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Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids
Sonali Nashine1, Marilyn Chwa1, Mina Kazemian2
1Gavin Herbert Eye Institute, University of California Irvine, Irvine, California, United States of America.
Purpose:
Variations in mitochondrial DNA (mtDNA) and abnormalities in the complement pathways have been implicated in the pathogenesis of age-related macular degeneration (AMD). This study was designed to determine the effects of mtDNA from AMD subjects on the complement pathway.
Methods:
Transmitochondrial cybrids were prepared by fusing platelets from AMD and age-matched Normal subjects with Rho0 (lacking mtDNA) human ARPE-19 cells. Quantitative PCR and Western blotting were performed to examine gene and protein expression profiles, respectively, of complement markers in these cybrids. Bioenergetic profiles of Normal and AMD cybrids were examined using the Seahorse XF24 flux analyzer.
Results:
Significant decreases in the gene and protein expression of complement inhibitors, along with significantly higher levels of complement activators, were found in AMD cybrids compared to Older-Normal cybrids. Seahorse flux data demonstrated that the bioenergetic profiles for Older-Normal and Older-AMD cybrid samples were similar to each other but were lower compared to Young-Normal cybrid samples.
Conclusion:
In summary, since all cybrids had identical nuclei and differed only in mtDNA content, the observed changes in components of complement pathways can be attributed to mtDNA variations in the AMD subjects, suggesting that mitochondrial genome and retrograde signaling play critical roles in this disease. Furthermore, the similar bioenergetic profiles of AMD and Older-Normal cybrids indicate that the signaling between mitochondria and nuclei are probably not via a respiratory pathway.
Insights
Mitochondrial DNA (mtDNA) variations in age-related macular degeneration (AMD) subjects alter complement pathways. This suggests the mitochondrial genome and retrograde signaling are critical in AMD pathogenesis.
Area of Science:
- Ophthalmology
- Genetics
- Immunology
Background:
- Mitochondrial DNA (mtDNA) variations and complement pathway abnormalities are linked to age-related macular degeneration (AMD) pathogenesis.
- Understanding the specific role of mtDNA in AMD is crucial for developing targeted therapies.
Purpose of the Study:
- To investigate the impact of mtDNA from AMD patients on the complement pathway.
- To elucidate the role of mitochondrial dysfunction in AMD.
Main Methods:
- Transmitochondrial cybrids were created by fusing AMD and normal subject platelets with mtDNA-deficient ARPE-19 cells.
- Quantitative PCR and Western blotting analyzed complement marker gene and protein expression.
- Seahorse XF24 flux analyzer assessed bioenergetic profiles of cybrids.
Main Results:
- AMD cybrids showed decreased complement inhibitor expression and increased complement activator levels compared to normal cybrids.
- Bioenergetic profiles of older normal and AMD cybrids were similar but lower than young normal cybrids.
- These findings implicate mtDNA variations in complement dysregulation in AMD.
Conclusions:
- Observed complement pathway changes in cybrids are attributable to AMD subject mtDNA, highlighting the mitochondrial genome's role.
- Retrograde signaling, not respiratory pathways, may mediate mitochondrial-nuclear communication in AMD.
- mtDNA variations are a significant factor in AMD pathogenesis, influencing complement system activity.
