Abstract
The death of a cell via apoptosis is characterized by morphological changes including cell shrinkage and nuclear condensation. Intracellularly, proteases, including caspases, are activated. In the present article we have compared the ability of three different neurotoxic agents to induce caspase activity in cerebellar granule cells (CGC). These compounds are the micro-tubule-disrupting agent colchicine and the oxidative stress-inducing agents hydrogen peroxide and meth-ylmercury (MeHg). We have previously shown that each of these agents causes nuclear changes that are consistent with apoptosis, i.e., induction of chromatin condensation and DNA cleavage into fragments of regular size (700, 300 and 50 kbp). However, only colchicine causes a large increase in caspase activity, as monitored by the ability of whole cell extracts to cleave the synthetic caspase substrate DEVD-MCA. In contrast, MeHg and hydrogen peroxide do not induce any significant increase of DEVDase activity as compared to control cells. Immunocytochemistry confirms that active caspase-3 is abundant only in colchicine-exposed cells. In agreement with these findings, the pan-caspase inhibitor, z-VAD-fmk, is efficient in protecting CGC against colchicine, but not against hydrogen peroxide or MeHg. These data suggest that in CGC the activation of caspases is not always required to induce morphological changes and pattern of DNA fragmentation consistent with apoptosis.
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Ahlbom, E., Grandison, L., Bonfoco, E., Zhivotovsky, B. and Ceccatelli, S. (1999) Androgen treatment of neonatal rats decreases susceptibility of cerebellar granule neurons to oxidative stress in vitro.Eur. J. Neurosci, 11, 1285–1291.
Ahlbom, E., Gogvadze, V., Chen M., Celsi, G. and Ceccatelli, S. (2000) Prenatal exposure to high levels of glucocorticoids increases the susceptibility of cerebellar granule cells to oxidative stress-induced cell death.Proc. Natl, Acad. Sri. (USA), 97, 14726–14730.
Ankarcrona, M., Dypbukt, J.M., Bonfoco, M., Zhivotovsky, B., Orrenius, S., Lipton, S.A. and Nicotera, P. (1995) Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function.Neuron, 15, 961–973.
Baker, A., Santos, B.D., Powis, G. (2000) Redox control of cas-pase-3 activity by thioredoxin and other reduced proteins.Biochem. Biophys. Res. Commun. 268, 78–81.
Behl, C, Skutella, T., Lezoualc’h, F., Post, A., Widmann, M., Newton, C.J. and Holsboer, F. (1997) Neuroprotection against oxidative stress by estrogens: structure-activity relationship.Mol. Pharmacol. 51, 535–541.
Behl, C. (1999) Alzheimer’s disease and oxidative stress: implications for novel therapeutic approaches.Prog. Neurobiol. 57, 301–323.
Bonfoco, E., Ceccatelli, S., Manzo, L. and Nicotera, P. (1995) Colchicine induces apoptosis in cerebellar granule cells.Exp. Cell Res. 218, 189–220.
Brunk, U. T., Dalen, H.; Roberg, K.; Hellquist, H. B. (1997) Photo-oxidative disruption of lysosomal membranes causes apoptosis of cultured human fibroblasts.Free Radic. Biol. Med. 23, 616–626.
Carmody, R.J. and Cotter, T.G. (2000) Oxidative stress induces caspase-independent retinal apoptosisin vitro. Cell Death Diff. 7, 282–291.
Castoldi, A.F., Barni, S., Turin, I, Gandini, C. and Manzo, L. (2000) Early acute necrosis, delayed apoptosis and cytoskeletal breakdown in cultured cerebellar granule neurons exposed to methylmercury.J. Neurosci. Res. 59, 775–787.
Cookson, M.R. and Shaw, P.J. (1999) Oxidative stress and motor neuron disease.Brain Pathol. 9, 165–186.
Dare, E., Gotz, M.E., Zhivotovsky, B., Manzo, L. and Ceccatelli, S. (2000) The antioxidants J811 and 17p-estradiol protect cerebellar granule cells from methylmer-cury-induced apoptotic cell death.J. Neurosci. Res. 62, 557–565.
Gorman, A.M., McGowan, A. and Cotter, T.G. (1996) Oxidative stress and apoptosis in neurodegeneration.J. Neurol. Sci. 139, S45–52.
Gorman, A.M., Orrenius, S. and Ceccatelli, S. (1998) Apoptosis in neuronal cells: role of caspases.Neuroreport 9, R49-R55.
Gorman, A.M., Bonfoco, E., Zhivotovsky, B., Orrenius, S. and Ceccatelli, S. (1999) Cytochromec release and caspase-3 activation during colchicine-induced apoptosis of cerebellar granule cells.Eur. J. Neurosci. 11, 1067–1073.
Gotz, M.E., Ahlbom, E., Zhivotovsky, B., Blum-Degen, D., Oettel, M., Romer, W., Riederer, P., Orrenius S. and Ceccatelli, S. (1999) Radical scavenging compound J 811 inhibits hydrogen peroxide-induced death of cerebellar granule cells.J. Neurosci. Res, 56, 420–426.
Hampton, M.B. and Orrenius S. (1997) Dual regulation of caspase activity by hydrogen peroxide: implications for apoptosis.FEBS Lett. 414, 552–556.
Hara, S., Halicka, H., Bruno, S., Gong, J., Traganos, F. and Darzynkiewicz, D. (1996) Effect of protease inhibitors on early events of apoptosis.Exp. Cell. Res. 223, 372–384.
Jenner, P. and Olanow, C.W. (1996) Oxidative stress and the pathogenesis of Parkinson’s disease.Neurology 47, S161–170.
Kaasik, A., Vassiljev, V., Poldoja, E., Kalda, A. and Zharko-vsky, A. (1999) Do nuclear condensation or fragmentation and DNA fragmentation reflect the mode of neuronal death?NeuroReport 10, 1937–1942.
Kerr, J.F.R., Wyllie, A.H. and Currie, A.R. (1972) Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics.Br. J. Cancer 26, 1790–1794.
Kouroku, Y., Urase, K., Fujita, E., Isahara, K., Ohsawa, Y., Uchiyama, Y., Momoi, M.Y. and Momoi, T. (1998) Detection of activated caspase-3 by a cleavage site-directed antiserum during naturally occurring DRG neurons apoptosis.Biochem. Biophys Res. Commun. 247, 780–784.
Kunimoto, M. (1994) Methylmercury induces apoptosis of rat cerebellar neurons in primary culture.Biochem. Biophys. Res. Commun. 204, 310–317.
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680–685.
Lankiewicz, S., Marc Luetjens, C, True Bui, N., Krohn, A.J., Poppe, M., Cole, G.M., Saido, T.C. and Prehn, J.H. (2000) Activation of calpain I converts excitotoxic neuron death into a caspase-independent cell death.J. Biol. Chem. 275, 17064–17071.
Lee, Y. and Shacter, E. (1999) Oxidative stress inhibits apoptosis in human lymphoma cells.J. Biol. Chem. 274, 19792–19798.
Marty, M.S. and Atchison, W.D. (1997) Pathways mediating Ca entry in rat cerebellar granule cells following in vitro exposure to methyl mercury.Toxicol. Appl. Pharmacol. 147, 319–330.
Mattson, M.P. (1992) Effects of microtubule stabilization and destabilization on tau immunoreactivity in cultured hip-pocampal neurons.Brain Res. 582, 107–118.
McGinnis, K.M., Gnegy, M.E., Park, Y.H., Mukerjee, N. and Wang, K.K. (1999) Procaspase-3 and poly(ADP)ribose polymerase (PARP) are calpain substrates.Biochem. Biophys. Res. Commun. 263, 94–99.
Mooradian, A.D. (1993) Antioxidant properties of steroids.J. Steroid Biochem. Mol. Biol. 45, 509–511.
Moosmann, B. and Behl, C. (1999) The antioxidant neuroprotective effects of estrogens and phenolic compounds are independent from their estrogenic properties.Proc. Natl. Acad. Sci. USA 96, 8867–8872.
Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays.J. Immunol. Meth. 65, 55–63.
Nakagawa, Y., Nakamura, S., Kasa, Y., Noguchi, T. and Ishi-hara, T. (1987) Colchicine lesions in the rat hippocampus mimic the alterations of several markers in Alzheimer’s disease.Brain Res. 408, 57–64.
Nath, R., Raser, K.J., Stafford, D., Hajimohammadreza, I., Pasner, A., Allen, H., Talanian, R.V., Yuen, P., Gilbert-sen, R.B. and Wang, K.K.W. (1996) Non-erythroid a-spectrin breakdown by calpain and interleukin lp-converting-enzyme-like protease(s) in apoptotic cells: contributory roles of both protease families in neuronal apoptosis.Biochem. J. 319, 683–690.
Nicholson D.W., Ali A., Thornberry N.A., Vaillancourt J.P., Ding C.K., Gallant M., Gareau Y., Griffin P.R., Labelle M., Lazebnik Y.A., Munday N.A., Raju A.M., Smulson M.E., Yamin T.T., Yu V.L. and Miller D.K. (1995) Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis.Nature 376, 37–43.
Nicholson, D.W. and Thornberry, N.A. (1997) Caspases: killer proteases.Trends Biochem. Sci. 22, 299–306.
Ollinger, K. and Brunk, U. T. (1995) Cellular injury induced by oxidative stress is mediated through lysosomal damage.Free Radic. Biol. Med. 19, 565–574.
Piwocka, K., Zablocki, K., Wieckowski, M.R., Skierski, J., Feiga, I., Szopa, J., Drela, N., Wojtczak, L. and Sikora, E. (1999) A novel apoptosis-like pathway, independent of mitochondria and caspases, induced by curcumin in human lymphoblastoid T (Jurkat) cells.Exp. Cell Res. 249, 299–307.
Roberg, K., Johansson, U. and Ollinger, K. (1999) Lysosomal release of cathepsin D precedes relocation of cytochrome c and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress.Free Radic. Biol. Med. 27, 1228–1237.
Romer, W., Oettel, M., Droescher, P. and Schwarz, S. (1997) Novel “scavestrogens” and their radical scavenging effects, iron-chelating and total antioxidative activities’. A8,9-dehydro derivatives of 17 a-estradiol.Steroids 62, 304–310.
Sarafian, T. and Verity, M.A. (1991) Oxidative mechanisms underlying methyl mercury neurotoxicity.Int. J. Dev. Neurosci. 9, 147–153.
Schaffner, W. and Weissmann, C. (1973) A rapid, sensitive and specific method for the determination of protein in dilute solution.Anal. Biochem. 56, 502–514.
Schousboe, A., Meier, E., Drejer, J. and Hertz, L. (1989) Preparation of cultures of mouse (rat) cerebellar granule cells. In: Shahar, A., de Vellis, J., Vernadakis, A. and Haber, B (Ed),Dissection and Tissue Culture Manual of the Nervous System (A.R. Liss, New York), pp. 203–206.
Slater, T.F., Sawyer, B. and StrSuli, U. (1963) Studies on succi-nate-tetrazolium reductase systems III. Points of coupling of four different tetrazolium salts.Biochim. Biophys. Acta 77, 383–393.
Sorimachi, H., Ishiura, S. and Suzuki, K. (1997) Structure and physiological function of calpains.Biochem. J. 328, 721–732.
Stridh, H., Kimland, M., Jones, D.P., Orrenius, S. and Hampton, M.B. (1998) Cytochrome c release and caspase activation in hydrogen peroxide- and tributyltin-induced apoptosis.FEBS Lett. 429, 351–355.
Stroh, C. and Schulze-Osthoff, K. (1998) Death by a thousand cuts: an ever increasing list of caspase substrates.Cell Death Differ. 5, 997–1000.
Sun, A.Y. and Chen, Y.M. (1998) Oxidative stress and neurodegenerative disorders.J. Biomed. Sci. 5, 401–414.
Susin, S.A., Lorenzo, H.K., Zamzami, N., Marzo, I., Snow, B.E., Brothers, G.M., Mangion, J., Jacotot, E., Constantini, P, Loeffler, M., Larochette, N., Goodlett, D.R., Aeber-sold, R., Siderovski, D., Penninger, J.M. and Kroemer, G. (1999) Molecular characterization of mitochondrial apoptosis-inducing factor.Nature 397, 441–446.
Thornberry, N.A. and Lazebnik, Y. (1998) Caspases: enemies within.Science 281, 1312–1316.
Urase, K., Fujita, E., Miho, Y., Kouroku, Y., Mukasa, T., Yagi, Y., Momoi, M.Y. and Momoi, T. (1998) Detection of activated Caspase-3 (CPP32) in the vertebrate nervous system during development by a cleavage site-directed antiserum.Dev. Brain Res. 111, 77–87.
Volbracht, C, Leist, M. and Nicotera P. (1999) ATP controls neuronal apoptosis triggered by microtubule breakdown or potassium deprivation.Mol. Med. 5, 477–89.
Wolf, B.B., Goldstein, J.C., Stennicke, H.R., Beere, H., Ama-rante-Mendes, G.P., Salvesen, G.S. and Green, D.R. (1999) Calpain functions in a caspase-independent manner to promote apoptosis-like events during platelet activation.Blood 94, 1683–1692.
Wyllie, A.H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation.Nature 284, 555–556.
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Dare, E., Gorman, A.M., Ahlbom, E. et al. Apoptotic morphology does not always require caspase activity in rat cerebellar granule neurons. neurotox res 3, 501–514 (2001). https://doi.org/10.1007/BF03033206
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DOI: https://doi.org/10.1007/BF03033206