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

Mallika Valapala, Christine Wilson, Stacey Hose, Imran A. Bhutto, Rhonda Grebe, Aling Dong, Seth Greenbaum, Limin Gu, Samhita Sengupta, Marisol Cano, Sean Hackett, Guotong Xu, Gerard A. Lutty, Lijin Dong, Yuri Sergeev, James T. Handa, Peter Campochiaro, Eric Wawrousek, J. Samuel Zigler, Debasish Sinha

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)480-496
Number of pages17
JournalAutophagy
Volume10
Issue number3
DOIs
StatePublished - Mar 2014

Keywords

  • Autophagy
  • Lysosomes
  • MTORC1
  • Phagocytosis
  • Retinal function
  • Retinal pigment epithelium
  • V-ATPase
  • βA3/A1-crystallin

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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