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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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Related in: MedlinePlus

Immunoblotting Measurement of Cytochrome c Oxidase in Differentiated and Undifferentiated RGC-5 Mitochondria. RGC-5 cells were differentiated with staurosporine. Isolated mitochondrial samples from undifferentiated and differentiated mitochondria standardized for protein content were compared to corresponding mitochondria depleted samples for the presence of the mitochondrial transmembrane protein cytochrome c oxidase (COX), complex IV. Samples were immunoblotted with antibody to subunit 1 of complex IV. There was considerable purification of mitochondria compared to the mitochondria depleted samples for differentiated and undifferentiated RGC-5 cells. Differentiated RGC-5 cells had 2.5 times more cytochrome c oxidase than undifferentiated RGC-5 cells.
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Figure 1: Immunoblotting Measurement of Cytochrome c Oxidase in Differentiated and Undifferentiated RGC-5 Mitochondria. RGC-5 cells were differentiated with staurosporine. Isolated mitochondrial samples from undifferentiated and differentiated mitochondria standardized for protein content were compared to corresponding mitochondria depleted samples for the presence of the mitochondrial transmembrane protein cytochrome c oxidase (COX), complex IV. Samples were immunoblotted with antibody to subunit 1 of complex IV. There was considerable purification of mitochondria compared to the mitochondria depleted samples for differentiated and undifferentiated RGC-5 cells. Differentiated RGC-5 cells had 2.5 times more cytochrome c oxidase than undifferentiated RGC-5 cells.

Mentions: Because RGCs are post-mitotic and are present in relatively small (105 cells/retina in the rat) numbers, it is impractical to biochemically study RGC mitochondria in bulk. Instead, we used the RGC-5 cell line, which when differentiated are phenotypically similar to RGCs [14]. RGC-5 cells were grown in tissue culture and mitochondria isolated. To assess purity of mitochondrial isolation, mitochondrial-enriched and cytosolic fractions were immunoblotted for cytochrome c oxidase, demonstrating significant enrichment (Figure 1). Degree of mitochondrial purification was similar among cell-types, based on quantitation with the fluorophore MitoTracker Green FM, which reacts with mitochondrial free sulfhydryls. Mitochondrial samples exhibited similar relative fluorescence values per mg protein (Figure 2).


Differential production of superoxide by neuronal mitochondria.

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

Immunoblotting Measurement of Cytochrome c Oxidase in Differentiated and Undifferentiated RGC-5 Mitochondria. RGC-5 cells were differentiated with staurosporine. Isolated mitochondrial samples from undifferentiated and differentiated mitochondria standardized for protein content were compared to corresponding mitochondria depleted samples for the presence of the mitochondrial transmembrane protein cytochrome c oxidase (COX), complex IV. Samples were immunoblotted with antibody to subunit 1 of complex IV. There was considerable purification of mitochondria compared to the mitochondria depleted samples for differentiated and undifferentiated RGC-5 cells. Differentiated RGC-5 cells had 2.5 times more cytochrome c oxidase than undifferentiated RGC-5 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Immunoblotting Measurement of Cytochrome c Oxidase in Differentiated and Undifferentiated RGC-5 Mitochondria. RGC-5 cells were differentiated with staurosporine. Isolated mitochondrial samples from undifferentiated and differentiated mitochondria standardized for protein content were compared to corresponding mitochondria depleted samples for the presence of the mitochondrial transmembrane protein cytochrome c oxidase (COX), complex IV. Samples were immunoblotted with antibody to subunit 1 of complex IV. There was considerable purification of mitochondria compared to the mitochondria depleted samples for differentiated and undifferentiated RGC-5 cells. Differentiated RGC-5 cells had 2.5 times more cytochrome c oxidase than undifferentiated RGC-5 cells.
Mentions: Because RGCs are post-mitotic and are present in relatively small (105 cells/retina in the rat) numbers, it is impractical to biochemically study RGC mitochondria in bulk. Instead, we used the RGC-5 cell line, which when differentiated are phenotypically similar to RGCs [14]. RGC-5 cells were grown in tissue culture and mitochondria isolated. To assess purity of mitochondrial isolation, mitochondrial-enriched and cytosolic fractions were immunoblotted for cytochrome c oxidase, demonstrating significant enrichment (Figure 1). Degree of mitochondrial purification was similar among cell-types, based on quantitation with the fluorophore MitoTracker Green FM, which reacts with mitochondrial free sulfhydryls. Mitochondrial samples exhibited similar relative fluorescence values per mg protein (Figure 2).

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