Glutathione Depletion and Parkinsonian Neurotoxin MPP+-Induced TRPM2 Channel Activation Play Central Roles in Oxidative Cytotoxicity and Inflammation in Microglia


Parkinson’s disease (PD) is one of most common neurodegenerative diseases. Environmental stressors such as oxidative stress (OS), calcium ion influx, apoptosis, and inflammation mechanisms are linked to activated microglia in patients with PD. The OS-dependent activated transient receptor potential melastatin 2 (TRPM2) channel is modulated in several neurons by glutathione (GSH). However, the cellular and molecular effects of GSH alteration on TRPM2 activation, OS, apoptosis, and inflammation in the microglia remain elusive. The microglia of TRPM2 wild-type (TRPM2-WT) and knockout (TRPM2-KO) mice were divided into control, PD model (MPP), l-buthionine sulfoximine (BSO), MPP + BSO and MPP + BSO + GSH groups. MPP-induced increases in apoptosis, death, OS, lipid peroxidation, PARP1, caspase-3 and caspase-9, inflammatory cytokines (IL-1β, TNF-α, IL-6), and intracellular free Zn2+ and Ca2+ levels in the microglia of TRPM2-WT mice were further increased by the BSO treatment, although they were diminished by the GSH treatment. Their levels were further reduced by PARP1 inhibitors (PJ34 and DPQ) and TRPM2 blockers (ACA and 2-APB). However, the effects of MPP and BSO were not observed in the microglia of TRPM2-KO mice. Taken together, our data demonstrate that maintaining GSH homeostasis is not only important for quenching OS in the microglia of patients with PD but also equally critical to modulating TRPM2, thus suppressing inflammatory responses elicited by environmental stressors.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8


[Ca2+]i :

Intracellular free calcium ion


2-Aminoethoxydiphenyl borate


N-(p-Amylcinnamoyl) anthranilic acid






Dorsal root ganglion








Oxidative stress


Poly[ADP-ribose] polymerase 1


Parkinson’s disease


Transient receptor potential melastatin 2


TRPM2 knockout


TRPM2 wild type


Voltage-gated calcium channels


  1. 1.

    Simon DK, Tanner CM, Brundin P (2020) Parkinson disease epidemiology, pathology, genetics, and pathophysiology. Clin Geriatr Med 36(1):1–12.

    Article  PubMed  Google Scholar 

  2. 2.

    Massaquoi MS, Liguore WA, Churchill MJ, Moore C, Melrose HL, Meshul CK (2020) Gait deficits and loss of striatal tyrosine hydroxlase/trk-b are restored following 7,8-dihydroxyflavone treatment in a progressive MPTP mouse model of Parkinson’s disease. Neuroscience. 433:53–71.

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Tamilselvam K, Braidy N, Manivasagam T, Essa MM, Prasad NR, Karthikeyan S, Thenmozhi AJ, Selvaraju S et al (2013) Neuroprotective effects of hesperidin, a plant flavanone, on rotenone-induced oxidative stress and apoptosis in a cellular model for Parkinson’s disease. Oxidative Med Cell Longev 2013:102741–102711.

    CAS  Article  Google Scholar 

  4. 4.

    Macchi B, Di Paola R, Marino-Merlo F, Felice MR, Cuzzocrea S, Mastino A (2015) Inflammatory and cell death pathways in brain and peripheral blood in Parkinson’s disease. CNS Neurol Disord Drug Targets 14(3):313–324.

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Nazıroglu M, Oz A, Yildizhan K (2020) Selenium and neurological diseases: focus on peripheral pain and TRP channels. Curr Neuropharmacol 18.

  6. 6.

    Kierdorf K, Prinz M (2013) Factors regulating microglia activation. Front Cell Neurosci 7:44.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Gutmann DH, Kettenmann H (2019) Microglia/brain macrophages as central drivers of brain tumor pathobiology. Neuron 104(3):442–449.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Zhang S, Wang R, Wang G (2019) Impact of dopamine oxidation on dopaminergic neurodegeneration. ACS Chem Neurosci 10(2):945–953.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Yoshioka Y, Sugino Y, Shibagaki F, Yamamuro A, Ishimaru Y, Maeda S (2020) Dopamine attenuates lipopolysaccharide-induced expression of proinflammatory cytokines by inhibiting the nuclear translocation of NF-kappaB p65 through the formation of dopamine quinone in microglia. Eur J Pharmacol 866:172826.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    de Araujo FM, Ferreira RS, Souza CS, Dos Santos CC, Rodrigues T, JHC ES, Gasparotto J, Gelain DP et al (2018) Aminochrome decreases NGF, GDNF and induces neuroinflammation in organotypic midbrain slice cultures. Neurotoxicology 66:98–106.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Zhang J, Culp ML, Craver JG, Darley-Usmar V (2018) Mitochondrial function and autophagy: integrating proteotoxic, redox, and metabolic stress in Parkinson’s disease. J Neurochem 144(6):691–709.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Bonilla-Porras AR, Jimenez-Del-Rio M, Velez-Pardo C (2019) N-Acetyl-cysteine blunts 6-hydroxydopamine- and L-buthionine-sulfoximine-induced apoptosis in human mesenchymal stromal cells. Mol Biol Rep 46(4):4423–4435.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Yang SJ, Yang JW, Na JM, Ha JS, Choi SY, Cho SW (2018) 3-(Naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride attenuates MPP-induced cytotoxicity by regulating oxidative stress and mitochondrial dysfunction in SH-SY5Y cells. BMB Rep 51(11):590–595

    CAS  Article  Google Scholar 

  14. 14.

    Korvers L, de Andrade Costa A, Mersch M, Matyash V, Kettenmann H, Semtner M (2016) Spontaneous Ca(2+) transients in mouse microglia. Cell Calcium 60(6):396–406.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Nazıroğlu M, Luckhoff A (2008) Effects of antioxidants on calcium influx through TRPM2 channels in transfected cells activated by hydrogen peroxide. J Neurol Sci 270(1–2):152–158.

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Hara Y, Wakamori M, Ishii M, Maeno E, Nishida M, Yoshida T, Yamada H, Shimizu S et al (2002) LTRPC2 Ca2+-permeable channel activated by changes in redox status confers susceptibility to cell death. Mol Cell 9(1):163–173.

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Nazıroglu M (2007) New molecular mechanisms on the activation of TRPM2 channels by oxidative stress and ADP-ribose. Neurochem Res 32(11):1990–2001.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Nazıroglu M (2012) Molecular role of catalase on oxidative stress-induced Ca(2+) signaling and TRP cation channel activation in nervous system. J Recept Signal Transduct Res 32(3):134–141.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Bak DW, Weerapana E (2015) Cysteine-mediated redox signalling in the mitochondria. Mol BioSyst 11(3):678–697.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Ovey IS, Naziroglu M (2015) Homocysteine and cytosolic GSH depletion induce apoptosis and oxidative toxicity through cytosolic calcium overload in the hippocampus of aged mice: involvement of TRPM2 and TRPV1 channels. Neuroscience 284:225–233.

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Belrose JC, Xie YF, Gierszewski LJ, MacDonald JF, Jackson MF (2012) Loss of glutathione homeostasis associated with neuronal senescence facilitates TRPM2 channel activation in cultured hippocampal pyramidal neurons. Mol Brain 5:11.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Ozgul C, Naziroglu M (2012) TRPM2 channel protective properties of N-acetylcysteine on cytosolic glutathione depletion dependent oxidative stress and Ca2+ influx in rat dorsal root ganglion. Physiol Behav 106(2):122–128.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Nazıroğlu M, Ozgul C, Cig B, Dogan S, Uguz AC (2011) Glutathione modulates Ca(2+) influx and oxidative toxicity through TRPM2 channel in rat dorsal root ganglion neurons. J Membr Biol 242(3):109–118.

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Lee M, Cho T, Jantaratnotai N, Wang YT, McGeer E, McGeer PL (2010) Depletion of GSH in glial cells induces neurotoxicity: relevance to aging and degenerative neurological diseases. FASEB J 24(7):2533–2545.

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Dauer W, Przedborski S (2003) Parkinson’s disease: mechanisms and models. Neuron 39(6):889–909

    CAS  Article  Google Scholar 

  26. 26.

    Yurekli VA, Gurler S, Nazıroğlu M, Uguz AC, Koyuncuoglu HR (2013) Zonisamide attenuates MPP+-induced oxidative toxicity through modulation of Ca2+ signaling and caspase-3 activity in neuronal PC12 cells. Cell Mol Neurobiol 33(2):205–212.

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Peng Z, Luchtman DW, Wang X, Zhang Y, Song C (2019) Activation of microglia synergistically enhances neurodegeneration caused by MPP(+) in human SH-SY5Y cells. Eur J Pharmacol 850:64–74.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Canals S, Casarejos MJ, de Bernardo S, Rodriguez-Martin E, Mena MA (2001) Glutathione depletion switches nitric oxide neurotrophic effects to cell death in midbrain cultures: implications for Parkinson’s disease. J Neurochem 79(6):1183–1195.

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Lee M, Kwon BM, Suk K, McGeer E, McGeer PL (2012) Effects of obovatol on GSH depleted glia-mediated neurotoxicity and oxidative damage. J NeuroImmune Pharmacol 7(1):173–186.

    Article  PubMed  Google Scholar 

  30. 30.

    Akhtar F, Rouse CA, Catano G, Montalvo M, Ullevig SL, Asmis R, Kharbanda K, Maffi SK (2017) Acute maternal oxidant exposure causes susceptibility of the fetal brain to inflammation and oxidative stress. J Neuroinflammation 14(1):195.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Ni M, Aschner M (2010) Neonatal rat primary microglia: isolation, culturing, and selected applications. Curr Protoc Toxicol Chapter 12: Unit 12 17.

  32. 32.

    Mortadza SS, Sim JA, Stacey M, Jiang LH (2017) Signalling mechanisms mediating Zn(2+)-induced TRPM2 channel activation and cell death in microglial cells. Sci Rep 7:45032.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Gordon R, Hogan CE, Neal ML, Anantharam V, Kanthasamy AG, Kanthasamy A (2011) A simple magnetic separation method for high-yield isolation of pure primary microglia. J Neurosci Methods 194(2):287–296.

    Article  PubMed  Google Scholar 

  34. 34.

    Yao S, Li L, Sun X, Hua J, Zhang K, Hao L, Liu L, Shi D et al (2019) FTY720 inhibits MPP(+)-induced microglial activation by affecting NLRP3 inflammasome activation. J NeuroImmune Pharmacol 14(3):478–492.

    Article  PubMed  Google Scholar 

  35. 35.

    Ozkaya D, Naziroglu M (2020) Curcumin diminishes cisplatin-induced apoptosis and mitochondrial oxidative stress through inhibition of TRPM2 channel signaling pathway in mouse optic nerve. J Recept Signal Transduct Res 40(2):97–108.

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Guzman JN, Ilijic E, Yang B, Sanchez-Padilla J, Wokosin D, Galtieri D, Kondapalli J, Schumacker PT et al (2018) Systemic isradipine treatment diminishes calcium-dependent mitochondrial oxidant stress. J Clin Invest 128(6):2266–2280.

    Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    An X, Fu Z, Mai C, Wang W, Wei L, Li D, Li C, Jiang LH (2019) Increasing the TRPM2 channel expression in human neuroblastoma SH-SY5Y cells augments the susceptibility to ROS-induced cell death. Cells 8(1).

  38. 38.

    Ataizi ZS, Ertilav K, Naziroglu M (2019) Mitochondrial oxidative stress-induced brain and hippocampus apoptosis decrease through modulation of caspase activity, Ca(2+) influx and inflammatory cytokine molecular pathways in the docetaxel-treated mice by melatonin and selenium treatments. Metab Brain Dis 34(4):1077–1089.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Yazgan Y, Naziroglu M (2017) Ovariectomy-induced mitochondrial oxidative stress, apoptosis, and calcium ion influx through TRPA1, TRPM2, and TRPV1 are prevented by 17beta-estradiol, tamoxifen, and raloxifene in the hippocampus and dorsal root ganglion of rats. Mol Neurobiol 54(10):7620–7638.

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Joshi DC, Bakowska JC (2011) Determination of mitochondrial membrane potential and reactive oxygen species in live rat cortical neurons. J Vis Exp 51.

  41. 41.

    Keil VC, Funke F, Zeug A, Schild D, Muller M (2011) Ratiometric high-resolution imaging of JC-1 fluorescence reveals the subcellular heterogeneity of astrocytic mitochondria. Pflugers Arch 462(5):693–708.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Zhou ZD, Lim TM (2010) Glutathione conjugates with dopamine-derived quinones to form reactive or non-reactive glutathione-conjugates. Neurochem Res 35(11):1805–1818.

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    Li X, Jiang LH (2018) Multiple molecular mechanisms form a positive feedback loop driving amyloid beta42 peptide-induced neurotoxicity via activation of the TRPM2 channel in hippocampal neurons. Cell Death Dis 9(2):195.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Hanamsagar R, Bilbo SD (2017) Environment matters: microglia function and dysfunction in a changing world. Curr Opin Neurobiol 47:146–155.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Fonfria E, Marshall IC, Benham CD, Boyfield I, Brown JD, Hill K, Hughes JP, Skaper SD et al (2004) TRPM2 channel opening in response to oxidative stress is dependent on activation of poly(ADP-ribose) polymerase. Br J Pharmacol 143(1):186–192.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Zhu T, Zhao Y, Hu H, Zheng Q, Luo X, Ling Y, Ying Y, Shen Z et al (2019) TRPM2 channel regulates cytokines production in astrocytes and aggravates brain disorder during lipopolysaccharide-induced endotoxin sepsis. Int Immunopharmacol 75:105836.

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    Aminzadeh M, Roghani M, Sarfallah A, Riazi GH (2018) TRPM2 dependence of ROS-induced NLRP3 activation in Alzheimer’s disease. Int Immunopharmacol 54:78–85.

    CAS  Article  PubMed  Google Scholar 

  48. 48.

    Moreno-Garcia ME, Sumoza-Toledo A, Lund FE, Santos-Argumedo L (2005) Localization of CD38 in murine B lymphocytes to plasma but not intracellular membranes. Mol Immunol 42(6):703–711.

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    Kraft R, Grimm C, Grosse K, Hoffmann A, Sauerbruch S, Kettenmann H, Schultz G, Harteneck C (2004) Hydrogen peroxide and ADP-ribose induce TRPM2-mediated calcium influx and cation currents in microglia. Am J Physiol Cell Physiol 286(1):C129–C137.

    CAS  Article  PubMed  Google Scholar 

  50. 50.

    Kraft R, Grimm C, Frenzel H, Harteneck C (2006) Inhibition of TRPM2 cation channels by N-(p-amylcinnamoyl)anthranilic acid. Br J Pharmacol 148(3):264–273.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. 51.

    McNaught KS, Jenner P (2000) Extracellular accumulation of nitric oxide, hydrogen peroxide, and glutamate in astrocytic cultures following glutathione depletion, complex I inhibition, and/or lipopolysaccharide-induced activation. Biochem Pharmacol 60(7):979–988.

    CAS  Article  PubMed  Google Scholar 

  52. 52.

    Thomas B, Banerjee R, Starkova NN, Zhang SF, Calingasan NY, Yang L, Wille E, Lorenzo BJ et al (2012) Mitochondrial permeability transition pore component cyclophilin D distinguishes nigrostriatal dopaminergic death paradigms in the MPTP mouse model of Parkinson’s disease. Antioxid Redox Signal 16(9):855–868.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Diaz-Hung ML, Yglesias-Rivera A, Hernandez-Zimbron LF, Orozco-Suarez S, Ruiz-Fuentes JL, Diaz-Garcia A, Leon-Martinez R, Blanco-Lezcano L et al (2016) Transient glutathione depletion in the substantia nigra compacta is associated with neuroinflammation in rats. Neuroscience 335:207–220.

    CAS  Article  PubMed  Google Scholar 

  54. 54.

    Franco R, Cidlowski JA (2009) Apoptosis and glutathione: beyond an antioxidant. Cell Death Differ 16(10):1303–1314.

    CAS  Article  PubMed  Google Scholar 

  55. 55.

    Roychowdhury S, Wolf G, Keilhoff G, Horn TF (2003) Cytosolic and mitochondrial glutathione in microglial cells are differentially affected by oxidative/nitrosative stress. Nitric Oxide 8(1):39–47.

    CAS  Article  PubMed  Google Scholar 

  56. 56.

    Sun Y, Sukumaran P, Selvaraj S, Cilz NI, Schaar A, Lei S, Singh BB (2018) TRPM2 promotes neurotoxin MPP(+)/MPTP-induced cell death. Mol Neurobiol 55(1):409–420.

    CAS  Article  PubMed  Google Scholar 

  57. 57.

    Togashi K, Inada H, Tominaga M (2008) Inhibition of the transient receptor potential cation channel TRPM2 by 2-aminoethoxydiphenyl borate (2-APB). Br J Pharmacol 153(6):1324–1330.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Gershkovitz M, Caspi Y, Fainsod-Levi T, Katz B, Michaeli J, Khawaled S, Lev S, Polyansky L et al (2018) TRPM2 mediates neutrophil killing of disseminated tumor cells. Cancer Res 78(10):2680–2690.

    CAS  Article  PubMed  Google Scholar 

  59. 59.

    Dringen R (2000) Metabolism and functions of glutathione in brain. Prog Neurobiol 62(6):649–671.

    CAS  Article  PubMed  Google Scholar 

  60. 60.

    Chatterjee S, Noack H, Possel H, Keilhoff G, Wolf G (1999) Glutathione levels in primary glial cultures: monochlorobimane provides evidence of cell type-specific distribution. Glia 27(2):152–161

    CAS  Article  Google Scholar 

  61. 61.

    Kumari A, Singh KP, Mandal A, Paswan RK, Sinha P, Das P, Ali V, Bimal S et al (2017) Intracellular zinc flux causes reactive oxygen species mediated mitochondrial dysfunction leading to cell death in Leishmania donovani. PLoS One 12(6):e0178800.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Zysk M, Gapys B, Ronowska A, Gul-Hinc S, Erlandsson A, Iwanicki A, Sakowicz-Burkiewicz M, Szutowicz A et al (2018) Protective effects of voltage-gated calcium channel antagonists against zinc toxicity in SN56 neuroblastoma cholinergic cells. PLoS One 13(12):e0209363.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Maret W (2019) The redox biology of redox-inert zinc ions. Free Radic Biol Med 134:311–326.

    CAS  Article  PubMed  Google Scholar 

Download references


The patch-clamp and LSC microscope analyses of the current study were from BSN Health, Analyses, Innovation, Consultancy, Organization, Agriculture and Industry Ltd., (Göller Bölgesi Teknokenti, Isparta, Turkey) by MN. Results of the current study were summarized from a PhD thesis of Kenan Yıldızhan.


The study was supported by Scientific Project Unit (BAP) of SDU, Isparta, Turkey (Project No: TDK-2019-7321. The coordinator of the project was Prof. Dr. Mustafa Nazıroğlu). There is no financial disclosure of the current study.

Author information




MN and KE formulated the hypothesis and MN were responsible for writing the report. KY was responsible for isolating the microglia and analyzing the intracellular Ca2+ concentration. MN was responsible for the LSC microscope analyses. KE was also responsible from plate reader analyses.

Corresponding author

Correspondence to Mustafa Nazıroğlu.

Ethics declarations

This article does not contain any studies with human participants performed by any of the authors. This study was approved by the Local Ethical Committee of Burdur Mehmet Akif University (BMAU), Burdur, Turkey (date: 15.05.2019, permit number: 2019-521). The mice were cared in accordance with the guidelines of the Animal Care Committee of BMAU.

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yıldızhan, K., Nazıroğlu, M. Glutathione Depletion and Parkinsonian Neurotoxin MPP+-Induced TRPM2 Channel Activation Play Central Roles in Oxidative Cytotoxicity and Inflammation in Microglia. Mol Neurobiol 57, 3508–3525 (2020).

Download citation


  • Apoptosis
  • Glutathione depletion
  • Oxidative stress
  • Parkinson’s disease
  • TRPM2 channel