Abstract
Purpose :
The effects of AMD mtDNA and HNG, a mitochondrial-derived peptide (MDP), on retinal cell death pathways have not yet been demonstrated. Therefore, in this study we tested the hypothesis that in an ARPE-19 transmitochondrial cybrid model, AMD mtDNA mediate cell death via apoptosis and autophagy, and that HNG protects the AMD cybrids against apoptosis and autophagy.
Methods :
Transmitochondrial cybrids were prepared by fusing platelets obtained from AMD patients and age-matched normal (NL) subjects, with Rho0 (lacking mtDNA) human ARPE-19 cells. All cybrids had identical nuclei and varied only in mtDNA content. The MTT assay, mt-GFP staining, and Mitosox assay were used to determine the cell viability, mitochondrial number, and mt-ROS levels, respectively. qRT-PCR and Western blotting were performed to examine gene and protein expression profiles, respectively, of apoptosis (Bax,Caspase-3,-7,-9, BCL2L13), and autophagy (LC3B, ATG5, MFN1, LAMP2) markers. AMD and NL cybrids were treated with 3.2 µM of HNG. Student’s t-test and ANOVA were used to measure statistical differences.
Results :
The number of viable cells was significantly decreased (p<0.05) at 24 h (18%), 48 h (17%), and 72h (22%) post incubation, in AMD vs NL cybrids (n=4-7). Elevated mt ROS (p<0.01) and reduction in mitochondrial number were observed in AMD cybrids (n=3). Up-regulation (p<0.05) of BAX (Fold change(FC)=1.3), CASPASE-3 (FC=1.8), CASPASE-7 (FC=6.3), CASPASE-9 (FC=1.7), LC3B (FC=3.1), ATG5 (FC=1.5), MFN1 (FC=1.4), and LAMP2 (FC=2.5) genes was observed in AMD vs NL cybrids (n=4-7). AMD cybrids (n=4) had higher (p<0.05) protein levels of CASPASE-3 and LC3B. Treatment of AMD cybrids with HNG caused a significant decrease (p<0.05) in the gene expression of BAX (FC=0.67), CASPASE- 3 (FC=0.75), CASPASE-7 (FC=0.71), CASPASE-9 (FC=0.71), BCL2L13 (FC=0.76), LC3B (FC=0.81), ATG5 (FC=0.77) compared to untreated AMD cybrids (n=3-4). No gene expression differences were found in untreated vs HNG-treated NL cybrids.
Conclusions :
Consistent with our hypothesis, the current study indicates that AMD mitochondria are significantly damaged and may act as biomarkers for AMD. It also highlights the protective effects of HNG against apoptosis and autophagy in AMD cybrids. Therefore, HNG could be a cell survival factor and a potential therapeutic target for treatment of AMD.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.