βA3/A1-crystallin is essential for lysosome-mediated waste clearance in retinal pigmented epithelial (RPE) cells

Abstract

Purpose: The RPE serves many physiological roles crucial to homeostasis of the retina. RPE is not only one of the most active phagocytic cell types in the body; it also, because of its high metabolic activity, has a high rate of autophagy, a process which degrades and recycles cellular constituents as part of normal cellular remodeling. The objective of this study was to analyze the involvement of βA3/A1-crystallin in the terminal stages of clearance by the lysosomal system in RPE during both autophagy and phagocytosis.

Methods: A conditional knockout (cKO) mouse was generated in which Cryba1 (gene encoding βA3/A1-crystallin) has been deleted specifically from RPE using the Cre-loxP system. Fundus photography and electroretinography (ERG) were performed. Cellular ultrastructure was studied by transmission electron microscopy (TEM). Lysosomal function was assessed by measuring acidification and activities of V-ATPase and Cathepsin D. Phagocytosis and autophagy were studied by using reporter-tagged markers and confocal microscopy.

Results: Fundus photographs showed drusen-like deposits. ERGs indicated loss of retinal function in the Cryba1 cKO mice. TEM of 2 month old mice revealed many vacuole-like structures with undigested photoreceptor outer segments and loss and truncation of basal infoldings in the Cryba1 cKO RPE. At 9 months Cryba1 cKO mice have larger vacuoles containing partially degraded cellular organelles. Lysosomal pH was elevated and Cathepsin D and V-ATPase activities markedly reduced in Cryba1 cKO RPE. The terminal stages of both phagocytosis and autophagy were disrupted in the Cryba1 cKO RPE. We show that the LKB1/AMPK/AKT/mTOR signaling pathway, which regulates the autophagic-lysosomal system, may be modulated by βA3/A1-crystallin.

Conclusions: Our studies suggest that perturbation of normal phagocytosis and/or autophagy may lead to RPE abnormalities similar to those seen in Age-related Macular Degeneration (AMD). Interestingly, βA3/A1-crystallin has been reported to be present in human drusen. Our present data provides novel clues that βA3/A1-crystallin is essential in the lysosomal system of the RPE during waste clearance and regulates this process via the LKB1/AMPK/AKT/mTOR signaling axis, thereby maintaining cellular homeostasis in RPE.

Keywords: 701 retinal pigment epithelium • 412 age-related macular degeneration • 645 phagocytosis and killing