Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

Abstract

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.

Fig 1. Overview of the complement cascade.

The classical pathway is triggered by interaction of C1 with immune or non-immune complexes leading to conformational changes in C1q, activation of C1r and C1s, and subsequent assembly of C3 convertase. The lectin pathway is activated by binding of mannose-binding lectin (MBL) to mannose residues on the pathogen surface, which activates the MBL-associated serine proteases (MASPs), followed by formation of C3 convertase. Spontaneous hydrolysis of C3 initiates the alternative complement pathway. All three pathways of the complement cascade converge on the classical C3 convertase, which cleaves and activates component C3, forming C3a and C3b. This triggers a series of further cleavage and activation events, leading to cleavage of C5 into C5a and C5b, and eventual formation of the membrane attack complex (MAC), consisting of C5b, C6, C7, C8, and C9. MAC is the terminal cytolytic complex of the complement pathway; it causes osmotic lysis of target cells by forming transmembrane channels that disrupt the phospholipid bilayer of target cells. The complement system is tightly regulated by the following complement control proteins: CFH, CFHR1, CFHR4, CFB, CD55, CD59, CD46, CFI, and CFP.

Fig 2. Western blot analysis of complement proteins.

Decreased protein levels of CFH, CFHL1, CD55, CD59, CFI, and CD46, and increased levels of CFP, CFB, CFHR4, and CFHR1 protein in AMD cybrids. (A, C, E, G, I, K, M, O, Q, S) Representative Western blots of CFH, CFHL1, CD55, CD59, CFI, CFP, CFB, CD46, CFHR4, and CFHR1 respectively. (B, D, F, H, J, L, N, P, R, T) Graphs showing quantitation of CFH, CFHL1, CD55, CD59, CFI, CFP, CFB, CD46, CFHR4, and CFHR1 proteins in Older-Normal and AMD cybrids. * P < 0.05, ** P < 0.01. n = 4–5. Data were analyzed using Student’s T-test.

Fig 3. Bioenergetic profiles for Normal and AMD cybrids.

(A) Representative bioenergetic profile measured by the Seahorse XF24 flux analyzer. This figure shows OCR and ECAR patterns after sequential treatment with oligomycin (1 μM), FCCP (1 μM) and Antimycin A plus Rotenone (1 μM), demonstrating the regions that define basal aerobic respiration, ATP turnover, maximal respiration, and spare respiratory capacity. (B), (C), (D), (E), (F) Graphs comparing the OCR, ECAR, ATP turnover, spare respiratory capacity, and proton leak values, respectively, in Young-Normal (NL), Old-Normal, and Old-AMD cybrids. These data are presented as a percent response from the basal readings, with non-mitochondrial respiration subtracted out and the basal respiratory rate normalized to 100%.