Nuclear gene expression changes due to mitochondrial dysfunction in ARPE-19 cells: implications for age-related macular degeneration.
OBJECTIVE
To measure changes in nuclear gene expression resulting from mitochondrial dysfunction in retinal pigment epithelial cells.


METHODS
ARPE-19 retinal pigment epithelial cells were depleted of their mitochondrial (mt)DNA by passaging in a low concentration of ethidium bromide. Loss of mitochondrial DNA was determined by uridine auxotrophy and quantitative real-time polymerase chain reaction of isolated DNA. Loss of mitochondrial membrane potential was estimated by uptake of JC-1. Changes in nuclear gene expression were determined by quantitative real-time reverse transcription-polymerase chain reaction of isolated total RNA from ethidium-bromide-treated and untreated cells. Morphologic and phenotypic changes were determined by phase-contrast microscopy, sensitivity to the oxidant tert-butyl hydroperoxide (tBH), and invasion assay.


RESULTS
ARPE-19 cells became auxotrophic for growth on uridine after eight passages in 50 ng/mL ethidium bromide. Quantitative PCR revealed almost complete loss of mitochondrial DNA (rho(0) cells). Uptake of JC-1 was reduced in the rho(0) cells, indicating reduction of mitochondrial membrane potential. Quantitative RT-PCR measured increased expression of genes coding for drusen components, lipid transport, extracellular matrix components, and responses to inflammation in the rho(0) cells. The rho(0) cells also exhibited an increased sensitivity to killing by tBH and increased migration and invasion through solubulized basement membrane-coated tissue culture inserts.


CONCLUSIONS
ARPE-19 cells respond to loss of mitochondrial function by changes in nuclear gene expression that resemble changes observed in age-related macular degeneration. The results lead to the hypothesis that loss of mitochondrial function with age and resultant changes in nuclear gene expression may explain some of the changes in the macula that are associated with the known clinical manifestations of age-related macular degeneration.
Miceli
Michael V
MV
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1901 Perdido Street, New Orleans, LA 70112, USA.
Jazwinski
S Michal
SM
D001562:Benzimidazoles / Q000378:metabolism; D002232:Carbocyanines / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D004272:DNA, Mitochondrial / Q000187:drug effects; D015536:Down-Regulation; D004996:Ethidium / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D008268:Macular Degeneration / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D028361:Mitochondrial Diseases / Q000378:metabolism* / Q000473:pathology; D009687:Nuclear Proteins / Q000235:genetics*; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
D001562:Benzimidazoles; D002232:Carbocyanines; D004272:DNA, Mitochondrial; D005456:Fluorescent Dyes; D009687:Nuclear Proteins; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D020122:tert-Butylhydroperoxide; D004996:Ethidium