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Differential production of superoxide by neuronal mitochondria.

Hoegger MJ, Lieven CJ, Levin LA - BMC Neurosci (2008)

Bottom Line: Cerebral but not RGC-5 or neuroblastoma cells increased superoxide production in response to the complex I inhibitor rotenone, while neuroblastoma but not cerebral or RGC-5 cells dramatically decreased superoxide production in response to the complex III inhibitor antimycin A.RGC-5 mitochondria produce superoxide at significantly lower rates than cerebral and neuroblastoma mitochondria, most likely as a result of differential expression of complex I components.Diversity in METC component expression and function could explain tissue specificity in diseases associated with inherited mtDNA abnormalities.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin Medical School, Madison, USA. markhoegger@gmail.com

ABSTRACT

Background: Mitochondrial DNA (mtDNA) mutations, which are present in all mitochondria-containing cells, paradoxically cause tissue-specific disease. For example, Leber's hereditary optic neuropathy (LHON) results from one of three point mutations mtDNA coding for complex I components, but is only manifested in retinal ganglion cells (RGCs), a central neuron contained within the retina. Given that RGCs use superoxide for intracellular signaling after axotomy, and that LHON mutations increase superoxide levels in non-RGC transmitochondrial cybrids, we hypothesized that RGCs regulate superoxide levels differently than other neuronal cells. To study this, we compared superoxide production and mitochondrial electron transport chain (METC) components in isolated RGC mitochondria to mitochondria isolated from cerebral cortex and neuroblastoma SK-N-AS cells.

Results: In the presence of the complex I substrate glutamate/malate or the complex II substrate succinate, the rate of superoxide production in RGC-5 cells was significantly lower than cerebral or neuroblastoma cells. Cerebral but not RGC-5 or neuroblastoma cells increased superoxide production in response to the complex I inhibitor rotenone, while neuroblastoma but not cerebral or RGC-5 cells dramatically decreased superoxide production in response to the complex III inhibitor antimycin A. Immunoblotting and real-time quantitative PCR of METC components demonstrated different patterns of expression among the three different sources of neuronal mitochondria.

Conclusion: RGC-5 mitochondria produce superoxide at significantly lower rates than cerebral and neuroblastoma mitochondria, most likely as a result of differential expression of complex I components. Diversity in METC component expression and function could explain tissue specificity in diseases associated with inherited mtDNA abnormalities.

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Superoxide Production in Cerebral, Neuroblastoma, and RGC-5 Mitochondria after Complex III inhibition. Mitochondria isolated from cerebral, neuroblastoma, and undifferentiated RGC-5 cells were normalized for protein content and superoxide production was analyzed after the addition of the complex II substrate succinate and the complex III inhibitor antimycin A. The mitochondrial superoxide production levels in cerebral cells were significantly higher than that of undifferentiated RGC-5 cells in the presence of succinate and after complex III inhibition with antimycin A. Both succinate and antimycin A elicited minimal superoxide production in undifferentiated RGC-5 cells, while in cerebral cells there was a near two-fold increase in superoxide production after the addition of antimycin A.
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Figure 4: Superoxide Production in Cerebral, Neuroblastoma, and RGC-5 Mitochondria after Complex III inhibition. Mitochondria isolated from cerebral, neuroblastoma, and undifferentiated RGC-5 cells were normalized for protein content and superoxide production was analyzed after the addition of the complex II substrate succinate and the complex III inhibitor antimycin A. The mitochondrial superoxide production levels in cerebral cells were significantly higher than that of undifferentiated RGC-5 cells in the presence of succinate and after complex III inhibition with antimycin A. Both succinate and antimycin A elicited minimal superoxide production in undifferentiated RGC-5 cells, while in cerebral cells there was a near two-fold increase in superoxide production after the addition of antimycin A.

Mentions: Mitochondria were incubated with succinate (10 mM), which yields FADH2 and serves as a substrate for complex II. In the presence of succinate, there was a small but significant increase in superoxide production in cerebral mitochondria (0.193 ± 0.027 to 0.216 ± 0.022; p = 0.04) and RGC-5 mitochondria (0.028 ± 0.005 to 0.031 ± 0.005; p = 0.024). Subsequent treatment with the complex III inhibitor antimycin A (0.5 μM) resulted in a dramatic increase in the rate of superoxide production in cerebral mitochondria (0.540 ± 0.116; p = .023 compared to substrate alone), but not RGC-5 mitochondria (0.024 ± 0.004; p = 0.278). The rates of superoxide production in all conditions were substantially greater in cerebral mitochondria than in RGC-5 mitochondria (Table 2). A typical experiment is depicted in Figure 4.


Differential production of superoxide by neuronal mitochondria.

Hoegger MJ, Lieven CJ, Levin LA - BMC Neurosci (2008)

Superoxide Production in Cerebral, Neuroblastoma, and RGC-5 Mitochondria after Complex III inhibition. Mitochondria isolated from cerebral, neuroblastoma, and undifferentiated RGC-5 cells were normalized for protein content and superoxide production was analyzed after the addition of the complex II substrate succinate and the complex III inhibitor antimycin A. The mitochondrial superoxide production levels in cerebral cells were significantly higher than that of undifferentiated RGC-5 cells in the presence of succinate and after complex III inhibition with antimycin A. Both succinate and antimycin A elicited minimal superoxide production in undifferentiated RGC-5 cells, while in cerebral cells there was a near two-fold increase in superoxide production after the addition of antimycin A.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2266764&req=5

Figure 4: Superoxide Production in Cerebral, Neuroblastoma, and RGC-5 Mitochondria after Complex III inhibition. Mitochondria isolated from cerebral, neuroblastoma, and undifferentiated RGC-5 cells were normalized for protein content and superoxide production was analyzed after the addition of the complex II substrate succinate and the complex III inhibitor antimycin A. The mitochondrial superoxide production levels in cerebral cells were significantly higher than that of undifferentiated RGC-5 cells in the presence of succinate and after complex III inhibition with antimycin A. Both succinate and antimycin A elicited minimal superoxide production in undifferentiated RGC-5 cells, while in cerebral cells there was a near two-fold increase in superoxide production after the addition of antimycin A.
Mentions: Mitochondria were incubated with succinate (10 mM), which yields FADH2 and serves as a substrate for complex II. In the presence of succinate, there was a small but significant increase in superoxide production in cerebral mitochondria (0.193 ± 0.027 to 0.216 ± 0.022; p = 0.04) and RGC-5 mitochondria (0.028 ± 0.005 to 0.031 ± 0.005; p = 0.024). Subsequent treatment with the complex III inhibitor antimycin A (0.5 μM) resulted in a dramatic increase in the rate of superoxide production in cerebral mitochondria (0.540 ± 0.116; p = .023 compared to substrate alone), but not RGC-5 mitochondria (0.024 ± 0.004; p = 0.278). The rates of superoxide production in all conditions were substantially greater in cerebral mitochondria than in RGC-5 mitochondria (Table 2). A typical experiment is depicted in Figure 4.

Bottom Line: Cerebral but not RGC-5 or neuroblastoma cells increased superoxide production in response to the complex I inhibitor rotenone, while neuroblastoma but not cerebral or RGC-5 cells dramatically decreased superoxide production in response to the complex III inhibitor antimycin A.RGC-5 mitochondria produce superoxide at significantly lower rates than cerebral and neuroblastoma mitochondria, most likely as a result of differential expression of complex I components.Diversity in METC component expression and function could explain tissue specificity in diseases associated with inherited mtDNA abnormalities.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin Medical School, Madison, USA. markhoegger@gmail.com

ABSTRACT

Background: Mitochondrial DNA (mtDNA) mutations, which are present in all mitochondria-containing cells, paradoxically cause tissue-specific disease. For example, Leber's hereditary optic neuropathy (LHON) results from one of three point mutations mtDNA coding for complex I components, but is only manifested in retinal ganglion cells (RGCs), a central neuron contained within the retina. Given that RGCs use superoxide for intracellular signaling after axotomy, and that LHON mutations increase superoxide levels in non-RGC transmitochondrial cybrids, we hypothesized that RGCs regulate superoxide levels differently than other neuronal cells. To study this, we compared superoxide production and mitochondrial electron transport chain (METC) components in isolated RGC mitochondria to mitochondria isolated from cerebral cortex and neuroblastoma SK-N-AS cells.

Results: In the presence of the complex I substrate glutamate/malate or the complex II substrate succinate, the rate of superoxide production in RGC-5 cells was significantly lower than cerebral or neuroblastoma cells. Cerebral but not RGC-5 or neuroblastoma cells increased superoxide production in response to the complex I inhibitor rotenone, while neuroblastoma but not cerebral or RGC-5 cells dramatically decreased superoxide production in response to the complex III inhibitor antimycin A. Immunoblotting and real-time quantitative PCR of METC components demonstrated different patterns of expression among the three different sources of neuronal mitochondria.

Conclusion: RGC-5 mitochondria produce superoxide at significantly lower rates than cerebral and neuroblastoma mitochondria, most likely as a result of differential expression of complex I components. Diversity in METC component expression and function could explain tissue specificity in diseases associated with inherited mtDNA abnormalities.

Show MeSH
Related in: MedlinePlus