/s12035-020-01974-7.
47. Yildizhan K, and Naziroglu M. (2022). Protective role of selenium on MPP(+) and homocysteine-induced TRPM2 channel activation in SH-SY5Y cells. J Recept Signal Transduct Res. 42 (4): 399-408. https://doi.org/10.1080/10799893.2021.1981381.
48. Yıldızhan K, and Nazıroğlu M. (2019). Microglia and its role in neurodegenerative diseases. Journal of Cellular Neuroscience and Oxidative Stress.
49. Zhang QS, Heng Y, Mou Z, Huang JY, Yuan YH, and Chen NH. (2017). Reassessment of subacute MPTP-treated mice as animal model of Parkinson's disease. Acta Pharmacol Sin. 38 (10): 1317-28. https://doi.org/10.1038/aps.2017.49.
50. Zhao TT, Shin KS, Kim KS, Park HJ, Kim HJ, Lee KE, and Lee MK. (2016). Effects of (-)-sesamin on motor and memory deficits in an MPTP-lesioned mouse model of Parkinson's disease treated with l-DOPA. Neuroscience. 339: 644-54. https://doi.org/10.1016/j.neuroscience.2016.10.042.
51. Zhao Y, Xiong S, Liu P, Liu W, Wang Q, Liu Y, Tan H, Chen X, Shi X, Wang Q, and Chen T. (2020). Polymeric Nanoparticles-Based Brain Delivery with Improved Therapeutic Efficacy of Ginkgolide B in Parkinson's Disease. Int J Nanomedicine. 15: 10453-67. https://doi.org/10.2147/IJN.S272831.
52. Zhu JH, Gusdon AM, Cimen H, Van Houten B, Koc E, and Chu CT. (2012). Impaired mitochondrial biogenesis contributes to depletion of functional mitochondria in chronic MPP+ toxicity: dual roles for ERK1/2. Cell Death Dis. 3 (5): e312. https://doi.org/10.1038/cddis.2012.46.