Neurochemical Research

, Volume 38, Issue 1, pp 186–200 | Cite as

Alteration in Glutathione Content and Associated Enzyme Activities in the Synaptic Terminals but not in the Non-synaptic Mitochondria from the Frontal Cortex of Parkinson’s Disease Brains

  • G. Harish
  • Anita Mahadevan
  • M. M. Srinivas Bharath
  • S. K. Shankar
Original Paper


Altered redox dynamics contribute to physiological aging and Parkinson’s disease (PD). This is reflected in the substantia nigra (SN) of PD patients as lowered antioxidant levels and elevated oxidative damage. Contrary to this observation, we previously reported that non-SN regions such as caudate nucleus and frontal cortex (FC) exhibited elevated antioxidants and lowered mitochondrial and oxidative damage indicating constitutive protective mechanisms in PD brains. To investigate whether the sub-cellular distribution of antioxidants could contribute to these protective effects, we examined the distribution of antioxidant/oxidant markers in the neuropil fractions [synaptosomes, non-synaptic mitochondria and cytosol] of FC from PD (n = 9) and controls (n = 8). In the control FC, all the antioxidant activities [Superoxide dismutase (SOD), glutathione (GSH), GSH peroxidase (GPx), GSH-S-transferase (GST)] except glutathione reductase (GR) were the highest in cytosol, but several fold lower in mitochondria and much lower in synaptosomes. However, FC synaptosomes from PD brains had significantly higher levels of GSH (p = 0.01) and related enzymes [GPx (p = 0.02), GR (p = 0.06), GST (p = 0.0001)] compared to controls. Conversely, mitochondria from the FC of PD cases displayed elevated SOD activity (p = 0.02) while the GSH and related enzymes were relatively unaltered. These changes in the neuropil fractions were associated with unchanged or lowered oxidative damage. Further, the mitochondrial content in the synaptosomes of both PD and control brains was ≥five-fold lower compared to the non-synaptic mitochondrial fraction. Altered distribution of oxidant/antioxidant markers in the neuropil fractions of the human brain during aging and PD has implications for (1) degenerative and protective mechanisms (2) distinct antioxidant mechanisms in synaptic terminals compared to other compartments.


Parkinson’s disease Frontal cortex Neuropil Synaptosomes Mitochondria Oxidative stress Glutathione 



Parkinson’s disease




Frontal cortex


Glutathione reduced


Postmortem interval




Glial fibrillary acidic protein


Superoxide Dismutase




Glutathione reductase


Xglutathione peroxidase

ns mito

Non-synaptic mitochondria

syn mito

Synaptic mitochondria




Citrate synthase


Malate dehydrogenase


Succinate dehydrogenase


3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide


  1. 1.
    Hoehn MM, Yahr MD (1967) Parkinsonism: onset, progression and mortality. Neurology 17(5):427–442PubMedCrossRefGoogle Scholar
  2. 2.
    Adams JD Jr, Chang ML, Klaidman L (2001) Parkinson’s disease–redox mechanisms. Curr Med Chem 8(7):809–814PubMedCrossRefGoogle Scholar
  3. 3.
    Beal MF (1992) Does impairment of energy metabolism result in excitotoxic neuronal death in neurodegenerative illnesses? Ann Neurol 31(2):119–130. doi:10.1002/ana.410310202 PubMedCrossRefGoogle Scholar
  4. 4.
    Jenner P, Dexter DT, Sian J, Schapira AH, Marsden CD (1992) Oxidative stress as a cause of nigral cell death in Parkinson’s disease and incidental Lewy body disease. The Royal Kings and Queens Parkinson’s Disease Research Group. Ann Neurol 32(Suppl):S82–S87PubMedCrossRefGoogle Scholar
  5. 5.
    Sayre LM, Smith MA, Perry G (2001) Chemistry and biochemistry of oxidative stress in neurodegenerative disease. Curr Med Chem 8(7):721–738PubMedCrossRefGoogle Scholar
  6. 6.
    Bharath S, Hsu M, Kaur D, Rajagopalan S, Andersen JK (2002) Glutathione, iron and Parkinson’s disease. Biochem Pharmacol 64(5–6):1037–1048. doi:10.1016/S0006-2952(02)01174-7 PubMedCrossRefGoogle Scholar
  7. 7.
    Alam ZI, Daniel SE, Lees AJ, Marsden DC, Jenner P, Halliwell B (1997) A generalised increase in protein carbonyls in the brain in Parkinson’s but not incidental Lewy body disease. J Neurochem 69(3):1326–1329PubMedCrossRefGoogle Scholar
  8. 8.
    Dexter DT, Wells FR, Lees AJ, Agid F, Agid Y, Jenner P, Marsden CD (1989) Increased nigral iron content and alterations in other metal ions occurring in brain in Parkinson’s disease. J Neurochem 52(6):1830–1836PubMedCrossRefGoogle Scholar
  9. 9.
    Floor E, Wetzel MG (1998) Increased protein oxidation in human substantia nigra pars compacta in comparison with basal ganglia and prefrontal cortex measured with an improved dinitrophenylhydrazine assay. J Neurochem 70(1):268–275PubMedCrossRefGoogle Scholar
  10. 10.
    Good PF, Hsu A, Werner P, Perl DP, Olanow CW (1998) Protein nitration in Parkinson’s disease. J Neuropathol Exp Neurol 57(4):338–342PubMedCrossRefGoogle Scholar
  11. 11.
    Sofic E, Paulus W, Jellinger K, Riederer P, Youdim MB (1991) Selective increase of iron in substantia nigra zona compacta of parkinsonian brains. J Neurochem 56(3):978–982PubMedCrossRefGoogle Scholar
  12. 12.
    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(2):381–389PubMedCrossRefGoogle Scholar
  13. 13.
    Yoritaka A, Hattori N, Uchida K, Tanaka M, Stadtman ER, Mizuno Y (1996) Immunohistochemical detection of 4-hydroxynonenal protein adducts in Parkinson disease. Proc Natl Acad Sci USA 93(7):2696–2701PubMedCrossRefGoogle Scholar
  14. 14.
    Alam ZI, Jenner A, Daniel SE, Lees AJ, Cairns N, Marsden CD, Jenner P, Halliwell B (1997) Oxidative DNA damage in the parkinsonian brain: an apparent selective increase in 8-hydroxyguanine levels in substantia nigra. J Neurochem 69(3):1196–1203PubMedCrossRefGoogle Scholar
  15. 15.
    Keeney PM, Xie J, Capaldi RA, Bennett JP Jr (2006) Parkinson’s disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled. J Neurosci 26(19):5256–5264. doi:10.1523/JNEUROSCI.0984-06.2006 PubMedCrossRefGoogle Scholar
  16. 16.
    Schapira AH, Cooper JM, Dexter D, Clark JB, Jenner P, Marsden CD (1990) Mitochondrial complex I deficiency in Parkinson’s disease. J Neurochem 54(3):823–827PubMedCrossRefGoogle Scholar
  17. 17.
    Braak H, Del Tredici K, Rub U, de Vos RA, Jansen Steur EN, Braak E (2003) Staging of brain pathology related to sporadic Parkinson’s disease. Neurobiol Aging 24(2):197–211PubMedCrossRefGoogle Scholar
  18. 18.
    Mythri RB, Venkateshappa C, Harish G, Mahadevan A, Muthane UB, Yasha TC, Srinivas Bharath MM, Shankar SK (2011) Evaluation of markers of oxidative stress, antioxidant function and astrocytic proliferation in the striatum and frontal cortex of Parkinson’s disease brains. Neurochem Res 36(8):1452–1463. doi:10.1007/s11064-011-0471-9 PubMedCrossRefGoogle Scholar
  19. 19.
    Mattson MP, Furukawa K (1998) Signaling events regulating the neurodevelopmental triad. Glutamate and secreted forms of beta-amyloid precursor protein as examples. Perspect Dev Neurobiol 5(4):337–352PubMedGoogle Scholar
  20. 20.
    Brown MR, Sullivan PG, Geddes JW (2006) Synaptic mitochondria are more susceptible to Ca2+ overload than nonsynaptic mitochondria. J Biol Chem 281(17):11658–11668. doi:10.1074/jbc.M510303200 PubMedCrossRefGoogle Scholar
  21. 21.
    Naga KK, Sullivan PG, Geddes JW (2007) High cyclophilin D content of synaptic mitochondria results in increased vulnerability to permeability transition. J Neurosci 27(28):7469–7475. doi:10.1523/jneurosci.0646-07.2007 PubMedCrossRefGoogle Scholar
  22. 22.
    Chandana R, Mythri RB, Mahadevan A, Shankar SK, Srinivas Bharath MM (2009) Biochemical analysis of protein stability in human brain collected at different post-mortem intervals. Indian J Med Res 129(2):189–199PubMedGoogle Scholar
  23. 23.
    Chinta SJ, Kommaddi RP, Turman CM, Strobel HW, Ravindranath V (2005) Constitutive expression and localization of cytochrome P-450 1A1 in rat and human brain: presence of a splice variant form in human brain. J Neurochem 93(3):724–736. doi:10.1111/j.1471-4159.2005.03061.x PubMedCrossRefGoogle Scholar
  24. 24.
    Cash AD, Perry G, Ogawa O, Raina AK, Zhu X, Smith MA (2002) Is Alzheimer’s disease a mitochondrial disorder? Neurosci Rev J Brin Neurobiol Neurol Psychiatry 8(5):489–496Google Scholar
  25. 25.
    Onyango I, Khan S, Miller B, Swerdlow R, Trimmer P, Bennett P Jr (2006) Mitochondrial genomic contribution to mitochondrial dysfunction in Alzheimer’s disease. J Alzheimers Dis 9(2):183–193PubMedGoogle Scholar
  26. 26.
    Reddy PH, Beal MF (2005) Are mitochondria critical in the pathogenesis of Alzheimer’s disease? Brain Res Brain Res Rev 49(3):618–632. doi:10.1016/j.brainresrev.2005.03.004 PubMedCrossRefGoogle Scholar
  27. 27.
    Forero DA, Casadesus G, Perry G, Arboleda H (2006) Synaptic dysfunction and oxidative stress in Alzheimer’s disease: emerging mechanisms. J Cell Mol Med 10(3):796–805PubMedCrossRefGoogle Scholar
  28. 28.
    Urano S, Asai Y, Makabe S, Matsuo M, Izumiyama N, Ohtsubo K, Endo T (1997) Oxidative injury of synapse and alteration of antioxidative defense systems in rats, and its prevention by vitamin E. Eur J Biochem 245(1):64–70PubMedCrossRefGoogle Scholar
  29. 29.
    Martinez M, Ferrandiz ML, Diez A, Miquel J (1995) Depletion of cytosolic GSH decreases the ATP levels and viability of synaptosomes from aged mice but not from young mice. Mech Ageing Dev 84(1):77–81PubMedCrossRefGoogle Scholar
  30. 30.
    Karunakaran S, Saeed U, Ramakrishnan S, Koumar RC, Ravindranath V (2007) Constitutive expression and functional characterization of mitochondrial glutaredoxin (Grx2) in mouse and human brain. Brain Res 1185:8–17. doi:10.1016/j.brainres.2007.09.019 PubMedCrossRefGoogle Scholar
  31. 31.
    Karunakaran S, Saeed U, Mishra M, Valli RK, Joshi SD, Meka DP, Seth P, Ravindranath V (2008) Selective activation of p38 mitogen-activated protein kinase in dopaminergic neurons of substantia nigra leads to nuclear translocation of p53 in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice. J Neurosci 28(47):12500–12509. doi:10.1523/jneurosci.4511-08.2008 PubMedCrossRefGoogle Scholar
  32. 32.
    Alladi PA, Mahadevan A, Yasha TC, Raju TR, Shankar SK, Muthane U (2009) Absence of age-related changes in nigral dopaminergic neurons of Asian Indians: relevance to lower incidence of Parkinson’s disease. Neuroscience 159(1):236–245. doi:10.1016/j.neuroscience.2008.11.051 PubMedCrossRefGoogle Scholar
  33. 33.
    Douglas MS (1983) Rapid, quantitative isolation of mitochondria from rat liver using Ficoll gradients in vertical rotors. Anal Biochem 131(2):453–457. doi:10.1016/0003-2697(83)90198-7 CrossRefGoogle Scholar
  34. 34.
    Frasca JM, Parks VR (1965) A routine technique for double-staining ultrathin sections using uranyl and lead salts. J Cell Biol 25:157–161PubMedCrossRefGoogle Scholar
  35. 35.
    Harish G, Venkateshappa C, Mahadevan A, Pruthi N, Bharath MM, Shankar SK (2012) Mitochondrial function in human brains is affected by pre and postmortem factors. Neuropathol Appl Neurobiol. doi:10.1111/j.1365-2990.2012.01285.x PubMedGoogle Scholar
  36. 36.
    Harish G, Venkateshappa C, Mahadevan A, Pruthi N, Bharath MM, Shankar SK (2012) Effect of pre and postmortem factors on the distribution and preservation of antioxidant activities in the cytosol and synaptosomes of human brains. Biopreserv Biobank 10(3):253–265. doi:10.1089/bio.2012.0001
  37. 37.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  38. 38.
    Jagatha B, Mythri RB, Vali S, Bharath MM (2008) Curcumin treatment alleviates the effects of glutathione depletion in vitro and in vivo: therapeutic implications for Parkinson’s disease explained via in silico studies. Free Radic Biol Med 44(5):907–917. doi:10.1016/j.freeradbiomed.2007.11.011 PubMedCrossRefGoogle Scholar
  39. 39.
    Bagnyukova TV, Storey KB, Lushchak VI (2003) Induction of oxidative stress in Rana ridibunda during recovery from winter hibernation. J Therm Biol 28:21–28CrossRefGoogle Scholar
  40. 40.
    Tietze F (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 27(3):502–522PubMedCrossRefGoogle Scholar
  41. 41.
    Hissin PJ, Hilf R (1976) A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem 74(1):214–226PubMedCrossRefGoogle Scholar
  42. 42.
    Flohe L, Gunzler WA (1984) Assays of glutathione peroxidase. Methods Enzymol 105:114–121PubMedCrossRefGoogle Scholar
  43. 43.
    Carlberg I, Mannervik B (1985) Glutathione reductase. Methods Enzymol 113:484–490PubMedCrossRefGoogle Scholar
  44. 44.
    Guthenberg C, Alin P, Mannervik B (1985) Glutathione transferase from rat testis. Methods Enzymol 113:507–510PubMedCrossRefGoogle Scholar
  45. 45.
    Cohen G, Farooqui R, Kesler N (1997) Parkinson disease: a new link between monoamine oxidase and mitochondrial electron flow. Proc Natl Acad Sci USA 94(10):4890–4894PubMedCrossRefGoogle Scholar
  46. 46.
    Srere PA (1969) [1] Citrate synthase: [EC Citrate oxaloacetate-lyase (CoA-acetylating)]. In: John ML (ed) Methods in enzymology, Vol 13. Academic Press, Waltham, pp 3–11Google Scholar
  47. 47.
    Pennington RJ (1961) Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. Biochem J 80:649–654PubMedGoogle Scholar
  48. 48.
    Kitto GB, Lewis RG (1967) Purification and properties of tuna supernatant and mitochondrial malate dehydrogenases. Biochimica et Biophysica Acta (BBA)-Enzymol 139(1):1–15. doi:10.1016/0005-2744(67)90107-6 CrossRefGoogle Scholar
  49. 49.
    Albers DS, Beal MF (2000) Mitochondrial dysfunction and oxidative stress in aging and neurodegenerative disease. J Neural Transm Suppl 59:133–154PubMedGoogle Scholar
  50. 50.
    Banerjee R, Starkov AA, Beal MF, Thomas B (2009) Mitochondrial dysfunction in the limelight of Parkinson’s disease pathogenesis. Biochim Biophys Acta 1792(7):651–663. doi:10.1016/j.bbadis.2008.11.007 PubMedCrossRefGoogle Scholar
  51. 51.
    Lin MT, Beal MF (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443(7113):787–795. doi:10.1038/nature05292 PubMedCrossRefGoogle Scholar
  52. 52.
    Choi SW, Gerencser AA, Lee DW, Rajagopalan S, Nicholls DG, Andersen JK, Brand MD (2011) Intrinsic bioenergetic properties and stress sensitivity of dopaminergic synaptosomes. J Neurosci 31(12):4524–4534. doi:10.1523/jneurosci.5817-10.2011 PubMedCrossRefGoogle Scholar
  53. 53.
    Ansari MA, Scheff SW (2010) Oxidative stress in the progression of Alzheimer disease in the frontal cortex. J Neuropathol Exp Neurol 69(2):155–167. doi:10.1097/NEN.0b013e3181cb5af4 PubMedCrossRefGoogle Scholar
  54. 54.
    Pocernich CB, Cardin AL, Racine CL, Lauderback CM, Butterfield DA (2001) Glutathione elevation and its protective role in acrolein-induced protein damage in synaptosomal membranes: relevance to brain lipid peroxidation in neurodegenerative disease. Neurochem Int 39(2):141–149PubMedCrossRefGoogle 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):39–47PubMedCrossRefGoogle Scholar
  56. 56.
    Schnellmann RG, Gilchrist SM, Mandel LJ (1988) Intracellular distribution and depletion of glutathione in rabbit renal proximal tubules. Kidney Int 34(2):229–233PubMedCrossRefGoogle Scholar
  57. 57.
    Radunovic A, Porto WG, Zeman S, Leigh PN (1997) Increased mitochondrial superoxide dismutase activity in Parkinson’s disease but not amyotrophic lateral sclerosis motor cortex. Neurosci Lett 239(2–3):105–108PubMedCrossRefGoogle Scholar
  58. 58.
    Harish G, Venkateshappa C, Mahadevan A, Pruthi N, Bharath MMS, Shankar SK (2011) Effect of storage time, postmortem interval, agonal state, and gender on the postmortem preservation of glial fibrillary acidic protein and oxidatively damaged proteins in human brains. Biopreserv Biobank 9(4):379–387. doi:10.1089/bio.2011.0033 CrossRefGoogle Scholar
  59. 59.
    Harish G, Venkateshappa C, Mahadevan A, Pruthi N, Srinivas Bharath MM, Shankar SK (2011) Glutathione metabolism is modulated by postmortem interval, gender difference and agonal state in postmortem human brains. Neurochem Int 59(7):1029–1042. doi:10.1016/j.neuint.2011.08.024 PubMedCrossRefGoogle Scholar
  60. 60.
    Gomez A, Ferrer I (2010) Involvement of the cerebral cortex in Parkinson disease linked with G2019S LRRK2 mutation without cognitive impairment. Acta Neuropathol 120(2):155–167. doi:10.1007/s00401-010-0669-y PubMedCrossRefGoogle Scholar
  61. 61.
    Riederer BM (1989) Antigen preservation tests for immunocytochemical detection of cytoskeletal proteins: influence of aldehyde fixatives. J Histochem Cytochem 37(5):675–681PubMedCrossRefGoogle Scholar
  62. 62.
    Sofic E, Lange KW, Jellinger K, Riederer P (1992) Reduced and oxidized glutathione in the substantia nigra of patients with Parkinson’s disease. Neurosci Lett 142(2):128–130PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • G. Harish
    • 1
  • Anita Mahadevan
    • 2
  • M. M. Srinivas Bharath
    • 1
  • S. K. Shankar
    • 2
  1. 1.Department of NeurochemistryNational Institute of Mental Health and NeurosciencesBangaloreIndia
  2. 2.Department of NeuropathologyNational Institute of Mental Health and NeurosciencesBangaloreIndia

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