Abstract
Purpose: :
Retinal pigment epithelium (RPE) cell dysfunction and death are hypothesized to play central roles in age-related macular degeneration (AMD), and reduction of RPE mitochondrial function also has been implicated in the pathogenesis of AMD. We sought to create a model of postnatal RPE degeneration due to loss of mitochondrial function and gain insight into the RPE response to respiratory chain deficiency and the resulting consequences for neighboring cells.
Methods: :
We generated mice homozygous for Tfamloxp, a conditional null allele of a nuclear gene essential for mitochondrial function, and heterozygous for a cre recombinase transgene driven by human BEST1 promoter (Tfamloxp/Tfamloxp, +/BEST1-cre), which should restrict ocular loss of mitochondrial function to the postnatal RPE.
Results: :
Immunoblot analysis demonstrated retinal cre expression beginning around Postnatal day (P) 10 and peaking around P63. RPE-restricted retinal cre expression with a patchy pattern was confirmed by immunofluorescence (IF). The fraction of cre-expressing RPE cells rapidly increased to ~75% by P28, eventually reaching a maximum of ~80%. We detected markedly reduced levels of mitochondrial-encoded Cox1 mRNA in ~75% of RPE cells at P35 by in situ hybridization, and cytochrome-c-oxidase (COX, containing mitochondrial-encoded catalytic subunits) enzyme activity was undetectable in the RPE of adjacent frozen sections, demonstrating a profound RPE-specific respiratory chain deficiency. Apoptotic markers were detected by immunohistochemistry in more than 60% of RPE cells at P84, whereas no RPE senescence was observed by that time. Surprisingly, while RPE cell morphology was clearly abnormal at P110, ~65% of RPE cells still remained. Adherens and tight junctions appeared preserved by IF, and there was minimal detectable RPE cell proliferation. Neither photoreceptors loss nor choroidal abnormalities were observed as late as P135, despite definitive evidence of RPE degeneration.
Conclusions: :
Mouse RPE cells are surprisingly resilient to loss of mitochondrial function. Cells persist for months despite little or no respiratory chain function and general epithelial integrity is maintained primarily by cell spreading, even with one-third of the cells gone. The remaining cells, while dysfunctional, appear sufficient to support the photoreceptors and choroid.
Keywords: retinal pigment epithelium • apoptosis/cell death • age-related macular degeneration