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Mallika Valapala, Christine Wilson, Imran Bhutto, Stacey Hose, Seth Greenbaum, Rhonda Grebe, James Handa, Lijin Dong, Eric Wawrousek, Debasish Sinha; βA3/A1-crystallin is essential for lysosome-mediated waste clearance in retinal pigmented epithelial (RPE) cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1786.
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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.
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.
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.
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.
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