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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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Quantitative Real-Time RT-PCR of Mitochondrial Components and Associated Proteins. Amplification curves for (A) VDAC, (B) Complex I, and (C) Complex IV. Solid black lines indicate undifferentiated RGC-5 cells, long dashes indicate differentiated RGC-5 cells, and short dashes indicate cerebral cells. The fuzzy line denotes the template-free control. The horizontal gray line indicates the calculated threshold value by which Ct is determined.
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Figure 6: Quantitative Real-Time RT-PCR of Mitochondrial Components and Associated Proteins. Amplification curves for (A) VDAC, (B) Complex I, and (C) Complex IV. Solid black lines indicate undifferentiated RGC-5 cells, long dashes indicate differentiated RGC-5 cells, and short dashes indicate cerebral cells. The fuzzy line denotes the template-free control. The horizontal gray line indicates the calculated threshold value by which Ct is determined.

Mentions: We used real-time RT-PCR to compare mRNA levels of mitochondrial electron transport chain complexes I, II, III, IV, V, SOD-2, PMP-70, and VDAC to see if cell type-specific gene transcription levels correlated with protein levels. When normalized to VDAC expression, all METC subunits were downregulated in both undifferentiated and differentiated RGC-5 cells (Table 6). In general, differences in mRNA levels between cell-types correlated with differences in protein levels. Surprisingly, complex I (alpha subunit, 9) transcription levels were higher in brain than RGC-5 cells, but this difference was not seen to the same degree in protein levels, measured by immunoblotting. On the other hand, complex IV (subunit 1) transcription levels were much lower than the difference in protein levels would predict (Figure 6). Differentiation of RGC-5 cells had a minimal effect on transcription levels of all genes studied.


Differential production of superoxide by neuronal mitochondria.

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

Quantitative Real-Time RT-PCR of Mitochondrial Components and Associated Proteins. Amplification curves for (A) VDAC, (B) Complex I, and (C) Complex IV. Solid black lines indicate undifferentiated RGC-5 cells, long dashes indicate differentiated RGC-5 cells, and short dashes indicate cerebral cells. The fuzzy line denotes the template-free control. The horizontal gray line indicates the calculated threshold value by which Ct is determined.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Quantitative Real-Time RT-PCR of Mitochondrial Components and Associated Proteins. Amplification curves for (A) VDAC, (B) Complex I, and (C) Complex IV. Solid black lines indicate undifferentiated RGC-5 cells, long dashes indicate differentiated RGC-5 cells, and short dashes indicate cerebral cells. The fuzzy line denotes the template-free control. The horizontal gray line indicates the calculated threshold value by which Ct is determined.
Mentions: We used real-time RT-PCR to compare mRNA levels of mitochondrial electron transport chain complexes I, II, III, IV, V, SOD-2, PMP-70, and VDAC to see if cell type-specific gene transcription levels correlated with protein levels. When normalized to VDAC expression, all METC subunits were downregulated in both undifferentiated and differentiated RGC-5 cells (Table 6). In general, differences in mRNA levels between cell-types correlated with differences in protein levels. Surprisingly, complex I (alpha subunit, 9) transcription levels were higher in brain than RGC-5 cells, but this difference was not seen to the same degree in protein levels, measured by immunoblotting. On the other hand, complex IV (subunit 1) transcription levels were much lower than the difference in protein levels would predict (Figure 6). Differentiation of RGC-5 cells had a minimal effect on transcription levels of all genes studied.

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