Changes in Genome Expression and Mitochondrial Fission/Fusion Dynamics in RPE J Haplogroup May be Linked to AMD Susceptibility

Abstract

Purpose : Mitochondrial DNA (mtDNA) have traceable sets of SNPs that define mtDNA haplogroups. European J haplogroup is linked to increased susceptibility and H to protection against AMD. In this study, we used RPE cytoplasmic hybrids (cybrids) containing J or H haplogroups to determine their effects on genome expression and resilence to oxidative stress.

Methods : RPE cybrids containing the J or H mt haplogroups were seeded out at 1x10⁵ cells/ml in DMEM and cells harvested at day 4. RNA was extracted, cDNA libraries constructed, and samples subjected to RNAseq using Illumina HiSeq 2500. De-multiplexed sequencing was extracted using Illumina Casava pipeline v1.8 and FPKM values calculated using Cufflinks v 2.0.2. Transcriptomes were compared and only significant reliably expressed genes with p<0.05, FC>1.5 were included in the analyses and validated by qPCR. RPE cybrids were further exposed to oxidative stress (tbH2O2; 100 mM) for 30min and mt dynamics studied. Tom20 staining and mitotracker uptake were used to visualize the organelles response to oxidative stress and mt function determined by fluctuations in the membrane potential (ΔΨm) and calcium uptake ([Ca²⁺]m) using flow cytometry. Stress-induced changes in levels of inflammatory and angiogenic markers were also measured using the bioplex xMAP technology.

Results : Fourteen genes were downregulated with FCs = -1.5-5.16 and forty-four upregulated with FCs = +1.5-3.1 in J compared to H cybrids. Genes involved in mt complex I and V function, mt fusion/fission events, cellular energy homeostasis, anti-oxidant defenses, and inflammatory responses were dysregulated in J cybrids. When exposed to oxidative stress, J cybrids mt showed pathology in fission and fusion events, increased [Ca²⁺]m uptake (p<0.05), and higher levels of IFN-γ, TNF-α and VEGF (p<0.01) when compared to H cybrids.

Conclusions : Our results suggest that RPE cells containing the mtDNA J haplogroup are associated with gene expression changes that alter mitochondrial function, resistance to oxidative stress, inflammatory environment and angiogenesis proclivity, which could all influence development of AMD-like pathologies in the retina.

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