Neurochemical Research

, Volume 35, Issue 10, pp 1575–1587

Liposomal-Glutathione Provides Maintenance of Intracellular Glutathione and Neuroprotection in Mesencephalic Neuronal Cells

  • Gail D. Zeevalk
  • Laura P. Bernard
  • F. T. Guilford
Original Paper

Abstract

A liposomal preparation of glutathione (GSH) was investigated for its ability to replenish intracellular GSH and provide neuroprotection in an in vitro model of Parkinson’s disease using paraquat plus maneb (PQMB) in rat mesencephalic cultures. In mixed neuronal/glial cultures depleted of intracellular GSH, repletion to control levels occurred over 4 h with liposomal-GSH or non-liposomal-GSH however, liposomal-GSH was 100-fold more potent; EC50s 4.75 μM and 533 μM for liposomal and non-liposomal-GSH, respectively. Liposomal-GSH utilization was also observed in neuronal cultures, but with a higher EC50 (76.5 μM), suggesting that glia facilitate utilization. Blocking γ-glutamylcysteine synthetase with buthionine sulfoxamine prevented replenishment with liposomal-GSH demonstrating the requirement for catabolism and resynthesis. Repletion was significantly attenuated with endosomal inhibition implicating the endosomal system in utilization. Liposomal-GSH provided dose-dependent protection against PQMB with an EC50 similar to that found for repletion. PQMB depleted intracellular GSH by 50%. Liposomal-GSH spared endogenous GSH during PQMB exposure, but did not require GSH biosynthesis for protection. No toxicity was observed with the liposomal preparation at 200-fold the EC50 for repletion. These findings indicate that glutathione supplied in a liposomal formulation holds promise as a potential therapeutic for neuronal maintenance.

Keywords

Glutathione Neurodegeneration Autism Schizophrenia Parkinson’s disease 

References

  1. 1.
    Graham DG (1978) Oxidatve pathways for catecholamines in the genesis of neuromelanin and cytotoxic quinones. Mol Pharmacol 14:633–643PubMedGoogle Scholar
  2. 2.
    Sayre LM, Smith MA, Perry G (2001) Chemistry and biochemistry of oxidative stress in neurodegenerative disease. Curr Med Chem 8:721–738PubMedGoogle Scholar
  3. 3.
    Emerit J, Edeas M, Bricaire F (2004) Neurodegenerative diseases and oxidative stress. Biomed Pharmacother 58:39–46CrossRefPubMedGoogle Scholar
  4. 4.
    Perry TL, Godin DV, Hansen S (1982) Parkinson’s disease: a disorder due to nigral glutathione deficiency? Neurosci Lett 33:305–310CrossRefPubMedGoogle Scholar
  5. 5.
    Jenner P, Dexter DT, Sian J, Schapira AHV, Marsden CD (1992) Oxidative stress as a cause of nigral cell death in Parkinson’s disease and incidental lewy body disease. Ann Neurol 32:S82–S87CrossRefPubMedGoogle Scholar
  6. 6.
    Riederer P, Sofie E, Rausch WD, Schmidt B, Reynolds GP, Jellinger K, Youdim MBH (1989) Transition metals, ferritin, glutathione and ascorbic acid in Parkinsonian brains. J Neurochem 52:515–520CrossRefPubMedGoogle Scholar
  7. 7.
    Do KQ, Trabesinger AH, Kirsten-Kruger M, Lauer CJ, Dydak U, Hell D, Holsboer F, Boesiger P, Cuenod M (2000) Schizophrenia: glutathione deficits in cerebrospinal fluid and prefrontal cortex in vivo. Eur J Neurosci 12:3721–3728CrossRefPubMedGoogle Scholar
  8. 8.
    Gysin R, Krafsik R, Sandell J, Bovet P, Chappuis C, Conus P, Deppen P, Preisig M, Ruiz V, Steullet P, Tosic M, Werge T, Cuenod M, Do KQ (2000) Impaired glutathione synthesis in schizophrenia: convergent genetic and functional evidence. Proc Natl Acad Sci 104:16621–16626CrossRefGoogle Scholar
  9. 9.
    Geier DA, Kern JK, Garver CR, Adams JB, Audhya T, Nataf R, Geier MR (2009) Biomarkers of environmental toxicity and susceptibility in autism. J Neurolog Sci 280:101–108CrossRefGoogle Scholar
  10. 10.
    James SJ, Cutler P, Melnyk S, Jernigan S, Janak L, Gaylor DW, Neubrander JA (2004) Metabolic biomarkers of increased oxidative stress and impaired methylation capacity in children with autism. Am J Clin Nutri 80:1611–1617Google Scholar
  11. 11.
    Pastore A, Tozzi G, Gaeta LM, Giannotti A, Bertini E, Federici G, Digilio MC, Piemonte F (2003) Glutathione metabolism and antioxidant enzymes in children with Down syndrome. J Pediat 142:583–585CrossRefPubMedGoogle Scholar
  12. 12.
    Dalla-Donne I, Rossi R, Giustarini D, Colombo R, Milzani A (2007) Glutathionylation in protein redox regulation. Free Rad Biol Med 43:883–898CrossRefGoogle Scholar
  13. 13.
    Ghezzi P (2005) Regulation of protein function by glutathionylation. Free Rad Res 39:573–580CrossRefGoogle Scholar
  14. 14.
    Zeevalk GD, Manzino L, Sonsalla PK, Bernard LP (2007) Characterization of intracellular elevation of glutathione (GSH) with glutathione monoethyl ester and GSH in brain and neuronal cultures: relevance to Parkinson’s disease. Exp Neur 203:512–520CrossRefGoogle Scholar
  15. 15.
    Wade LA, Brady HM (1981) Cysteine and cystine transport at the blood-brain barrier. J Neurochem 37:730–734CrossRefPubMedGoogle Scholar
  16. 16.
    Sagara J, Miura K, Bannai S (1993) Maintenance of neuronal glutathione by glial cells. J Neurochem 61:1677–1684CrossRefGoogle Scholar
  17. 17.
    Olney JW, Zorumski C, Price MT, Labruyere J (1990) L-cysteine, a bicarbonate-sensitive endogenous excitotoxin. Science 248:596–599CrossRefPubMedGoogle Scholar
  18. 18.
    Puka-Sundvall M, Eriksson P, Nilsson M, Sandberg M, Lehmann A (1995) Neurotoxicity of cysteine: interaction with glutamate. Brain Res 705:65–70CrossRefPubMedGoogle Scholar
  19. 19.
    Munoz AM, Rey P, Soto-Otero R, Guerra MJ, Labandeira-Garcia JL (2004) Systemic administration of N-acetylcysteine protects dopaminergic neurons against 6-hydroxydopamine-induced degeneration. J Neurosci Res 76:551–562CrossRefPubMedGoogle Scholar
  20. 20.
    Sekhon B, Sekhon C, Khan M, Patel SJ, Singh I, Singh AK (2003) N-Acetyl cysteine protects against injury in a rat model of focal cerebral ischemia. Brain Res 971:1–8Google Scholar
  21. 21.
    Gillissen A, Joworska M, Orth M, Coffiner M, Maes P, App EM (2006) Nacystelyn, a novel lysine salt of N-acetylcysteine, to augment cellular antioxidant defence in vitro. Respir Med 91:159–168CrossRefGoogle Scholar
  22. 22.
    Janaky R, Ogita K, Pasqualotto BA, Bains JS, Oja SS, Yondea Y, Shaw CA, (1999) Glutathione and signal transduction in the mammalian CNS. J Neurochem 73:889–902Google Scholar
  23. 23.
    Regan R, Guo Y (1999) Extracellular reduced glutathioine increases neuronal vulnerability to combined chemical hypoxia and glucose deprivation. Brain Res 817:145–150CrossRefPubMedGoogle Scholar
  24. 24.
    Regan RF, Guo YP (1999) Potentiation of excitotoxic injury by high concentrations of extracellular reduced glutathione. Neuroscience 91:463–470CrossRefPubMedGoogle Scholar
  25. 25.
    Rosenblat M, Volkova N, Coleman R, Aviram M (2007) Anti-oxidant and anti-artherogenic properties of liposomal glutathione: studies in vitro, and in the atherosclerotic apolipoprotein E-deficient mice. Atherosclerosis 195:e61–e68CrossRefPubMedGoogle Scholar
  26. 26.
    Thiruchelvam M, Richfield EK, Baggs RB, Tank AW, Cory-Slechta DA (2000) The nigrostriatal dopaminergic system as a preferential target of repeated exposures to combined paraquat and maneb: Implications for Parkinson’s disease. J Neurosci 20:9207–9214PubMedGoogle Scholar
  27. 27.
    Domico LM, Zeevalk GD, Bernard LP, Cooper KR (2006) Acute neurotoxic effects of mancozeb and maneb in mesencephalic neuronal cultures are associated with mitochondrial dysfunction. NeuroToxicol 27:816–825CrossRefGoogle Scholar
  28. 28.
    Domico LM, Cooper KR, Bernard LP, Zeevalk GD (2007) Reactive oxygen species generation by the ethylene-bis-dithiocarbamate (EBDC) fungicide mancozeb and its contribution to neuronal toxicity in mesencephalic cells. NeuroToxicol 28:1079–1091CrossRefGoogle Scholar
  29. 29.
    Wood TK, Sullivan AM, McDermott KW (2003) Viability of dopaminergic neurons is influenced by serum and astroglial cells in vitro. J Neurocytol 32:97–103CrossRefPubMedGoogle Scholar
  30. 30.
    Zeevalk GD, Bernard LP, Nicklas WJ (1998) Role of oxidative stress and the glutathione system in loss of dopamine neurons due to impairment of energy metabolism. J Neurochem 70:1421–1430CrossRefPubMedGoogle Scholar
  31. 31.
    Zeevalk GD, Bernard LP (2005) Energy status, ubiquitin proteasomal function and oxidative stress during chronic and acute complex I inhibition with rotenone in mesencephalic cells. Antioxid Redox Signal 7:662–672CrossRefPubMedGoogle Scholar
  32. 32.
    Zeevalk GD, Bernard LP, Sinha C, Ehrhart J, Nicklas WJ (1998) Excitotoxicity and oxidative stress during inhibition of energy metabolism. Dev Neurosci 20:444–453CrossRefPubMedGoogle Scholar
  33. 33.
    Lee K-D, Nir S, Papahadjopoulos D (1993) Quantitative analysis of liposome-cell interactions in vitro: rate constants of binding and endocytosis with suspension and adherent J774 cells and human monocytes. Biochemistry 32:889–899CrossRefPubMedGoogle Scholar
  34. 34.
    Luzio JP, Rous BA, Bright NA, Pryor PR, Mullock BM, Piper RC (2000) Lysosome-endosome fusion and lysosome biogenesis. J Cell Sci 113:1515–1524PubMedGoogle Scholar
  35. 35.
    Gibson AE, Noel RJ, Herlihy JT, Ward WF (1989) Phenylarsine oxide inhibition of endocytosis: effects on asiolofetuin internalization, Am. J Physiol Cell Physiol 257:C182–C184Google Scholar
  36. 36.
    Edelson PJ, Cohn ZA (1974) Effects of concanavalin A on mouse peritoneal macrophages I. Stimulation of endocytic activity and inhibition of phago-lysosome formation. J Exp Med 140:1364–1386CrossRefPubMedGoogle Scholar
  37. 37.
    Zeevalk GD, Razmpour R, Bernard LP (2008) Glutathione and Parkinson’s disease: is this the elephant in the room? Biomed Pharmacother 62:236–249CrossRefPubMedGoogle Scholar
  38. 38.
    Liou HH, Tsai MC, Chen CJ, Jeng JS, Chang YC, Chen SY, Chen RC (1997) Environmental risk factors and Parkinson’s disease: a case control study in Taiwan. Neurology 48:1583–1588PubMedGoogle Scholar
  39. 39.
    Meco G, Bonifati V, Vanacore N, Fabrizio E (1994) Parkinsonism after chronic exposure to the fungicide maneb (manganese ethylene-bis-dithiocarbamate. Scan J Work Environ Health 20:301–305Google Scholar
  40. 40.
    Thiruchelvam M, Prokopenko O, Cory-Slechta DA, Richfield EK, Buckley B, Mirochnitchenko O (2005) Overexpression of superoxide dismutase or glutathione peroxidase protects against the paraquat plus maneb-induced Parkinson disease phenotype. J Biol Chem 280:22530–22539CrossRefPubMedGoogle Scholar
  41. 41.
    Dringen R (2000) Metabolism and functions of glutathione in brain. Prog Neurobiol 62:649–671CrossRefPubMedGoogle Scholar
  42. 42.
    Byrd A, Sikorska M, Walker PR, Sandhu JK (2004) Effects of glutathione depletion on the viability of human NT2-derived neuronal and astroglial cultures. Neuron Glia Biol 1:317–326CrossRefPubMedGoogle Scholar
  43. 43.
    Zeevalk GD, Bernard L, Nicklas WJ (1998) The role of oxidative stress and the glutathione system in the loss of dopamine neurons due to impairment of energy metabolism. J Neurochem 70:1421–1430CrossRefPubMedGoogle Scholar
  44. 44.
    Bains JS, Shaw CA (1997) Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death. Brain Res Rev 25:335–358CrossRefPubMedGoogle Scholar
  45. 45.
    Pagano RE, Weinstein JN (1978) Interaction of liposomes with mammalian cells. Annu Rev Biophys Bioeng 7:435–468CrossRefPubMedGoogle Scholar
  46. 46.
    Chonn A, Cullis PR (1995) Recent advances in liposomal drug-delivery systems. Curr Opin Biotech 6:698–708CrossRefPubMedGoogle Scholar
  47. 47.
    Sagara J, Makino N, Bannai S (1996) Glutathione efflux from cultured astrocytes. J Neurochem 66:1876–1881CrossRefPubMedGoogle Scholar
  48. 48.
    Dringen R, Kranich O, Hamprecht B (1997) The gamma-glutamyl transpeptidase inhibitor acivicin preserves glutathione released by astroglial cells in culture. Neurochem Res 22:727–733CrossRefPubMedGoogle Scholar
  49. 49.
    Tolleshaug H, Abdelnour M, Berg T (1980) Binding of concanavalin A to isolated hepatocytes and its effect on uptake and degradation of asialo-fetuin by the cells. Biochem J 190:697–703PubMedGoogle Scholar
  50. 50.
    Shanker G, Allen JW, Mutkus LA, Aschner M (2001) The uptake of cysteine in cultured primary astrocytes and neurons. Brain Res 902:156–163CrossRefPubMedGoogle Scholar
  51. 51.
    Chen Y, Swanson RA (2003) The glutamate transporters EAAT2 and EAAT3 mediate cysteine uptake in cortical neuron cultures. J Neurochem 84:1332–1339CrossRefPubMedGoogle Scholar
  52. 52.
    Olanow CW, Tatton WG (1999) Etiology and pathogenesis of Parkinson’s disease. Annu Rev Neurosci 22:123–144CrossRefPubMedGoogle Scholar
  53. 53.
    Tanner CM, Ottman R, Goldman SM, Ellenberg J, Chan P, Mayeux R, Langston JW (1999) Parkinson disease in twins: an etiologic study. JAMA 281:341–346CrossRefPubMedGoogle Scholar
  54. 54.
    Martinsson J, Meister A (1991) Glutathione deficiency decreases tissue ascorbate levels in newborn rats: ascorbate spares glutathione and protects. Proc Natl Acad Sci 88:4656–4660CrossRefGoogle Scholar
  55. 55.
    Sechi G, Deledda MG, Bua G, Satta WM, Deiana GA, Pes GM, Rosati G (1996) Reduced intravenous glutathione in the treatment of early Parkinson’s disease. Psychopharmacol Biol Psychiat 20:1159–1170CrossRefGoogle Scholar
  56. 56.
    Hauser RA, Lyons KE, McClain T, Carter S, Perlmutter D (2009) Randomized, double-blind, pilot evaluation of intravenous glutathione in Parkinson’s disease. Mov Disord 24:979–983CrossRefPubMedGoogle Scholar
  57. 57.
    Kannan R, Kuhlenkamp JF, Jeandidler E, Trinh H, Ookhetens M, Kkaplowitz N (1990) Evidence for carrier-mediated transport of glutathione across the blood brain barrier in the rat. J Clin Invest 85:2009–2013Google Scholar
  58. 58.
    Cornford EM, Braun LD, Crane PD, Oldendorf WH (1978) Blood-brain barrier restriction of peptides and the low uptake of enkephalins. Endocrinology 103:1297–1303CrossRefPubMedGoogle Scholar
  59. 59.
    Sian J, Dexter DT, Lees AJ, Daniel S, Jenner P, Marsden CD (1994) Glutathione-related enzymes in brain in Parkinson’s Disease. Ann Neurol 36:356–361CrossRefPubMedGoogle Scholar
  60. 60.
    Dringen R, Pfeiffer B, Hamprecht B (1999) Synthesis of the antioxidant glutathione in neurons: supply by astrocytes of CysGly as precursor for neuronal glutathione. J Neurosci 19:562–569PubMedGoogle Scholar
  61. 61.
    Carlsson A, Fornstedt B (1991) Catechol metabolites in the cerebrospinal fluid as possible markers in the early diagnosis of Parkinson’s disease. Neurology 41:50–52PubMedGoogle Scholar
  62. 62.
    Cheng FC, Kuo JS, Chia LG, Dryhurst G (1996) Elevated 5-S-cysteinyldopamine/homovanillic acid ratio and reduced homovanillic acid in cerebrospinal fluid: possible markers for and potential insights into the pathoetiology of Parkinson’s disease. J Neural Transmission 103:433–446CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Gail D. Zeevalk
    • 1
  • Laura P. Bernard
    • 1
  • F. T. Guilford
    • 2
  1. 1.Department of NeurologyUMDNJ-Robert Wood Johnson Medical SchoolPiscatawayUSA
  2. 2.Your Energy Systems, LLCPalo AltoUSA

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