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Two caspase-mediated apoptotic pathways induced by rotenone toxicity in cortical neuronal cells¹

Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

³Correspondence: Department of Neurology, University of Pittsburgh School of Medicine, S526, Biomedical Science Tower, 3500, Terrace St., Pittsburgh, PA 15261, USA. E-mail: jun{at}med.pitt.edu

SPECIFIC AIM

We wanted to elucidate all the apoptotic pathways elicited by a sublethal dosage of rotenone treatment to primary cortical neurons. We also addressed the controversy of the chronological and casual relationship between the two events: cytochrome c release from the mitochondria and mitochondrial permeability transition (MPT).

PRINCIPAL FINDINGS

Rotenone-induced cell death can be attributed to oxidative stress and mitochondrial permeability transition
Upon establishing that treatment with 0.5 µM of rotenone for 24 h elicited mainly apoptosis and the presence of protein synthesis inhibitor were able to attenuate the cell death from 60% to 20%, we examined the oxidative component of the degenerative pathway. We have found that there is an increase in the ROS production as early as after 2–6 h of rotenone treatment. However, in the presence of antioxidants N-acetyl-L-cysteine (NAC) and N-t-butyl--phenyl-nitrone (BPN), the percentage cell death of the cortical neurons is significantly decreased from 60% to 30%. Quantification of cell death by trypan blue exclusion method and Hoechst 33342 staining agree, suggesting oxidative stress to be a major cause of cell death for the primary cortical neurons. After 24 h of rotenone treatment, depolarization of the mitochondria causes the emergence of MPT as indicated by the increase in the florescence signal corresponding to the monomeric form of JC-1 probe. These results in concert suggest oxidative stress as the primary cause of cell death. Nevertheless, prolonged exposure to the stress appears to instigate MPT, which could have a secondary detrimental effect on cell viability.

Cytochrome c release is not a consequence of the emergence of MPT
The apoptotic cell death resulting from oxidative stress and MPT typically involved release of cytochrome c from the mitochondria to the cytoplasm. However, the chronological and causal relationship between this latter event and MPT is still uncertain. We wanted to determine whether cytochrome c release is a direct result of ROS elevation or a consequence of MPT. We have charted the localization pattern and temporal emergence of cytochrome c in the cytoplasm over a time course of 24 h and the results are presented in Fig. 1 A, B. By immunocytochemistry, we observed that cytochrome c (represented by the green signals in Fig. 1A ) migrated from the mitochondria to the cytoplasm as early as after 2–6 h of rotenone treatment. This is again confirmed by Western blot, indicating the emergence of cytochrome c in the cytoplasm within the same time frame. Since the emergence of MPT is 24 h, the release of cytochrome c is at least 18 h earlier, rendering it impossible for MPT to be the cause of cytochrome c release. Figure 1C shows that the release of cytochrome c is inhibited by antioxidants NAC and BPN but not by the MPT inhibitor cyclosporin, again suggesting the causal independence of these two events in our study.

View larger version (30K): [in this window] [in a new window]   Figure 1. Significant rescue from death of cortical neurons under the cytotoxicty of rotenone in the presence of caspase-1 inhibitor (z-YVAD-fmk) and caspase-3 inhibitor (z-DEVD-fmk). The percentage of cell death was quantified by measuring the ratio of the LDH release, the ratio of the number of dead cells by the trypan blue exclusion method, the ratio of the amount of DNA damage, and the ratio of the number of cells exhibiting nuclear condensation between the challenged cell population and the control cell population. Data are the means ± SE pf at least nine measurements per data point, from three separate experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 derived from ANOVA and post-hoc PLSD tests.

Activation of both caspase-3 and caspase-1 leads to cell death
Finally, we would like to elucidate the apoptotic pathway elicited by cortical neurons when challenged with rotenone. It is clear from the first two panels of Fig. 2 that the presence of caspase-3 and caspase-1 inhibitors can attenuate the degeneration process significantly. Based on what we know, the activation of caspase-3 is expected and is likely to result from the release of cytochrome c. However, the involvement of caspase-1 in the degeneration process was unexpected. The expected significant attenuation of DNA damage and nuclear condensation in the presence of caspase-3 inhibitor but not in the presence of caspase-1 inhibitor suggest that the caspase-1-mediated cell death pathway is mechanistically distinct from that mediated by caspase-3. Therefore, our results suggested that two caspase-mediated pathways are used by cortical neurons to degenerate under the cytotoxicity of rotenone.

View larger version (31K): [in this window] [in a new window]   Figure 2. Evidence showing the release of cytochrome c from the mitochondria is an early event and its response to antioxidants and MPT inhibitor. In addition, changes in caspase 3 activity in the presence of antioxidants, caspase 3 inhibitor and MPT inhibitor are also presented. A) Panels a-d showing cytochrome c release from the mitochondria in dying cells after 0, 2, 6, and 24 hours of rotenone treatment. The localization pattern is visualized by double labelling with an anti-cytochrome c antibody and staining with propidium iodide. B) Western blot of the cytosolic protein of cortical neurons after 2, 6, and 24 hours of rotenone treatment. In addition, the cytochrome c release pattern in the presence of antioxidants NAC and BPN as well as MPT inhibitor, cyclosporin A are also presented. The population of the cortical neurons that are cultured for 6 hours upon the instigation of the experiments are used as control. The actin band on the blot was used as a normalization indicator for this set of experiments. C) The response of caspase 3 activity under the cytotoxicity of rotenone. In panel a, the western blot indicates the significant decrease in the cleaved form of caspase 3 in the presence of caspase 3 inhibitor DEVD and antioxidants BPN and NAC as compared to control showed as the first lane in the blot. Panel b showed again the attenuation of the caspase 3 activity in the presence of caspase 3 inhibitor and antioxidants (NAC and BPN) but not in the presence of MPT inhibitor cyclosporin A (CsA). Data are the mean ± SE of at least 9 measurements per data point, from three independent experiments. *P < 0.05 derived from ANOVA and post hoc PLSD tests.

CONCLUSIONS AND SIGNIFICANCE

The results from our study have again indicated the importance of the cytochrome c-induced, caspase-9/caspase-3-mediated cell death pathway when cells are challenged with pharmacological agents that elevate the intracellular oxidative stress. Our results suggest that cytochrome c release is an independent process from MPT. However, what is unexpected is the involvement of caspase-1 in a mechanistic distinct pathway used by primary cortical neurons to degenerate. The notion that caspase-1 as an effecter molecule for cell death goes against the traditional role of caspase-1 in the context of apoptosis. Nevertheless, the apoptotic mechanism of caspase-1-mediated cell death pathway is yet to be determined. From the therapeutic viewpoint, it is important to document and elucidate all the apoptotic pathways in order to develop an effective agent to nullify cell death. Hence, our results have provided new information to be taken into consideration in the development of a good therapeutic agent.

View larger version (30K): [in this window] [in a new window]   Figure 3. Schematic diagram

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0653fje; to cite this article, use FASEB J. (January 27, 2003) 10.1096/fj.02-0653fje

² Both authors contributed equally to this manuscript.

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