- Email: [email protected]
Rotenone induces disassembly of the Golgi apparatus in the rat dopaminergic neuroblastoma B65 cell line
Neuroscience Letters 354 (2004) 59–63 www.elsevier.com/locate/neulet
Rotenone induces disassembly of the Golgi apparatus in the rat dopaminergic neuroblastoma B65 cell line Francisco J. Diaz-Corrales, Masato Asanuma*, Ikuko Miyazaki, Norio Ogawa Department of Brain Science, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikatacho, Okayama 700-8558, Japan Received 27 August 2003; received in revised form 12 September 2003; accepted 16 September 2003
Abstract It has been reported that the Golgi apparatus (GA) is fragmented in some neurodegenerative diseases. However, the significance of the GA fragmentation or disassembly in neurodegeneration is still obscure. To clarify the involvement of this organelle in apoptosis of neuronal cells, we examined the morphological changes in the GA induced by rotenone, a pesticide that produces selective dopaminergic neurodegeneration. In dopaminergic neuroblastoma B65 cells, a 5-day rotenone treatment (50 nM) promoted cell damage. Rotenonetreated cells showed round nuclei, diffuse signals of the GA and cytosolic redistribution of cytochrome c. Nevertheless, these type of cells without nuclear fragmentation did not show any caspase-3 expression. These results indicate that rotenone induces disassembly of the GA in the early stages of the apoptotic process. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Rotenone; Disassembly of the Golgi apparatus; Endoplasmic reticulum stress; Apoptosis; Neurodegeneration; Parkinsonism
Rotenone is a selective inhibitor of mitochondrial complex I activity, and this pesticide can reproduce many features of Parkinson’s disease [1]. Nevertheless, the exact mechanisms of dopaminergic neuron-specific degeneration induced by rotenone are not completely understood. Evidence supports the hypothesis that production of reactive oxygen species is not the only way to induce cell death by rotenone [17]. Moreover, Ryu et al. have reported that endoplasmic reticulum (ER) stress can contribute to rotenone-induced toxicity [13]. In dopaminergic neuroblastoma cells, rotenone treatment produces apoptotic cells and protein aggregation of both asynuclein and ubiquitin [15]. One of the mechanisms proposed for this protein aggregation is that a reduction in energy production generates an inadequate defense against unfolded proteins [8]. It has recently been reported that prefibrillar a-synuclein aggregates can induce fragmentation of the Golgi apparatus (GA) [6]. On the other hand, disassembly of this important organelle produces an accumulation of unfolded proteins and can subsequently promote ER stress and apoptosis [2,7]. The GA is a specialized membranous cytoplasmic * Corresponding author. Tel.: þ 81-86-235-7410; fax: þ81-86-235-7412. E-mail address: [email protected] (M. Asanuma).
organelle, which is conformed by perinuclear aggregates of both stacked cisternal membranes and different vesicles [9]. This organelle is localized beside the centrosome and maintains a dynamic equilibrium with the ER [11]. The GA takes part in different metabolic pathways such as carbohydrate synthesis and protein trafficking [10]. Fragmentation of the GA has been linked to the pathogenesis of several representative neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer’s disease, and multiple system atrophy [5,14]. However, the significance of the GA disassembly in apoptosis and its relationship with neurodegeneration remain obscure. Thus, to clarify the involvement of GA disassembly in apoptosis of neuronal cells, we examined the morphological changes in the GA in dopaminergic neuroblastoma cells treated with rotenone. The rat dopaminergic neuroblastoma B65 cell line was obtained from the European Collection of Cell Culture (ECACC # 85042305, Salisbury, UK) and continuously cultured in Dulbecco’s Modified Eagle Medium (Gibco BRL, Rockville, MD) supplemented with 10% (v/v) fetal bovine serum, 2 mM L -glutamine, and 60 mg/ml kanamycin sulfate. Cells were plated at a density of 1 £ 104 cells/cm2 onto six-well culture plates and four-chamber glass culture slides (Becton Dickinson Labware, Franklin Lakes, NJ) for the viability assay and morphological study, respectively.
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.09.059
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F.J. Diaz-Corrales et al. / Neuroscience Letters 354 (2004) 59–63
Cells were maintained in the culture medium at 37 8C in a 5% to 95% CO2/air gas mixture. B65 cells were grown for 6 days (up to a cell confluence of 90 –95%), with the medium being changed every 2 – 3 days. After the first 24 h of incubation, cells were exposed to 50 nM rotenone (SigmaAldrich Corp., St. Louis, MO) diluted in dimethyl sulfoxide (DMSO) for 5 days (the final concentration of DMSO was 0.01%). The cell viability was then determined by a Trypan Blue exclusion assay. In the immunohistochemistry, B65 cells on the chamber slides were washed with 10 mM phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer for 10 min. Nonspecific antigens were blocked by 2.5% normal donkey serum with 0.1% Triton X100 in 10 mM PBS for 20 min. The culture slides were then incubated overnight at 4 8C with rabbit anti-g-adaptin antibodies (1:200), and goat anti-cytochrome c (1:100) or goat anti-caspase-3 p11 antibodies (1:100) (Santa Cruz Biotechnology, Santa Cruz, CA). The g-adaptin protein is a component of clathrin-coated vesicles of the GA [12]. The cells were then reacted with rhodamine-conjugated antirabbit IgG (1:200) and FITC-conjugated anti-goat IgG (1:200) (Chemicon International, Temecula, CA) for 2 h. Finally, cells were stained with 10 mg/ml Hoechst 33342 (Molecular Probes, Eugene, OR) for 2 min to visualize cell nuclei and chromatin condensation. Slides were analyzed under a fluorescence microscope (Olympus BX50-FLA, Tokyo, Japan). Photos of the immunostained cells were taken at a magnification of £ 400. Three independent experiments were conducted, and four random pictures were taken of either the vehicle- or rotenone-treated group in each experiment. The number of cells showing apoptotic changes or disassembly of the GA were counted in each picture, which corresponded to a 0.07 mm2 area. Cells with disassembly of the GA were defined as cells with a dispersion of compact perinuclear-foci of gadaptin-positive signals. Results are expressed as the total number of cells per mm2 and represent the mean ^ standard error (SEM). The statistical significance between the vehicle- and rotenone-treated groups was determined by the two-tailed Student’s t-test. Rotenone (50 nM) induced cell death in the dopaminergic neuroblastoma B65 cell line after 5 days of continuous treatment. The mean of the cell viability was 50.9 ^ 6.6% compared with the vehicle group (data not shown). At this time point, rotenone-treated cells showed a significant increase in both apoptotic changes and disassembly of the GA (Fig. 1). The number of apoptotic cells in the rotenonetreated group was increased by three fold in relation to the vehicle-treated group; however, the increment of cells showing disassembly of the GA after rotenone treatment was greater than the increment of cells showing apoptotic changes. Approximately 59% of cells with disassembly of the GA did not show clear apoptotic nuclei. Disassembly of the GA was observed in rotenone-treated cells with or without nuclear fragmentation (Fig. 2). Most of
Fig. 1. Effects of rotenone on the number of cells showing apoptotic changes or disassembly of the GA. B65 cells were treated with vehicle and 50 nM rotenone (Rot) for 5 days, then stained by Hoechst 33342 dye and gadaptin antibodies. The number of cells with either an apoptotic nucleus or disassembly of the GA was counted as described in the text. Values are expressed as the number of cells per mm2 and represent the mean ^ SEM of 12 observations in three independent experiments. *P , 0:01, **P , 0:001 compared with vehicle-treated groups (two-tailed Student’s t-test).
the cells in the vehicle-treated group presented oval nuclei and single strong foci of the GA signal located beside the nucleus (Fig. 2A). The rotenone treatment showed two types of damaged cells: one having a round nucleus without nuclear fragmentation (Fig. 2B,C), and another having a shrunken apoptotic nucleus with either fragmentation by budding (Fig. 2D) or complete nuclear fragmentation (Fig. 2E). In some cells after rotenone treatment, the nuclei became circular with apparently intact chromatins and diffuse g-adaptin-positive signals (Fig. 2B). Clear round nuclei with brightly stained chromatin condensation were also observed (Fig. 2C). In this stage, the GA signal was weak and appeared as a thin halo of small fragments distributed around the nucleus (Fig. 2C). Cells showing nuclear fragmentation by budding after rotenone treatment presented fragments of the GA dispersed throughout the cytosol (Fig. 2D). Finally, a complete disappearance of GA signals was observed in rotenone-treated cells with clear fragmentation of the nucleus (Fig. 2E). Double fluorescence immunohistochemistry for g-adaptin and cytochrome c or caspase-3 was performed to clarify when disassembly of the GA is involved in the apoptosis cascade (Fig. 3). Normal cells with compact perinuclear-foci of GA signals showed diffuse cytochrome c signals (Fig. 3A) but no caspase-3 expression (Fig. 3B). Cells without evidence of nuclear fragmentation with round nuclei and dispersion of the g-adaptin signals (as in Fig. 2B,C) presented a strong cytosolic redistribution of the cytochrome c signal (Fig. 3C), while they showed no caspase-3 expression (Fig.
F.J. Diaz-Corrales et al. / Neuroscience Letters 354 (2004) 59–63
61
Fig. 2. Immunostaining of the GA in both normal and apoptotic cells. B65 cells were treated with vehicle (A) or rotenone (50 nM) for 5 days (B– E). Nuclei were visualized using Hoechst 33342 dye (blue), and the GA was immunostained by g-adaptin antibodies (red). The normal morphology of cells with oval nuclei (A, left) and one strong foci of the GA signal are located near the nucleus (A, right: arrows). Rotenone-treated cells showed different stages of the apoptotic process (B –E, left) and disassembly of the GA (B–E, right). In some cells, nuclear fragmentation by budding was evident (D, left: arrowhead). Scale bar, 10 mm.
3D). In contrast, cells with fragmented nuclei and an almost complete loss of g-adaptin fluorescence (as in Fig. 2D,E) showed weak cytochrome c signals (Fig. 3E), but strong caspase-3 expression (Fig. 3F). Although disassembly of the GA remains obscure in the pathogenesis of parkinsonism, evidence has demonstrated that fragmentation of this organelle can be seen in the lesions of several neurodegenerative diseases with ubiquitinated cytoplasmic inclusions [5,14]. This evidence suggests that disassembly of the GA could be a common
phenomenon in some ubiquitinated cytoplasmic-inclusion diseases. The present study has revealed that rotenone, a common pesticide used to produce experimental models of parkinsonism [1,15], induces disassembly of the GA in dopaminergic neuroblastoma B65 cells. We observed a significant increase in the number of cells with disassembly of the GA in the rotenone-treated group. Most of the cells with disassembly of the GA after rotenone treatment did not show nuclear fragmentation. Moreover, cells having round nuclei showed a dispersion of the GA signal. Round nuclei,
62
F.J. Diaz-Corrales et al. / Neuroscience Letters 354 (2004) 59–63
Fig. 3. Double immunostaining of the GA and cytochrome c or caspase-3. B65 cells were treated with vehicle (A,B) and rotenone (50 nM) for 5 days (C –F). The left panels (A,C,E) show immunofluorescence of the GA by g-adaptin antibodies (red) and cytochrome c (green). The right panels (B,D,F) show the GA signals (red) and caspase-3 (green). Nuclei were visualized using Hoechst 33342 dye (blue). Cells with normal nuclei having a single strong signal of the GA (A,B), a diffused cytochrome c fluorescence (A), and negative caspase-3 expression (B). Rotenone-treated cells at different stages of the apoptotic process and disassembly of the GA (C–F). The cytosolic cytochrome c signal was strongly positive in cells with round nuclei (C: arrows). Caspase-3 expression was only evident in cells having completed fragmentation of the nucleus (F: arrowhead). Scale bar, 10 mm.
which were seen in the present study, have also been described as representing the initial change in nuclear shape during the apoptotic process [4]. As such, these results suggest that disassembly of the GA could occur before nuclear fragmentation in the initial stages of the apoptosis pathway induced by rotenone. Nevertheless, these morphological changes do not provide enough evidence to demonstrate chronological events in the apoptotic process. To elucidate the sequence of events in these cells, we performed double immunostaining of g-adaptin and cytochrome c or caspase-3. The release of cytochrome c from the mitochondria precedes the activation of caspase-3 [16]. In the present study, rotenone-treated cells with round nuclei and dispersion of the GA showed a redistribution of the cytochrome c signal, but not caspase-3 expression. These findings support the conclusion that disassembly of the GA induced by rotenone occurs in the early stages of the apoptotic process. The cause of the GA disassembly in rotenone-treated cells is unclear. It has been demonstrated that proteins of the GA can be cleaved by effector caspases such as caspase-3 [3]. However, we found dispersion of the GA in cells without expression of caspase-3, suggesting another possible reason for disassembly of this organelle. It has been reported that proteins of the GA can also be cleaved as well by caspase-2, which is an initiator caspase localized in both the GA and the nucleus [3,9]. An increase in the number of unfolded proteins and the amount of ER stress can activate caspase-2 [9]. Recently, ER stress has been considered as another alternative mechanism in rotenone-induced toxicity [13]. Moreover, it
has been found that aggregation of prefibrillar a-synuclein can induce fragmentation of the GA [6]. Taken together, these findings suggest that ER stress or an increase in the number of aggregated proteins may be considered as possible causes of GA disassembly at the early stages of rotenone-induced apoptotic cell damage.
Acknowledgements This work was supported in part by Grants-in-Aid for Research on Psychiatric and Neurological Diseases and Mental Health, for Comprehensive Research on Aging and Health, and for Research on Pharmaceutical and Medical Safety from the Japanese Ministry of Health, Labour, and Welfare, and Grants-in-Aid for Scientific Research (C), and for Young Scientists (B) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology.
References [1] R. Betarbet, T.B. Sherer, G. MacKenzie, M. Garcia-Osuna, A.V. Panov, J.T. Greenamyre, Chronic systemic pesticide exposure reproduces features of Parkinson’s disease, Nat. Neurosci. 3 (2000) 1301–1306. [2] L. Chen, X. Gao, Neuronal apoptosis induced by endoplasmic reticulum stress, Neurochem. Res. 27 (2002) 891–898. [3] R. Chiu, L. Novikov, S. Mukherjee, D. Shields, A caspase cleavage fragment of p115 induces fragmentation of the Golgi apparatus and apoptosis, J. Cell Biol. 159 (2002) 637– 648. [4] L. Dini, S. Coppola, M.T. Ruzittu, L. Ghibelli, Multiple pathways for apoptotic nuclear fragmentation, Exp. Cell Res. 223 (1996) 340 –347.
F.J. Diaz-Corrales et al. / Neuroscience Letters 354 (2004) 59–63 [5] N.K. Gonatas, J.O. Gonatas, A. Stieber, The involvement of Golgi apparatus in the pathogenesis of amyotrophic lateral sclerosis, Alzheimer’s disease, and ricin intoxication, Histochem. Cell Biol. 109 (1998) 591– 600. [6] N. Gosavi, H.-J. Lee, J.S. Lee, S. Patel, S.-J. Lee, Golgi fragmentation occurs in the cells with prefibrillar a-synuclein aggregates and precedes the formation of fibrillar inclusion, J. Biol. Chem. 277 (2002) 48984–48992. [7] S. Kikuchi, K. Shinpo, S. Tsuji, I. Yabe, M. Niino, K. Tashiro, Brefeldin A-induced neurotoxicity in cultured spinal cord neurons, J. Neurosci. Res. 71 (2003) 591–599. [8] H.-J. Lee, S.Y. Shin, C. Choi, Y.H. Lee, S.-J. Lee, Formation and removal of a-synuclein aggregates in cells exposed to mitochondrial inhibitors, J. Biol. Chem. 277 (2002) 5411–5417. [9] C.E. Machamer, Golgi disassembly in apoptosis: cause or effect?, Trends Cell Biol. 13 (2003) 279–281. [10] K. Nozawa, C.A. Casiano, J.C. Hamel, C. Molinaro, M.J. Fritzler, E.K.L. Chan, Fragmentation of Golgi complex and Golgi autoantigens during apoptosis and necrosis, Arthritis Res. 4 (2002) R3. [11] R.M. Rios, M. Bornens, The Golgi apparatus at the cell centre, Curr. Opin. Cell Biol. 15 (2003) 60–66. [12] M.S. Robinson, Cloning and expression of g-adaptin, a component of
[13]
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[16]
[17]
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clathrin-coated vesicles associated with the Golgi apparatus, J. Cell Biol. 111 (1990) 2319– 2326. E.J. Ryu, H.P. Harding, J.M. Angelastro, O.V. Vitolo, D. Ron, L.A. Greene, Endoplasmic reticulum stress and the unfolded protein response in cellular models of Parkinson’s disease, J. Neurosci. 22 (2002) 10690– 10698. A. Sakurai, K. Okamoto, M. Yaguchi, Y. Fujita, Y. Mizuno, Y. Nakazato, N.K. Gonatas, Pathology of the inferior olivary nucleus in patients with multiple system atrophy, Acta Neuropathol. 103 (2002) 550–554. T.B. Sherer, R. Betarbet, A.K. Stout, S. Lund, M. Baptista, A.V. Panov, M.R. Cookson, J.T. Greenamyre, An in vitro model of Parkinson’s disease: linking mitochondrial impairment to altered asynuclein metabolism and oxidative damage, J. Neurosci. 22 (2002) 7006– 7015. W.G. Tatton, R. Chalmers-Redman, D. Brown, N. Tatton, Apoptosis in Parkinson’s disease: signals for neuronal degradation, Ann. Neurol. 53 (2003) S61–S72. V.N. Uversky, J. Li, A.L. Fink, Pesticides directly accelerate the rate of a-synuclein fibril formation: a possible factor in Parkinson’s disease, FEBS Lett. 500 (2001) 105– 108.
Rotenone induces disassembly of the Golgi apparatus in the rat dopaminergic neuroblastoma B65 cell line Francisco J. Diaz-Corrales, Masato Asanuma*, Ikuko Miyazaki, Norio Ogawa Department of Brain Science, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikatacho, Okayama 700-8558, Japan Received 27 August 2003; received in revised form 12 September 2003; accepted 16 September 2003
Abstract It has been reported that the Golgi apparatus (GA) is fragmented in some neurodegenerative diseases. However, the significance of the GA fragmentation or disassembly in neurodegeneration is still obscure. To clarify the involvement of this organelle in apoptosis of neuronal cells, we examined the morphological changes in the GA induced by rotenone, a pesticide that produces selective dopaminergic neurodegeneration. In dopaminergic neuroblastoma B65 cells, a 5-day rotenone treatment (50 nM) promoted cell damage. Rotenonetreated cells showed round nuclei, diffuse signals of the GA and cytosolic redistribution of cytochrome c. Nevertheless, these type of cells without nuclear fragmentation did not show any caspase-3 expression. These results indicate that rotenone induces disassembly of the GA in the early stages of the apoptotic process. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Rotenone; Disassembly of the Golgi apparatus; Endoplasmic reticulum stress; Apoptosis; Neurodegeneration; Parkinsonism
Rotenone is a selective inhibitor of mitochondrial complex I activity, and this pesticide can reproduce many features of Parkinson’s disease [1]. Nevertheless, the exact mechanisms of dopaminergic neuron-specific degeneration induced by rotenone are not completely understood. Evidence supports the hypothesis that production of reactive oxygen species is not the only way to induce cell death by rotenone [17]. Moreover, Ryu et al. have reported that endoplasmic reticulum (ER) stress can contribute to rotenone-induced toxicity [13]. In dopaminergic neuroblastoma cells, rotenone treatment produces apoptotic cells and protein aggregation of both asynuclein and ubiquitin [15]. One of the mechanisms proposed for this protein aggregation is that a reduction in energy production generates an inadequate defense against unfolded proteins [8]. It has recently been reported that prefibrillar a-synuclein aggregates can induce fragmentation of the Golgi apparatus (GA) [6]. On the other hand, disassembly of this important organelle produces an accumulation of unfolded proteins and can subsequently promote ER stress and apoptosis [2,7]. The GA is a specialized membranous cytoplasmic * Corresponding author. Tel.: þ 81-86-235-7410; fax: þ81-86-235-7412. E-mail address: [email protected] (M. Asanuma).
organelle, which is conformed by perinuclear aggregates of both stacked cisternal membranes and different vesicles [9]. This organelle is localized beside the centrosome and maintains a dynamic equilibrium with the ER [11]. The GA takes part in different metabolic pathways such as carbohydrate synthesis and protein trafficking [10]. Fragmentation of the GA has been linked to the pathogenesis of several representative neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer’s disease, and multiple system atrophy [5,14]. However, the significance of the GA disassembly in apoptosis and its relationship with neurodegeneration remain obscure. Thus, to clarify the involvement of GA disassembly in apoptosis of neuronal cells, we examined the morphological changes in the GA in dopaminergic neuroblastoma cells treated with rotenone. The rat dopaminergic neuroblastoma B65 cell line was obtained from the European Collection of Cell Culture (ECACC # 85042305, Salisbury, UK) and continuously cultured in Dulbecco’s Modified Eagle Medium (Gibco BRL, Rockville, MD) supplemented with 10% (v/v) fetal bovine serum, 2 mM L -glutamine, and 60 mg/ml kanamycin sulfate. Cells were plated at a density of 1 £ 104 cells/cm2 onto six-well culture plates and four-chamber glass culture slides (Becton Dickinson Labware, Franklin Lakes, NJ) for the viability assay and morphological study, respectively.
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.09.059
60
F.J. Diaz-Corrales et al. / Neuroscience Letters 354 (2004) 59–63
Cells were maintained in the culture medium at 37 8C in a 5% to 95% CO2/air gas mixture. B65 cells were grown for 6 days (up to a cell confluence of 90 –95%), with the medium being changed every 2 – 3 days. After the first 24 h of incubation, cells were exposed to 50 nM rotenone (SigmaAldrich Corp., St. Louis, MO) diluted in dimethyl sulfoxide (DMSO) for 5 days (the final concentration of DMSO was 0.01%). The cell viability was then determined by a Trypan Blue exclusion assay. In the immunohistochemistry, B65 cells on the chamber slides were washed with 10 mM phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer for 10 min. Nonspecific antigens were blocked by 2.5% normal donkey serum with 0.1% Triton X100 in 10 mM PBS for 20 min. The culture slides were then incubated overnight at 4 8C with rabbit anti-g-adaptin antibodies (1:200), and goat anti-cytochrome c (1:100) or goat anti-caspase-3 p11 antibodies (1:100) (Santa Cruz Biotechnology, Santa Cruz, CA). The g-adaptin protein is a component of clathrin-coated vesicles of the GA [12]. The cells were then reacted with rhodamine-conjugated antirabbit IgG (1:200) and FITC-conjugated anti-goat IgG (1:200) (Chemicon International, Temecula, CA) for 2 h. Finally, cells were stained with 10 mg/ml Hoechst 33342 (Molecular Probes, Eugene, OR) for 2 min to visualize cell nuclei and chromatin condensation. Slides were analyzed under a fluorescence microscope (Olympus BX50-FLA, Tokyo, Japan). Photos of the immunostained cells were taken at a magnification of £ 400. Three independent experiments were conducted, and four random pictures were taken of either the vehicle- or rotenone-treated group in each experiment. The number of cells showing apoptotic changes or disassembly of the GA were counted in each picture, which corresponded to a 0.07 mm2 area. Cells with disassembly of the GA were defined as cells with a dispersion of compact perinuclear-foci of gadaptin-positive signals. Results are expressed as the total number of cells per mm2 and represent the mean ^ standard error (SEM). The statistical significance between the vehicle- and rotenone-treated groups was determined by the two-tailed Student’s t-test. Rotenone (50 nM) induced cell death in the dopaminergic neuroblastoma B65 cell line after 5 days of continuous treatment. The mean of the cell viability was 50.9 ^ 6.6% compared with the vehicle group (data not shown). At this time point, rotenone-treated cells showed a significant increase in both apoptotic changes and disassembly of the GA (Fig. 1). The number of apoptotic cells in the rotenonetreated group was increased by three fold in relation to the vehicle-treated group; however, the increment of cells showing disassembly of the GA after rotenone treatment was greater than the increment of cells showing apoptotic changes. Approximately 59% of cells with disassembly of the GA did not show clear apoptotic nuclei. Disassembly of the GA was observed in rotenone-treated cells with or without nuclear fragmentation (Fig. 2). Most of
Fig. 1. Effects of rotenone on the number of cells showing apoptotic changes or disassembly of the GA. B65 cells were treated with vehicle and 50 nM rotenone (Rot) for 5 days, then stained by Hoechst 33342 dye and gadaptin antibodies. The number of cells with either an apoptotic nucleus or disassembly of the GA was counted as described in the text. Values are expressed as the number of cells per mm2 and represent the mean ^ SEM of 12 observations in three independent experiments. *P , 0:01, **P , 0:001 compared with vehicle-treated groups (two-tailed Student’s t-test).
the cells in the vehicle-treated group presented oval nuclei and single strong foci of the GA signal located beside the nucleus (Fig. 2A). The rotenone treatment showed two types of damaged cells: one having a round nucleus without nuclear fragmentation (Fig. 2B,C), and another having a shrunken apoptotic nucleus with either fragmentation by budding (Fig. 2D) or complete nuclear fragmentation (Fig. 2E). In some cells after rotenone treatment, the nuclei became circular with apparently intact chromatins and diffuse g-adaptin-positive signals (Fig. 2B). Clear round nuclei with brightly stained chromatin condensation were also observed (Fig. 2C). In this stage, the GA signal was weak and appeared as a thin halo of small fragments distributed around the nucleus (Fig. 2C). Cells showing nuclear fragmentation by budding after rotenone treatment presented fragments of the GA dispersed throughout the cytosol (Fig. 2D). Finally, a complete disappearance of GA signals was observed in rotenone-treated cells with clear fragmentation of the nucleus (Fig. 2E). Double fluorescence immunohistochemistry for g-adaptin and cytochrome c or caspase-3 was performed to clarify when disassembly of the GA is involved in the apoptosis cascade (Fig. 3). Normal cells with compact perinuclear-foci of GA signals showed diffuse cytochrome c signals (Fig. 3A) but no caspase-3 expression (Fig. 3B). Cells without evidence of nuclear fragmentation with round nuclei and dispersion of the g-adaptin signals (as in Fig. 2B,C) presented a strong cytosolic redistribution of the cytochrome c signal (Fig. 3C), while they showed no caspase-3 expression (Fig.
F.J. Diaz-Corrales et al. / Neuroscience Letters 354 (2004) 59–63
61
Fig. 2. Immunostaining of the GA in both normal and apoptotic cells. B65 cells were treated with vehicle (A) or rotenone (50 nM) for 5 days (B– E). Nuclei were visualized using Hoechst 33342 dye (blue), and the GA was immunostained by g-adaptin antibodies (red). The normal morphology of cells with oval nuclei (A, left) and one strong foci of the GA signal are located near the nucleus (A, right: arrows). Rotenone-treated cells showed different stages of the apoptotic process (B –E, left) and disassembly of the GA (B–E, right). In some cells, nuclear fragmentation by budding was evident (D, left: arrowhead). Scale bar, 10 mm.
3D). In contrast, cells with fragmented nuclei and an almost complete loss of g-adaptin fluorescence (as in Fig. 2D,E) showed weak cytochrome c signals (Fig. 3E), but strong caspase-3 expression (Fig. 3F). Although disassembly of the GA remains obscure in the pathogenesis of parkinsonism, evidence has demonstrated that fragmentation of this organelle can be seen in the lesions of several neurodegenerative diseases with ubiquitinated cytoplasmic inclusions [5,14]. This evidence suggests that disassembly of the GA could be a common
phenomenon in some ubiquitinated cytoplasmic-inclusion diseases. The present study has revealed that rotenone, a common pesticide used to produce experimental models of parkinsonism [1,15], induces disassembly of the GA in dopaminergic neuroblastoma B65 cells. We observed a significant increase in the number of cells with disassembly of the GA in the rotenone-treated group. Most of the cells with disassembly of the GA after rotenone treatment did not show nuclear fragmentation. Moreover, cells having round nuclei showed a dispersion of the GA signal. Round nuclei,
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F.J. Diaz-Corrales et al. / Neuroscience Letters 354 (2004) 59–63
Fig. 3. Double immunostaining of the GA and cytochrome c or caspase-3. B65 cells were treated with vehicle (A,B) and rotenone (50 nM) for 5 days (C –F). The left panels (A,C,E) show immunofluorescence of the GA by g-adaptin antibodies (red) and cytochrome c (green). The right panels (B,D,F) show the GA signals (red) and caspase-3 (green). Nuclei were visualized using Hoechst 33342 dye (blue). Cells with normal nuclei having a single strong signal of the GA (A,B), a diffused cytochrome c fluorescence (A), and negative caspase-3 expression (B). Rotenone-treated cells at different stages of the apoptotic process and disassembly of the GA (C–F). The cytosolic cytochrome c signal was strongly positive in cells with round nuclei (C: arrows). Caspase-3 expression was only evident in cells having completed fragmentation of the nucleus (F: arrowhead). Scale bar, 10 mm.
which were seen in the present study, have also been described as representing the initial change in nuclear shape during the apoptotic process [4]. As such, these results suggest that disassembly of the GA could occur before nuclear fragmentation in the initial stages of the apoptosis pathway induced by rotenone. Nevertheless, these morphological changes do not provide enough evidence to demonstrate chronological events in the apoptotic process. To elucidate the sequence of events in these cells, we performed double immunostaining of g-adaptin and cytochrome c or caspase-3. The release of cytochrome c from the mitochondria precedes the activation of caspase-3 [16]. In the present study, rotenone-treated cells with round nuclei and dispersion of the GA showed a redistribution of the cytochrome c signal, but not caspase-3 expression. These findings support the conclusion that disassembly of the GA induced by rotenone occurs in the early stages of the apoptotic process. The cause of the GA disassembly in rotenone-treated cells is unclear. It has been demonstrated that proteins of the GA can be cleaved by effector caspases such as caspase-3 [3]. However, we found dispersion of the GA in cells without expression of caspase-3, suggesting another possible reason for disassembly of this organelle. It has been reported that proteins of the GA can also be cleaved as well by caspase-2, which is an initiator caspase localized in both the GA and the nucleus [3,9]. An increase in the number of unfolded proteins and the amount of ER stress can activate caspase-2 [9]. Recently, ER stress has been considered as another alternative mechanism in rotenone-induced toxicity [13]. Moreover, it
has been found that aggregation of prefibrillar a-synuclein can induce fragmentation of the GA [6]. Taken together, these findings suggest that ER stress or an increase in the number of aggregated proteins may be considered as possible causes of GA disassembly at the early stages of rotenone-induced apoptotic cell damage.
Acknowledgements This work was supported in part by Grants-in-Aid for Research on Psychiatric and Neurological Diseases and Mental Health, for Comprehensive Research on Aging and Health, and for Research on Pharmaceutical and Medical Safety from the Japanese Ministry of Health, Labour, and Welfare, and Grants-in-Aid for Scientific Research (C), and for Young Scientists (B) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology.
References [1] R. Betarbet, T.B. Sherer, G. MacKenzie, M. Garcia-Osuna, A.V. Panov, J.T. Greenamyre, Chronic systemic pesticide exposure reproduces features of Parkinson’s disease, Nat. Neurosci. 3 (2000) 1301–1306. [2] L. Chen, X. Gao, Neuronal apoptosis induced by endoplasmic reticulum stress, Neurochem. Res. 27 (2002) 891–898. [3] R. Chiu, L. Novikov, S. Mukherjee, D. Shields, A caspase cleavage fragment of p115 induces fragmentation of the Golgi apparatus and apoptosis, J. Cell Biol. 159 (2002) 637– 648. [4] L. Dini, S. Coppola, M.T. Ruzittu, L. Ghibelli, Multiple pathways for apoptotic nuclear fragmentation, Exp. Cell Res. 223 (1996) 340 –347.
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