Mitochondria from AMD subjects can mediate RNA and protein levels of complement markers in transmitochondrial cybrids

Abstract

Purpose : Mitochondrial (mt) dysfunction, variations in mtDNA and abnormalities in complement pathways have been implicated in the pathogenesis of age-related macular degeneration (AMD). Our hypothesis is that retrograde signaling from mitochondria of AMD subjects can mediate expression of genes in the complement pathway and alter the respiratory bioenergetic profile compare to age-matched Normal (NL) subjects.

Methods : Cybrids were prepared by fusing platelets from AMD, age-matched Older-NL subjects or Younger-NL subjects with Rho0 (lacking mtDNA) human ARPE-19 cells. Gene expression and protein levels were measured by Q-PCR and Western blotting. Bioenergetic profiles of Young-NL (n = 4), Older-NL (n = 6) and AMD (n = 8) cybrids were examined using the Seahorse XF24 flux analyzer. Student’s t-test was used for statistical analyses (P < 0.05 was statistically significant).

Results : The AMD cybrids had significant decrease in the expression levels of complement inhibitors (CFH, 0.71-fold, P = 0.02; CD55, 0.50-fold, P = 0.03; CD59, 0.66-fold, P = 0.02; CD46, 0.72-fold, P = 0.04 and CFI, 0.55-fold, P = 0.03) and an increased expression of CFP (2.18-fold, P = 0.03) and CFHR4 (24.16-fold, P = 0.01), both positive regulators of the complement pathway. Seahorse flux data demonstrated that the AMD cybrids and Older-NL cybrids had OCR-ECAR ratios, ATP Turnover Rates, and Spare Respiratory Capacities similar to each other but both groups were significantly lower than the values for the Younger-NL cybrids (P < 0.001).

Conclusions : Since all cybrids have identical nuclei and differ 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 the signaling between mitochondria and nuclei are probably not via a respiratory pathway. Finally, mitochondria in the AMD cybrids were originally from platelets, suggesting that in AMD subjects, mitochondrial dysfunctions are occurring at a systemic level and are not relegated only to the retina.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.