mTOR-mediated dedifferentiation of the retinal pigment epithelium initiates photoreceptor degeneration in mice

Abstract

Retinal pigment epithelial (RPE) cell dysfunction plays a central role in various retinal degenerative diseases, but knowledge is limited regarding the pathways responsible for adult RPE stress responses in vivo. RPE mitochondrial dysfunction has been implicated in the pathogenesis of several forms of retinal degeneration. Here we have shown that postnatal ablation of RPE mitochondrial oxidative phosphorylation in mice triggers gradual epithelium dedifferentiation, typified by reduction of RPE-characteristic proteins and cellular hypertrophy. The electrical response of the retina to light decreased and photoreceptors eventually degenerated. Abnormal RPE cell behavior was associated with increased glycolysis and activation of, and dependence upon, the hepatocyte growth factor/met proto-oncogene pathway. RPE dedifferentiation and hypertrophy arose through stimulation of the AKT/mammalian target of rapamycin (AKT/mTOR) pathway. Administration of an oxidant to wild-type mice also caused RPE dedifferentiation and mTOR activation. Importantly, treatment with the mTOR inhibitor rapamycin blunted key aspects of dedifferentiation and preserved photoreceptor function for both insults. These results reveal an in vivo response of the mature RPE to diverse stressors that prolongs RPE cell survival at the expense of epithelial attributes and photoreceptor function. Our findings provide a rationale for mTOR pathway inhibition as a therapeutic strategy for retinal degenerative diseases involving RPE stress. IntroductionThe retinal pigment epithelium (RPE) is a polarized, cuboidal epithelial cell layer situated in the outer retina between the photoreceptors and choroidal vasculature. The RPE supplies an estimated 60% of the glucose consumed by the neural retina (1) and performs a variety of other functions crucial for retinal homeostasis, including delivery of amino acids and docosahexaenoic acid for photoreceptor protein and membrane synthesis; transport, storage, and enzymatic conversion of retinoids essential for phototransduction; regulation of fluid and ion balance in the subretinal space; maintenance of the blood retinal barrier; secretion of growth factors; and phagocytosis of shed photoreceptor outer segment membranes (2). The RPE is a postmitotic tissue, so RPE cells must carry out these functions for the life of an individual.The retinal degenerative consequences of mutations in RPEexpressed genes illustrate the importance of the RPE for photoreceptor viability in humans. Mutations that impair production of the chromophore 11-cis retinal cause Leber congenital amaurosis, retinitis pigmentosa (RP), and allied disorders (3). Disruption of RPE phagocytosis causes RP and rod/cone dystrophy (4, 5). Mutations that affect ion channel function cause disease of the specialized retinal region necessary for high-acuity vision (the macula) (6) as well as RP (7), while mutations in genes encoding the RPE-secreted proteins TIMP3 (8) and EFEMP1 (9) cause lateronset macular disease.

Transcription Factor SOX9 Plays a Key Role in the Regulation of Visual Cycle Gene Expression in the Retinal Pigment Epithelium

“…A similar down-regulation of visual cycle genes was observed after retinal detachment created in mice (8). Injection of sodium iodate (NaIO 3 ), an oxidizing agent known to induce selective RPE cell death followed by retinal degeneration, resulted in down-regulation of RPE65, LRAT, and RLBP1 within 2 days (9,10). These findings support the idea that pan-down-regulation of visual cycle genes may be a general response of RPE cells to stress (8).…”

The Retinal Pigment Epithelium Utilizes Fatty Acids for Ketogenesis

“…In addition to intracellular metabolites, we also analyzed the Ringer's solution in the apical and basolateral compartments of these hfRPE for 13 C metabolites. We found that hfRPE cells incubated with [1][2][3][4][5][6][7][8][9][10][11][12][13] C]palmitate alone released significantly more (7-fold) labeled ␤-HB (75.2 Ϯ 49.7 pmol/cm 2 RPE in a 500-l volume over 3 h) into the apical chamber compared with the basal chamber (10.5 Ϯ 18.3 pmol/cm 2 RPE in a 1500-l volume over 3 h) (Fig. 3E).…”

“…Analysis of the publicly available mouse microarray data (GSE10246, Ref. 6) revealed that mouse RPE expresses particularly high levels of mitochondrial HMG-CoA synthase 2 (Hmgcs2), the key enzyme required for ketone production (7). Hmgcs2 mRNA is also abundant in two other tissues, liver and colon, both of which are known sites of ketogenesis (8).…”

Reductive carboxylation is a major metabolic pathway in the retinal pigment epithelium

“…RPE cells are exposed to ongoing oxidative stress from the combined effects of light, choroidal O 2 , polyunsaturated fatty acids, and retinoids (6). The resulting impairment in RPE energy metabolism and function by oxidative stress is one likely mechanism for the pathogenesis of AMD (3,(7)(8)(9)(10)(11).…”

“…Mitochondria support the active energy metabolism of the RPE (10)(11)(12). A recent report showed that RPE is less stable and less able to support the retina when it is forced to rely on glycolytic rather than mitochondrial metabolism (13).…”