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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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Immunoblotting of Mitochondrial Components. Isolated mitochondrial samples standardized for protein content were compared to corresponding mitochondria depleted samples for the presence of the mitochondrial membrane proteins Complexes I-IV, and VDAC. Standardized mitochondrial preparations were also investigated for the peroxisomal marker PMP70. Samples were subject to an electrophoresis and were transferred overnight to nitrocellulose. Samples were incubated with a mouse a primary antibody corresponding the protein of interest, and subsequently treated with an HRP-conjugated antibody. Each mitochondrial protein shows that there is considerable purification of mitochondria compared to the mitochondria depleted samples for cerebral, neuroblastoma, and undifferentiated RGC-5 cells. There is a nearly 18-fold increase in the expression of complex I in cerebral cells compared to RGC-5. Similar levels of expression in PMP70 and complex II were seen in all three samples.
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Figure 5: Immunoblotting of Mitochondrial Components. Isolated mitochondrial samples standardized for protein content were compared to corresponding mitochondria depleted samples for the presence of the mitochondrial membrane proteins Complexes I-IV, and VDAC. Standardized mitochondrial preparations were also investigated for the peroxisomal marker PMP70. Samples were subject to an electrophoresis and were transferred overnight to nitrocellulose. Samples were incubated with a mouse a primary antibody corresponding the protein of interest, and subsequently treated with an HRP-conjugated antibody. Each mitochondrial protein shows that there is considerable purification of mitochondria compared to the mitochondria depleted samples for cerebral, neuroblastoma, and undifferentiated RGC-5 cells. There is a nearly 18-fold increase in the expression of complex I in cerebral cells compared to RGC-5. Similar levels of expression in PMP70 and complex II were seen in all three samples.

Mentions: Most mitochondrial superoxide is produced by the METC. To determine if differences in superoxide production among mitochondria from different neuronal subtypes could be caused by differences in the METC, we measured the relative amounts of METC complexes for protein concentration-corrected mitochondrial isolates. Immunoblots against specific METC complex subunits were scanned by densitometry and normalized to concentrations from cerebral mitochondria (Figure 5 and Table 5). The most surprising finding was that RGC-5 mitochondria contained only one-sixteenth as much METC complex I as was present in cerebral cells. Complex II, III, and IV components were expressed at a molar concentration in RGC-5 mitochondria about half that of cerebral mitochondria. Neuroblastoma mitochondria had similar levels of complexes I-IV, ranging from 16% to 46% that of brain.


Differential production of superoxide by neuronal mitochondria.

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

Immunoblotting of Mitochondrial Components. Isolated mitochondrial samples standardized for protein content were compared to corresponding mitochondria depleted samples for the presence of the mitochondrial membrane proteins Complexes I-IV, and VDAC. Standardized mitochondrial preparations were also investigated for the peroxisomal marker PMP70. Samples were subject to an electrophoresis and were transferred overnight to nitrocellulose. Samples were incubated with a mouse a primary antibody corresponding the protein of interest, and subsequently treated with an HRP-conjugated antibody. Each mitochondrial protein shows that there is considerable purification of mitochondria compared to the mitochondria depleted samples for cerebral, neuroblastoma, and undifferentiated RGC-5 cells. There is a nearly 18-fold increase in the expression of complex I in cerebral cells compared to RGC-5. Similar levels of expression in PMP70 and complex II were seen in all three samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Immunoblotting of Mitochondrial Components. Isolated mitochondrial samples standardized for protein content were compared to corresponding mitochondria depleted samples for the presence of the mitochondrial membrane proteins Complexes I-IV, and VDAC. Standardized mitochondrial preparations were also investigated for the peroxisomal marker PMP70. Samples were subject to an electrophoresis and were transferred overnight to nitrocellulose. Samples were incubated with a mouse a primary antibody corresponding the protein of interest, and subsequently treated with an HRP-conjugated antibody. Each mitochondrial protein shows that there is considerable purification of mitochondria compared to the mitochondria depleted samples for cerebral, neuroblastoma, and undifferentiated RGC-5 cells. There is a nearly 18-fold increase in the expression of complex I in cerebral cells compared to RGC-5. Similar levels of expression in PMP70 and complex II were seen in all three samples.
Mentions: Most mitochondrial superoxide is produced by the METC. To determine if differences in superoxide production among mitochondria from different neuronal subtypes could be caused by differences in the METC, we measured the relative amounts of METC complexes for protein concentration-corrected mitochondrial isolates. Immunoblots against specific METC complex subunits were scanned by densitometry and normalized to concentrations from cerebral mitochondria (Figure 5 and Table 5). The most surprising finding was that RGC-5 mitochondria contained only one-sixteenth as much METC complex I as was present in cerebral cells. Complex II, III, and IV components were expressed at a molar concentration in RGC-5 mitochondria about half that of cerebral mitochondria. Neuroblastoma mitochondria had similar levels of complexes I-IV, ranging from 16% to 46% that of brain.

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