Abstract
Defects in the proton-translocating NADH-quinone oxidoreductase (complex I) of mammalian mitochondria are linked to neurodegenerative disorders. The mechanism leading to cell death elicited by complex I deficiency remains elusive. We have shown that expression of a rotenone-insensitive yeast NADH-quinone oxidoreductase (Ndi1) can rescue mammalian cells from complex I dysfunction. By using the Ndi1 enzyme, we have investigated the key events in the process of cell death using a rat dopaminergic cell line, PC12. We found that complex I inhibition provokes the following events: 1) activation of specific kinase pathways; 2) release of mitochondrial proapoptotic factors, apoptosis inducing factor, and endonuclease G. AS601245, a kinase inhibitor, exhibited significant protection against these apoptotic events. The traditional caspase pathway does not seems to be involved because caspase 3 activation was not observed. Our data suggest that overproduction of reactive oxygen species (ROS) caused by complex I inhibition is responsible for triggering the kinase activation, for the release of the proapoptotic factors, and then for cell death. Nearly perfect prevention of apoptotic cell death by Ndi1 agrees with our earlier observation that the presence of Ndi1 diminishes rotenone-induced ROS generation from complex I. In fact, this study demonstrated that Ndi1 keeps the redox potential high even in the presence of rotenone. Under these conditions, ROS formation by complex I is known to be minimal. Possible use of our cellular model is discussed with regard to development of therapeutic strategies for neurodegenerative diseases caused by complex I defects.
Highlights
In the case of MPTP mouse Parkinson disease (PD) models, it has been reported that apoptosis is involved in the neurodegeneration
PC12 and PC12-NDI1 cells were incubated with increasing concentrations of rotenone for 4 days, and the extent of cell death was assessed by staining the cells with trypan blue (Fig. 1)
In addition to their function as the major energy source, mitochondria play a central role in cellular processes such as apoptosis [36, 37]
Summary
Materials—Antibodies against AIF and EndoG were purchased from Chemicon International (Temecula, CA). To evaluate the AIF/ EndoG relocalization, cells were incubated in DMEM with 1% serum with or without 1 M rotenone for 48 h and were harvested in 750 l of isotonic mitochondria buffer (210 mM mannitol, 70 mM sucrose, 1 mM EDTA, 10 mM HEPES, pH 7.5) supplemented with a complete mixture of protease inhibitors. PC12 and PC12-NDI1 cells were grown in 12-well plates, and the medium was replaced with DMEM with 1% serum for 18 h before the start of the experiment. Primary mouse antibody anti-8-oxo-dG (1/300) was added to the cells in TBS with 2.5% FBS, 2.5% horse serum, and 0.05% Triton X-100 overnight at 4 °C. Cells were incubated with 1 M rotenone in DMEM with 1% serum for up to 6 h and washed twice with warm medium. Any variations from the procedures and other details are described in the figure legends
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