Altered mTOR signaling in senescent retinal pigment epithelium

Advanced age is a major risk factor for a number of vision-threatening eye diseases, including cataract, glaucoma, diabetic retinopathy, and age-related macular degeneration (AMD). Although the mechanistic link between aging and AMD remains elusive, age-dependent degeneration of the RPE is a hallmark pathologic change of AMD, especially in the atrophic form.1–3 The RPE is part of the blood-retina barrier and is responsible for nutrient support and metabolism.4 With aging, the RPE monolayer loses cells, whereas the remaining cells are subjected to increased metabolic demand.5,6 Decreased mitochondrial function and compromised antioxidant capacity are evident in the aged RPE.7 The production of neurotrophic and angiogenic factors secreted by the RPE shows age-related changes.8,9 Recently, it has been shown that the RPE/choroid complex becomes immunoreactive with aging10; this may be associated with chronic inflammation and abnormal immune responses under certain genetically susceptible backgrounds such as complement factor H mutation. The molecular mechanisms underlying these age-related changes are largely unknown. The process of aging is determined by a genetically programmed molecular clock and is subjected to regulation by multiple pathways in which TOR-mediated signaling is one of the central players.11,12 TOR is a large serine/threonine protein kinase that integrates upstream signals from nutrients, growth factors, and energy levels to control cell growth and proliferation.13,14 In mammals, mTOR resides in two distinct signaling complexes (mTORC): mTORC1 (mTOR/Raptor/GβL) and mTORC2 (mTOR/Rictor/GβL/SIN1). The rapamycin-sensitive mTORC1 regulates translation and protein synthesis largely through the downstream effector proteins S6K and 4E-BP1.15 The phosphorylation status of these two proteins and substrate S6, further downstream, are commonly used as indicators of mTORC1 activity. In contrast to mTORC1, mTORC2 is resistant to short-term exposure to rapamycin16 and is a principal kinase responsible for the phosphorylation of Akt at Ser473.17–19 Negative regulation of aging by TOR signaling has been firmly established in various invertebrate models.12 Very recently, it has been shown that knocking out S6K increases life span in mice.20 Consistently, reduced mTOR/S6K activity has been observed in mice with extended longevity.21 However, whether mTOR regulates tissue-specific degeneration of the retina has not been well studied. In the present study, we have performed an initial characterization of the mTOR pathway in both immortalized ARPE-19 cells and human RPE cells isolated from donor eye tissues. Our results demonstrate that the RPE cells contain mTOR complexes that can be activated by various upstream stimuli. Cultured primary RPE cells entered replicative senescence after serial passages, and downregulation of mTORC1 signaling relieved senescence-associated phenotypical changes. Furthermore, we found that the response of mTORC1 to nutrient signal was upregulated in the aged RPE cells. Our data indicate that changes in the mTOR-related signaling network will likely contribute to degeneration of the RPE.