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Amino acid starvation induced autophagic cell death in PC-12 cells: Evidence for activation of caspase-3 but not calpain-1

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Abstract

While the apoptotic and necrotic cell death pathways have been well studied, there lacks a comprehensive understanding of the molecular events involving autophagic cell death. We examined the potential roles of the apoptosis-linked caspase-3 and the necrosis/apoptosis-linked calpain-1 after autophagy induction under prolonged amino acid (AA) starvation conditions in PC-12 cells. Autophagy induction was observed as early as three hours following amino acid withdrawal. Cell death, measured by lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays occurred within 24 h following starvation and was accompanied by an upregulation in caspase-3 activity but not calpain-1. The cell death that occurred following AA starvation was significantly alleviated by treatment with the autophagy inhibitor 3-methyl adenine but not with the broad spectrum caspase inhibitors. Thus, this study demonstrates that 3-methyladenine-sensitive autophagic cell death due to AA starvation in PC-12 cells is mechanistically and biochemically similar to, yet distinct from, classic caspase dependent apoptosis.

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References

  1. Baehrecke EH (2003) Autophagic programmed cell death in Drosophila. Cell Death Differ 10:940–945

    Article  PubMed  CAS  Google Scholar 

  2. Liu X, Van Vleet T, Schnellmann RG (2004) The role of calpain in oncotic cell death. Annu Rev Pharmacol Toxicol 44:349–370

    Article  PubMed  CAS  Google Scholar 

  3. Hengartner MO (2000) The biochemistry of apoptosis. Nature 407:770–776

    Article  PubMed  CAS  Google Scholar 

  4. Meijer AJ, Codogno P (2004) Regulation and role of autophagy in mammalian cells. Int J Biochem Cell Biol 36:2445–2462

    Article  PubMed  CAS  Google Scholar 

  5. Clarke PG (1990) Developmental cell death: morphological diversity and multiple mechanisms. Anat Embryol (Berl) 181:195–213

    CAS  Google Scholar 

  6. Lockshin RA, Zakeri Z (2004) Apoptosis, autophagy, and more. Int J Biochem Cell Biol 36:2405–2419

    Article  PubMed  CAS  Google Scholar 

  7. Larsen KE, Sulzer D (2002) Autophagy in neurons: A review. Histol Histopathol 17:897–908

    PubMed  CAS  Google Scholar 

  8. Xue L, Fletcher GC, Tolkovsky AM (1999) Autophagy is activated by apoptotic signalling in sympathetic neurons: An alternative mechanism of death execution. Mol Cell Neurosci 14:180–198

    Article  PubMed  CAS  Google Scholar 

  9. Levine B, Klionsky DJ (2004) Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell 6:463–477

    Article  PubMed  CAS  Google Scholar 

  10. Stefanis L, Larsen KE, Rideout HJ, Sulzer D, Greene LA (2001) Expression of A53T mutant but not wild-type alpha-synuclein in PC12 cells induces alterations of the ubiquitin-dependent degradation system, loss of dopamine release, and autophagic cell death. J Neurosci 21:9549–9560

    PubMed  CAS  Google Scholar 

  11. Diskin T, Tal-Or P, Erlich S, et al (2005) Closed head injury induces upregulation of Beclin 1 at the cortical site of injury. J Neurotrauma 22:750–762

    Article  PubMed  Google Scholar 

  12. Ravikumar B, Vacher C, Berger Z, et al (2004) Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet 36:585–595

    Article  PubMed  CAS  Google Scholar 

  13. Sikorska B, Liberski PP, Giraud P, Kopp N, Brown P (2004) Autophagy is a part of ultrastructural synaptic pathology in Creutzfeldt-Jakob disease: A brain biopsy study. Int J Biochem Cell Biol 36:2563–2573

    Article  PubMed  CAS  Google Scholar 

  14. Yu WH, Kumar A, Peterhoff C, Shapiro Kulnane L, et al (2004) Autophagic vacuoles are enriched in amyloid precursor protein-secretase activities: Implications for beta-amyloid peptide over-production and localization in Alzheimer’s disease. Int J Biochem Cell Biol 36:2531–2540

    Article  PubMed  CAS  Google Scholar 

  15. Chan PH (2004) Mitochondria and neuronal death/survival signaling pathways in cerebral ischemia. Neurochem Res 29:1943–1949

    Article  PubMed  CAS  Google Scholar 

  16. Green DR (2005) Apoptotic pathways: Ten minutes to dead. Cell 121:671–674

    Article  PubMed  CAS  Google Scholar 

  17. Wang KKW (2000) Calpain and caspase: Can you tell the difference? TINS 23:20–26

    PubMed  Google Scholar 

  18. Stefanis L (2005) Caspase-dependent and -independent neuronal death: Two distinct pathways to neuronal injury. Neuroscientist 11:50–62

    Article  PubMed  CAS  Google Scholar 

  19. Boya P, Gonzalez-Polo RA, Casares N, et al (2005) Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol 25:1025–1040

    Article  PubMed  CAS  Google Scholar 

  20. Canu N, Tufi R, Serafino AL, Amadoro G, Ciotti MT, Calissano P (2005) Role of the autophagic-lysosomal system on low potassium-induced apoptosis in cultured cerebellar granule cells. J Neurochem 92:1228–1242

    Article  PubMed  CAS  Google Scholar 

  21. Gozuacik D, Kimchi A (2004) Autophagy as a cell death and tumor suppressor mechanism. Oncogene 23:2891–2906

    Article  PubMed  CAS  Google Scholar 

  22. Yu L, Lenardo MJ, Baehrecke EH (2004) Autophagy and caspases: A new cell death program. Cell Cycle 3:1124–1126

    PubMed  CAS  Google Scholar 

  23. Hajimohammadreza I, Raser KJ, Nath R, Nadimpalli R, Scott M, Wang KKW (1997) Neuronal nitric oxide synthase and calmodulin-dependent protein kinase IIalpha undergo neurotoxin-induced proteolysis. J Neurochem 69:1006–1013

    Article  PubMed  CAS  Google Scholar 

  24. Koh JY, Choi DW (1987) Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay. J Neurosci Methods 20:83–90

    Article  PubMed  CAS  Google Scholar 

  25. Posmantur R, McGinnis K, Nadimpalli R, Gilbertsen RB, Wang KK (1997) Characterization of CPP32-like protease activity following apoptotic challenge in SH-SY5Y neuroblastoma cells. J Neurochem 68:2328–2337

    Article  PubMed  CAS  Google Scholar 

  26. Biederbick A, Kern HF, Elsasser HP (1995) Monodansylcadaverine (MDC) is a specificin vivo marker for autophagic vacuoles. Eur J Cell Biol 66:3–14

    PubMed  CAS  Google Scholar 

  27. Dunn WA Jr. (1990) Studies on the mechanisms of autophagy: maturation of the autophagic vacuole. J Cell Biol 110:1935–1945

    Article  PubMed  CAS  Google Scholar 

  28. Kabeya Y, Mizushima N, Ueno T, et al (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. Embo J 19:5720–5728

    Article  PubMed  CAS  Google Scholar 

  29. Nath R, Raser KJ, Stafford D, et al (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(3): 683–690

    PubMed  CAS  Google Scholar 

  30. Pike BR, Zhao X, Newcomb JK, Posmantur RM, Wang KK, Hayes RL (1998) Regional calpain and caspase-3 proteolysis of alpha-spectrin after traumatic brain injury. Neuroreport 9:2437–2442

    PubMed  CAS  Google Scholar 

  31. Hengartner M (2005) Cell biology: Divide and conquer Nature 433:692–694

    Article  PubMed  CAS  Google Scholar 

  32. Cuervo AM (2004) Autophagy: In sickness and in health. Trends Cell Biol 14:70–77

    Article  PubMed  CAS  Google Scholar 

  33. Melendez A, Talloczy Z, Seaman M, Eskelinen EL, Hall DH, Levine B (2003) Autophagy genes are essential for dauer development and life-span extension in C. elegans. Science 301:1387–1391

    Article  PubMed  CAS  Google Scholar 

  34. Otto GP, Wu MY, Kazgan N, Anderson OR, Kessin RH (2003) Macroautophagy is required for multicellular development of the social amoeba Dictyostelium discoideum. J Biol Chem 278:17636–17645

    Article  PubMed  CAS  Google Scholar 

  35. Wang CW, Klionsky DJ (2003) The molecular mechanism of autophagy. Mol Med 9:65–76

    PubMed  Google Scholar 

  36. Mizushima N (2004) Methods for monitoring autophagy. Int J Biochem Cell Biol 36:2491–2502

    Article  PubMed  CAS  Google Scholar 

  37. Munafo DB, Colombo MI (2001) A novel assay to study autophagy: Regulation of autophagosome vacuole size by amino acid deprivation. J Cell Sci 114:3619–3629

    PubMed  CAS  Google Scholar 

  38. Polster BM, Basanez G, Etxebarria A, Hardwick JM, Nicholls DG (2005) Calpain I induces cleavage and release of apoptosis-inducing factor from isolated mitochondria. J Biol Chem 280:6447–6454

    Article  PubMed  CAS  Google Scholar 

  39. Kroemer G, Jäättellä M (2005) Lysosomes and autophagy in cell death control. Nat Rev Cancer 5:886–897

    Article  PubMed  CAS  Google Scholar 

  40. Guicciardi ME, Deussing J, Miyoshi H, Bronk SF, Svingen PA, Peters C, Kaufmann SH, Gores GJ (2000) Cathepsin B contributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c. J Clin Invest 106:1127–1137

    Article  PubMed  CAS  Google Scholar 

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Correspondence to Kevin K. W. Wang.

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Shankar Sadasivan and Anu Waghray have contributed equally to this work.

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Sadasivan, S., Waghray, A., Larner, S.F. et al. Amino acid starvation induced autophagic cell death in PC-12 cells: Evidence for activation of caspase-3 but not calpain-1. Apoptosis 11, 1573–1582 (2006). https://doi.org/10.1007/s10495-006-7690-6

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  • DOI: https://doi.org/10.1007/s10495-006-7690-6

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