βA3/A1-crystallin is essential for autophagic lysosomal reformation (ALR) in the retinal pigmented epithelial (RPE) cells

Abstract

Purpose : We have previously shown that βA3/A1-crystallin is a lysosomal lumenal protein in RPE cells that regulates the activity of mechanistic target of rapamycin complex 1 (mTORC1) by modulating V-ATPase (vacuolar-type H⁺-ATPase). The focus of this study was to investigate whether βA3/A1-crystallin modulates the process of ALR in RPE cells. ALR is an mTORC1-dependent process whereby prolonged starvation induces activation and formation of tubular structures on autolysosomal membranes that pinch off to yield protolysosomes.

Methods : Transmission electron microscopy (TEM) was carried out to observe the cellular ultrastructure of RPE cells from both Cryba1 KO and Cryba1^fl/fl mice. RPE flat-mounts, retinal cryosections and RBCC (RPE-Bruch’s membrane-Choriocapillary complex) protein lysates were used from fresh eyes of Cryba1^fl/fl and Cryba1 KO mice to detect the expression of specific autophagy markers either by immunofluoresecence or western analyses. Lysosomes were labeled either with Lamp1, Cathepsin D (CTSD) or lysotracker DND-99. Nuclear and cytosolic extraction and western analyses were carried out to detect Transcription Factor EB (TFEB). Genes from the coordinated lysosomal expression and regulation (CLEAR) network of lysosomal genes were assayed by qPCR.

Results : Our data suggest that even when autophagy is induced in RPE cells by starvation in vivo, absence of βA3/A1-crystallin causes TFEB to remain in the cytosol, thereby preventing activation of CLEAR genes. TEM data showed an increased number of autolysosomes in KO RPE due to impaired lysosome-mediated degradation and thereby inhibited ALR. The depletion of functional lysosomes results in the accumulation of undegraded material in the RPE, finally causing retinal degeneration. Large vacuoles, undigested cell debris, basal laminar deposits and subretinal deposits were found in 20-month old Cryba1 KO mice, but not in age-matched controls. As Cryba1 KO mice age, some RPE cells gradually lose the regular hexagonal shape, as well as RPE65 and LRAT, two essential enzymes in the visual cycle. RPE cells in Cryba1 KO mice also have reduced CTSD expression relative to control mice, which have increased CTSD expression as they age.

Conclusions : Our studies provide novel evidence that βA3/A1-crystallin is essential for regulating the V-ATPase/mTORC1/TFEB signaling axis as well as the reformation of lysosomes in RPE cells.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.