Neurotoxicity Research

, Volume 19, Issue 3, pp 374–388

Calpain Plays a Central Role in 1-Methyl-4-phenylpyridinium (MPP(+))-Induced Neurotoxicity in Cerebellar Granule Neurons

  • Richard A. Harbison
  • Kristen R. Ryan
  • Heather M. Wilkins
  • Emily K. Schroeder
  • F. Alexandra Loucks
  • Ron J. Bouchard
  • Daniel A. Linseman
Article

Abstract

1-Methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity has previously been attributed to either caspase-dependent apoptosis or caspase-independent cell death. In the current study, we found that MPP(+) induces a unique, non-apoptotic nuclear morphology coupled with a caspase-independent but calpain-dependent mechanism of cell death in primary cultures of rat cerebellar granule neurons (CGNs). Using a terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay in CGNs exposed to MPP(+), we observed that these neurons are essentially devoid of caspase-dependent DNA fragments indicative of apoptosis. Moreover, proteolysis of a well recognized caspase-3 substrate, poly (ADP ribose) polymerase (PARP), was not observed in CGNs exposed to MPP(+). In contrast, calpain-dependent proteolysis of fodrin and pro-caspases-9 and -3 occurred in this model coupled with inhibition of caspase-3/-7 activities. Notably, several key members of the Bcl-2 protein family appear to be prominent calpain targets in MPP(+)-treated CGNs. Bid and Bax were proteolyzed to truncated forms thought to have greater pro-death activity at mitochondria. Moreover, the pro-survival Bcl-2 protein was degraded to a form predicted to be inactive at mitochondria. Cyclin E was also cleaved by calpain to an active low MW fragment capable of facilitating cell cycle re-entry. Finally, MPP(+)-induced neurotoxicity in CGNs was significantly attenuated by a cocktail of calpain and caspase inhibitors in combination with the antioxidant glutathione. Collectively, these results demonstrate that caspases do not play a central role in CGN toxicity induced by exposure to MPP(+), whereas calpain cleavage of key protein targets, coupled with oxidative stress, plays a critical role in MPP(+)-induced neurotoxicity. Our findings underscore the complexity of MPP(+)-induced neurotoxicity and suggest that calpain may play a fundamental role in causing neuronal death downstream of mitochondrial oxidative stress and dysfunction.

Keywords

Cerebellar granule neurons MPP(+) Calpain Oxidative stress Neurotoxicity Bcl-2 

References

  1. Alvira D, Tajes M, Verdaguer E, de Arriba SG, Allgaier C, Matute C, Trullas R, Jiménez A, Pallàs M, Camins A (2007) Inhibition of cyclin-dependent kinases is neuroprotective in 1-methyl-4-phenylpyridinium-induced apoptosis in neurons. Neuroscience 146:350–365CrossRefPubMedGoogle Scholar
  2. Anantharam V, Kaul S, Song C, Kanthasamy A, Kanthasamy AG (2007) Pharmacological inhibition of neuronal NADPH oxidase protects against 1-methyl-4-phenylpyridinium (MPP +)-induced oxidative stress and apoptosis in mesencephalic dopaminergic neuronal cells. Neurotoxicology 28:988–997CrossRefPubMedGoogle Scholar
  3. Bizat N, Hermel JM, Humbert S, Jacquard C, Créminon C, Escartin C, Saudou F, Krajewski S, Hantraye P, Brouillet E (2003) In vivo calpain/caspase cross-talk during 3-nitropropionic acid-induced striatal degeneration: implication of a calpain-mediated cleavage of active caspase-3. J Biol Chem 278:43245–43253CrossRefPubMedGoogle Scholar
  4. Blomgren K, Zhu C, Wang X, Karlsson JO, Leverin AL, Bahr BA, Mallard C, Hagberg H (2001) Synergistic activation of caspase-3 by m-calpain after neonatal hypoxia-ischemia. J Biol Chem 276:10191–10198CrossRefPubMedGoogle Scholar
  5. Bo J, Ming BY, Gang LZ, Lei C, Jia AL (2005) Protection by puerarin against MPP+-induced neurotoxicity in PC12 cells mediated by inhibiting mitochondrial dysfunction and caspase-3-like activation. Neurosci Res 53:183–188CrossRefPubMedGoogle Scholar
  6. Cao X, Deng X, May WS (2003) Cleavage of Bax to p18 Bax accelerates stress-induced apoptosis, and a cathepsin-like protease may rapidly degrade p18 Bax. Blood 102:2605–2614CrossRefPubMedGoogle Scholar
  7. Chen M, He H, Zhan S, Krajewski S, Reed JC, Gottlieb RA (2001) Bid is cleaved by calpain to an active fragment in vitro and during myocardial ischemia/reperfusion. J Biol Chem 276:30724–30728CrossRefPubMedGoogle Scholar
  8. Cheng EH, Wei MC, Weiler S, Flavell RA, Mak TW, Lindsten T, Korsmeyer SJ (2001) BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol Cell 8:705–711CrossRefPubMedGoogle Scholar
  9. Cheng YF, Zhu GQ, Wang M, Cheng H, Zhou A, Wang N, Fang N, Wang XC, Xiao XQ, Chen ZW, Li QL (2009) Involvement of ubiquitin proteasome system in protective mechanisms of puerarin to MPP(+)-elicited apoptosis. Neurosci Res 63:52–58CrossRefPubMedGoogle Scholar
  10. Choi WS, Lee EH, Chung CW, Jung YK, Jin BK, Kim SU, Oh TH, Saido TC, Oh YJ (2001) Cleavage of Bax is mediated by caspase-dependent or–independent calpain activation in dopaminergic neuronal cells: protective role of Bcl-2. J Neurochem 77:1531–1541CrossRefPubMedGoogle Scholar
  11. Choi WS, Lee E, Lim J, Oh YJ (2008) Calbindin-D28K prevents drug-induced dopaminergic neuronal death by inhibiting caspase and calpain activity. Biochem Biophys Res Commun 371:127–131CrossRefPubMedGoogle Scholar
  12. Chu CT, Zhu JH, Cao G, Signore A, Wang S, Chen J (2005) Apoptosis inducing factor mediates caspase-independent 1-methyl-4-phenylpyridinium toxicity in dopaminergic cells. J Neurochem 94:1685–1695CrossRefPubMedGoogle Scholar
  13. Chua BT, Guo K, Li P (2000) Direct cleavage by the calcium-activated protease calpain can lead to inactivation of caspases. J Biol Chem 275:5131–5135CrossRefPubMedGoogle Scholar
  14. D’Mello SR, Galli C, Ciotti T, Calissano P (1993) Induction of apoptosis in cerebellar granule neurons by low potassium: inhibition of death by insulin-like growth factor I and cAMP. Proc Natl Acad Sci USA 90:10989–10993CrossRefPubMedGoogle Scholar
  15. Ding WX, Ni HM, DiFrancesca D, Stolz DB, Yin XM (2004) Bid-dependent generation of oxygen radicals promotes death receptor activation-induced apoptosis in murine hepatocytes. Hepatology 40:403–413CrossRefPubMedGoogle Scholar
  16. Domingues AF, Esteves AR, Swerdlow RH, Oliveira CR, Cardoso SM (2008) Calpain-mediated MPP+ toxicity in mitochondrial DNA depleted cells. Neurotox Res 13:31–38CrossRefPubMedGoogle Scholar
  17. Du YS, Dodel RC, Bales KR, Jemmerson R, Hamilton-Byrd E, Paul SM (1997) Involvement of a caspase-3-like cysteine protease in 1-methyl-4-phenypyridinium-mediated apoptosis of cultured cerebellar granule neurons. J Neurochem 69:1382–1388CrossRefPubMedGoogle Scholar
  18. Ekshyyan O, Aw TY (2004) Apoptosis: a key in neurodegenerative disorders. Curr Neurovasc Res 1:355–371CrossRefPubMedGoogle Scholar
  19. Gao G, Dou QP (2000) N-terminal cleavage of bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes bcl-2-independent cytochrome c release and apoptotic cell death. J Cell Biochem 80:53–72CrossRefPubMedGoogle Scholar
  20. González-Polo RA, Mora A, Clemente N, Sabio G, Centeno F, Soler G, Fuentes JM (2001) Mechanisms of MPP(+) incorporation into cerebellar granule cells. Brain Res Bull 56:119–123CrossRefPubMedGoogle Scholar
  21. González-Polo RA, Soler G, Alonso JC, Rodríguez-Martín A, Fuentes JM (2003a) MPP(+) causes inhibition of cellular energy supply in cerebellar granule cells. Neurotoxicology 24:219–225CrossRefPubMedGoogle Scholar
  22. González-Polo RA, Soler G, Alvarez A, Fabregat I, Fuentes JM (2003b) Vitamin E blocks early events induced by 1-methyl-4-phenylpyridinium (MPP +) in cerebellar granule cells. J Neurochem 84:305–315CrossRefPubMedGoogle Scholar
  23. González-Polo RA, Soler G, Fuentes JM (2004) MPP+: mechanism for its toxicity in cerebellar granule cells. Mol Neurobiol 30:253–264CrossRefPubMedGoogle Scholar
  24. Han BS, Hong HS, Choi WK, Markelonis GJ, Oh TH, Oh YJ (2003) Caspase-dependent and-independent cell death pathways in primary cultures of mesencephalic dopaminergic neurons after neurotoxin treatment. J Neurosci 23:5069–5078PubMedGoogle Scholar
  25. Harwell RM, Porter DC, Danes C, Keyomarsi K (2000) Processing of cyclin E differs between normal and tumor breast cells. Cancer Res 60:481–489PubMedGoogle Scholar
  26. Itoh T, Itoh H, Pleasure D (2003) Bcl-2-related protein family gene expression during oligodendroglial differentiation. J Neurochem 85:1500–1512CrossRefPubMedGoogle Scholar
  27. Janumyan YM, Sansam CG, Chattopadhyay A, Cheng N, Soucie EL, Penn LZ, Andrews D, Knudson CM, Yang E (2003) Bcl-xL/Bcl-2 coordinately regulates apoptosis, cell cycle arrest and cell cycle entry. EMBO J 22:5459–5470CrossRefPubMedGoogle Scholar
  28. Jordan-Sciutto KL, Malaiyandi LM, Bowser R (2002) Altered distribution of cell cycle transcriptional regulators during Alzheimer disease. J Neuropathol Exp Neurol 61:358–367PubMedGoogle Scholar
  29. Jourdi H, Hamo L, Oka T, Seegan A, Baudry M (2009) BDNF mediates the neuroprotective effects of positive AMPA receptor modulators against MPP(+)-induced toxicity in cultured hippocampal and mesencephalic slices. Neuropharmacology 56:876–885CrossRefPubMedGoogle Scholar
  30. Keyomarsi K, Herliczek TW (1997) The role of cyclin E in cell proliferation, development and cancer. Prog Cell Cycle Res 3:171–191PubMedGoogle Scholar
  31. Keyomarsi K, Pardee AB (1993) Redundant cyclin overexpression and gene amplification in breast cancer cells. Proc Natl Acad Sci USA 90:1112–1116CrossRefPubMedGoogle Scholar
  32. Keyomarsi K, Conte D Jr, Toyofuku W, Fox MP (1995) Deregulation of cyclin E in breast cancer. Oncogene 11:941–950PubMedGoogle Scholar
  33. Kim NK, Choi BH, Huang X, Snyder BJ, Bukhari S, Kong TH, Park H, Park HC, Park SR, Ha Y (2009) Granulocyte-macrophage colony-stimulating factor promotes survival of dopaminergic neurons in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-induced murine Parkinson’s disease model. Eur J Neurosci 29:891–900CrossRefPubMedGoogle Scholar
  34. Kroemer G, Galluzi L, Vandenabeele P, Abrams J, Alneri ES, Baehrecke EH, Blagosklonny MV, El-Deiry WS, Golstein P, Green DR, Hengartner M, Knight RA, Kumar S, Lipton SA, Malorni W, Nuñez G, Peter ME, Tscopp J, Yuan J, Piacentini M, Zhivotovsky B, Melino G (2009) Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ 16:3–11CrossRefPubMedGoogle Scholar
  35. Leist M, Volbracht C, Fava E, Nicotera P (1998) 1-Methyl-4-phenylpyridinium induces autocrine excitotoxicity, protease activation, and neuronal apoptosis. Mol Pharmacol 54:789–801PubMedGoogle Scholar
  36. Liang LP, Patel M (2004) Mitochondrial oxidative stress and increased seizure susceptibility in Sod2−/+ mice. Free Radic Biol Med 36:542–554CrossRefPubMedGoogle Scholar
  37. Libertini SJ, Robinson BS, Dhillon NK, Glick D, George M, Dandekar S, Gregg JP, Sawai E, Mudryj M (2005) Cyclin E both regulates and is regulated by calpain 2, a protease associated with metastatic breast cancer phenotype. Cancer Res 65:10700–10708CrossRefPubMedGoogle Scholar
  38. Linseman DA, Phelps RA, Bouchard RJ, Le SS, Laessig TA, McClure ML, Heidenreich KA (2002) Insulin-like growth factor-I blocks Bcl-2 interacting mediator of cell death (Bim) induction and intrinsic death signaling in cerebellar granule neurons. J Neurosci 22:9287–9297PubMedGoogle Scholar
  39. Linseman DA, Butts BD, Precht TA, Phelps RA, Le SS, Laessig TA, Bouchard RJ, Florez-McClure ML, Heidenreich KA (2004) Glycogen synthase kinase-3beta phosphorylates Bax and promotes its mitochondrial localization during neuronal apoptosis. J Neurosci 24:9993–10002CrossRefPubMedGoogle Scholar
  40. Loucks FA, Le SS, Zimmermann AK, Ryan KR, Barth H, Aktories K, Linseman DA (2006) Rho family GTPase inhibition reveals opposing effects of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase and Janus kinase/signal transducer and activator of transcription signaling cascades on neuronal survival. J Neurochem 97:957–967CrossRefPubMedGoogle Scholar
  41. Loucks FA, Schroeder EK, Zommer AE, Hilger S, Kelsey NA, Bouchard RJ, Blackstone C, Brewster JL, Linseman DA (2009) Caspases indirectly regulate cleavage of the mitochondrial fusion GTPase OPA1 in neurons undergoing apoptosis. Brain Res 1250:63–74CrossRefPubMedGoogle Scholar
  42. Mandic A, Viktorsson K, Strandberg L, Heiden T, Hansson J, Linder S, Shoshan MC (2002) Calpain-mediated Bid cleavage and calpain-independent Bak modulation: two separate pathways in cisplatin-induced apoptosis. Mol Cell Biol 22:3003–3013CrossRefPubMedGoogle Scholar
  43. Martin SJ, Green DR, Cotter TG (1994) Dicing with death: dissecting the components of the apoptosis machinery. Trends Biochem Sci 19:26–30CrossRefPubMedGoogle Scholar
  44. McGinnis KM, Gnegy ME, Park YH, Mukerjee N, Wang KK (1999) Procaspase-3 and poly(ADP)ribose polymerase (PARP) are calpain substrates. Biochem Biophys Res Commun 263:94–99CrossRefPubMedGoogle Scholar
  45. Nath R, Raser KJ, Stafford D, Hajimohammadreza I, Posner A, Allen H, Talanian RV, Yuen P, Gilbertsen RB, Wang KK (1996) Non-erythroid alpha-spectrin breakdown by calpain and interleukin 1 beta-converting-enzyme-like protease(s) in apoptotic cells: contributory roles of both protease families in neuronal apoptosis. Biochem J 319(Pt 3):683–690PubMedGoogle Scholar
  46. Neumar RW, Xu YA, Gada H, Guttmann RP, Siman R (2003) Cross-talk between calpain and caspase proteolytic systems during neuronal apoptosis. J Biol Chem 278:14162–14167CrossRefPubMedGoogle Scholar
  47. O’Brien MA, Moravec RA, Riss TL (2001) Poly (ADP-ribose) polymerase cleavage monitored in situ in apoptotic cells. Biotechniques 30:886–891PubMedGoogle Scholar
  48. Okouchi M, Ekshyyan O, Maracine M, Aw TY (2007) Neuronal apoptosis in neurodegeneration. Antioxid Redox Signal 9:1059–1096CrossRefPubMedGoogle Scholar
  49. Rubin LL, Gatchalian CL, Rimon G, Brooks SF (1994) The molecular mechanisms of neuronal apoptosis. Curr Opin Neurobiol 4:696–702CrossRefPubMedGoogle Scholar
  50. Saez ME, Ramirez-Lorca R, Moron FJ, Ruiz A (2006) The therapeutic potential of the calpain family: new aspects. Drug Discov Today 11:917–923CrossRefPubMedGoogle Scholar
  51. Shang T, Uihlein AV, Van Asten J, Kalyanaraman B, Hillard CJ (2003) 1-Methyl-4-phenylpyridinium accumulates in cerebellar granule neurons via organic cation transporter 3. J Neurochem 85:358–367CrossRefPubMedGoogle Scholar
  52. Starkov AA, Polster BM, Fiskum G (2002) Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax. J Neurochem 83:220–228CrossRefPubMedGoogle Scholar
  53. Tompa P, Buzder-Lantos P, Tantos A, Farkas A, Szilágyi A, Bánóczi Z, Hudecz F, Friedrich P (2004) On the sequential determinants of calpain cleavage. J Biol Chem 279:20775–20785CrossRefPubMedGoogle Scholar
  54. Wales SQ, Laing JM, Chen L, Aurelian L (2008) ICP10PK inhibits calpain-dependent release of apoptosis-inducing factor and programmed cell death in response to the toxin MPP+. Gene Ther 15:1397–1409CrossRefPubMedGoogle Scholar
  55. Wang XJ, Xu JX (2005) Salvianic acid A protects human neuroblastoma SH-SY5Y cells against MPP+-induced cytotoxicity. Neurosci Res 51:129–138CrossRefPubMedGoogle Scholar
  56. Wang XD, Rosales JL, Magliocco A, Gnanakumar R, Lee KY (2003) Cyclin E in breast tumors is cleaved into its low molecular weight forms by calpain. Oncogene 22:769–774CrossRefPubMedGoogle Scholar
  57. Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ, Roth KA, MacGregor GR, Thompson CB, Korsmeyer SJ (2001) Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292:727–730CrossRefPubMedGoogle Scholar
  58. Wesierska-Gadek J, Gueorguieva M, Wojciechowski J, Tudzarova-Trajkovska S (2004) In vivo activated caspase-3 cleaves PARP-1 in rat liver after administration of the hepatocarcinogen N-nitrosomomorpholine (NNM) generating the 85 kDa fragment. J Cell Biochem 93:774–787CrossRefPubMedGoogle Scholar
  59. Willis SN, Adams JM (2005) Life in the balance: how BH3-only proteins induce apoptosis. Curr Opin Cell Biol 17:617–625CrossRefPubMedGoogle Scholar
  60. Wood DE, Newcomb EW (2000) Cleavage of Bax enhances its cell death function. Exp Cell Res 256:375–382CrossRefPubMedGoogle Scholar
  61. Yang Y, Geldmacher DS, Herrup K (2001) DNA replication precedes neuronal cell death in Alzheimer’s disease. J Neurosci 21:2661–2668PubMedGoogle Scholar
  62. Yang Y, Mufson EJ, Herrup K (2003) Neuronal cell death is preceded by cell cycle events at all stages of Alzheimer’s disease. J Neurosci 23:2557–2563PubMedGoogle Scholar
  63. Yang L, Zhao K, Calingasan NY, Luo G, Szeto HH, Beal F (2009) Mitochondria targeted peptides protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity. Antioxid Redox Signal. Epub 2009 Feb 9Google Scholar
  64. Zatz M, Starling A (2005) Calpains and disease. N Engl J Med 352:2413–2423CrossRefPubMedGoogle Scholar
  65. Zhang Z, Larner SF, Liu MC, Zheng W, Hayes RL, Wang KKW (2009) Multiple alphaII-spectrin breakdown products distinguish calpain and caspase dominated necrotic and apoptotic cell death pathways. Apoptosis 14:1289–1298CrossRefPubMedGoogle Scholar
  66. Zimmermann AK, Loucks FA, Schroeder EK, Bouchard RJ, Tyler KL, Linseman DA (2007) Glutathione binding to the Bcl-2 homology-3 domain groove: a molecular basis for Bcl-2 antioxidant function at mitochondria. J Biol Chem 282:29296–29304CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Richard A. Harbison
    • 1
  • Kristen R. Ryan
    • 2
  • Heather M. Wilkins
    • 1
  • Emily K. Schroeder
    • 3
  • F. Alexandra Loucks
    • 4
  • Ron J. Bouchard
    • 3
  • Daniel A. Linseman
    • 1
    • 3
    • 5
  1. 1.Department of Biological Sciences and Eleanor Roosevelt InstituteUniversity of DenverDenverUSA
  2. 2.Toxicology Program, Department of Pharmaceutical SciencesUniversity of Colorado DenverAuroraUSA
  3. 3.Research ServiceVeterans Affairs Medical CenterDenverUSA
  4. 4.Neuroscience ProgramUniversity of California San FranciscoSan FranciscoUSA
  5. 5.Division of Clinical Pharmacology and Toxicology, Department of MedicineUniversity of Colorado DenverAuroraUSA

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