Lysosomal-mediated waste clearance in retinal pigment epithelial cells is regulated by CRYBA1/βA3/A1-crystallin via V-ATPase-MTORC1 signaling

Abstract

In phagocytic cells, including the retinal pigment epithelium (RPE), acidic compartments of the endolysosomal system are regulators of both phagocytosis and autophagy, thereby helping to maintain cellular homeostasis. The acidification of the endolysosomal system is modulated by a proton pump, the V-ATPase, but the mechanisms that direct the activity of the V-ATPase remain elusive. We found that in RPE cells, CRYBA1/βA3/A1-crystallin, a lens protein also expressed in RPE, is localized to lysosomes, where it regulates endolysosomal acidification by modulating the V-ATPase, thereby controlling both phagocytosis and autophagy. We demonstrated that CRYBA1 coimmunoprecipitates with the ATP6V0A1/V0-ATPase a1 subunit. Interestingly, in mice when Cryba1 (the gene encoding both the βA3- and βA1-crystallin forms) is knocked out specifically in RPE, V-ATPase activity is decreased and lysosomal pH is elevated, while cathepsin D (CTSD) activity is decreased. Fundus photographs of these Cryba1 conditional knockout (cKO) mice showed scattered lesions by 4 months of age that increased in older mice, with accumulation of lipid-droplets as determined by immunohistochemistry. Transmission electron microscopy (TEM) of cryba1 cKO mice revealed vacuole-like structures with partially degraded cellular organelles, undigested photoreceptor outer segments and accumulation of autophagosomes. Further, following autophagy induction both in vivo and in vitro, phospho-AKT and phospho-RPTOR/Raptor decrease, while pMTOR increases in RPE cells, inhibiting autophagy and AKT-MTORC1 signaling. Impaired lysosomal clearance in the RPE of the cryba1 cKO mice also resulted in abnormalities in retinal function that increased with age, as demonstrated by electroretinography. Our findings suggest that loss of CRYBA1 causes lysosomal dysregulation leading to the impairment of both autophagy and phagocytosis.

The marine n-3 PUFA DHA evokes cytoprotection against oxidative stress and protein misfolding by inducing autophagy and NFE2L2 in human retinal pigment epithelial cells

“…32 These findings suggest that autophagy is an important cytoprotective mechanism that counteracts the development of several age-related diseases, especially proteinopathies, by clearence of damaged proteins and organelles. 33 Lysosomal-mediated clearance is also important in RPE cells 34 supporting the idea that also in RPE cells, autophagy is critical to maintain cellular homeostasis. Whether autophagy could be induced as a disease preventive mechanism in these cells is still uncertain.…”

Subcellular Distribution and Activity of Mechanistic Target of Rapamycin in Aged Retinal Pigment Epithelium

“…Our finding is consistent with results from a recent study by Sinha's group that showed mTORC1 pathway was involved in regulating lysosomal clearance in RPE of Cryba1 conditional knockout mice. 43 Aging is controlled by multiple levels of genetic and epigenetic mechanisms. 73 Particularly to the RPE cells, oxidative stress is an important contributing factor to their aging.…”

“…Cultured RPE cells have been an invaluable tool for characterizing their unique physiological functions such as tight junction, 40,41 phagocytosis and autophagy 42,43 and immune regulation. 44,45 One of the goals of our current study was to develop an in vitro culture model that can be used to probe mechanisms of RPE aging.…”

Deletion of autophagy inducerRB1CC1results in degeneration of the retinal pigment epithelium

“…[16][17][18][19][20][21] For example, markers of autophagy and exosomes have been found in the RPE of aged mice as well as in drusen from cadaveric eyes from people with age-related macular degeneration (AMD), 16 and there has been increasing evidence for dysregulation of autophagy in the RPE leading to accumulation of lipofuscin and reduced ability to clear intracellular debris. [22][23][24][25] However, the causal relationship between autophagy dysfunction and RPE degeneration has not been directly demonstrated in vivo. One method for assessing the role of a process in a specific cell type is to reduce or eliminate that function and assess the effect on the phenotype.…”