Lysosomal-mediated waste clearance in retinal pigment epithelial cells is regulated by CRYBA1/βA3/A1-crystallin via V-ATPase-MTORC1 signaling.
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.
Wilmer Eye Institute; Johns Hopkins University School of Medicine; Baltimore, MD USA.
Mallika
M
Valapala
Wilmer Eye Institute; Johns Hopkins University School of Medicine; Baltimore, MD USA.
Christine
C
Wilson
Wilmer Eye Institute; Johns Hopkins University School of Medicine; Baltimore, MD USA.
Stacey
S
Hose
Wilmer Eye Institute; Johns Hopkins University School of Medicine; Baltimore, MD USA.
Imran A
IA
Bhutto
Wilmer Eye Institute; Johns Hopkins University School of Medicine; Baltimore, MD USA.
Rhonda
R
Grebe
Wilmer Eye Institute; Johns Hopkins University School of Medicine; Baltimore, MD USA.
Aling
A
Dong
Wilmer Eye Institute; Johns Hopkins University School of Medicine; Baltimore, MD USA.
Seth
S
Greenbaum
Wilmer Eye Institute; Johns Hopkins University School of Medicine; Baltimore, MD USA; Department of Ophthalmology of Shanghai Tenth People's Hospital and Tongji Eye Institute; Tongji University School of Medicine; Shanghai, China.
Limin
L
Gu
Wilmer Eye Institute; Johns Hopkins University School of Medicine; Baltimore, MD USA.
Samhita
S
Sengupta
Wilmer Eye Institute; Johns Hopkins University School of Medicine; Baltimore, MD USA.
Marisol
M
Cano
Wilmer Eye Institute; Johns Hopkins University School of Medicine; Baltimore, MD USA.
Sean
S
Hackett
Department of Ophthalmology of Shanghai Tenth People's Hospital and Tongji Eye Institute; Tongji University School of Medicine; Shanghai, China.
Guotong
G
Xu
Wilmer Eye Institute; Johns Hopkins University School of Medicine; Baltimore, MD USA.
Gerard A
GA
Lutty
National Eye Institute; National Institutes of Health; Bethesda, MD USA.
Lijin
L
Dong
National Eye Institute; National Institutes of Health; Bethesda, MD USA.
Yuri
Y
Sergeev
Wilmer Eye Institute; Johns Hopkins University School of Medicine; Baltimore, MD USA.
James T
JT
Handa
Wilmer Eye Institute; Johns Hopkins University School of Medicine; Baltimore, MD USA.
Peter
P
Campochiaro
National Eye Institute; National Institutes of Health; Bethesda, MD USA.
Eric
E
Wawrousek
Wilmer Eye Institute; Johns Hopkins University School of Medicine; Baltimore, MD USA.
J Samuel
JS
Zigler
Wilmer Eye Institute; Johns Hopkins University School of Medicine; Baltimore, MD USA.
Debasish
D
Sinha
D000818:Animals; D001343:Autophagy / Q000235:genetics / Q000502:physiology*; D003459:Crystallins / Q000235:genetics / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D000076222:Mechanistic Target of Rapamycin Complex 1; D051379:Mice; D018345:Mice, Knockout; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D010587:Phagocytosis / Q000502:physiology; D010588:Phagosomes / Q000378:metabolism; D051381:Rats; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*
D003459:Crystallins; D046912:Multiprotein Complexes; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; C000595959:ATP6V0A1 protein, mouse; D025262:Vacuolar Proton-Translocating ATPases
MTORC1; V-ATPase; autophagy; lysosomes; phagocytosis; retinal function; retinal pigment epithelium; βA3/A1-crystallin