June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
βA3/A1-crystallin modulates endolysosomal acidification in astrocytes by preventing phosphorylation of the V0 a1 subunit of V-ATPase
Author Affiliations & Notes
  • Subrata H Mishra
    Ophthalmology , Wilmer Eye Institute, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States
  • Meysam Yazdankhah
    Ophthalmology , Wilmer Eye Institute, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States
  • Peng Shang
    Ophthalmology , Wilmer Eye Institute, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States
  • J. Samuel Zigler, Jr.
    Ophthalmology , Wilmer Eye Institute, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States
  • Debasish Sinha
    Ophthalmology , Wilmer Eye Institute, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Subrata Mishra, None; Meysam Yazdankhah, None; Peng Shang, None; J. Samuel Zigler, Jr., None; Debasish Sinha, None
  • Footnotes
    Support  Research to Prevent Blindness (an unrestricted grant to the Wilmer Eye Institute)
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 5243. doi:
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      Subrata H Mishra, Meysam Yazdankhah, Peng Shang, J. Samuel Zigler, Jr., Debasish Sinha; βA3/A1-crystallin modulates endolysosomal acidification in astrocytes by preventing phosphorylation of the V0 a1 subunit of V-ATPase
      . Invest. Ophthalmol. Vis. Sci. 2017;58(8):5243.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Our studies have shown that the abnormal migration of astrocytes, implicated in Persistent Fetal Vasculature (PFV) disease, is correlated to elevated endolysosomal pH, decreased autophagy and enhanced mTORC1 signaling. We have previously shown that the interaction between βA3/A1-crystallin and V0 a1 subunit of the V-ATPase, the lysosomal proton pump, is key in maintaining pH in astrocytes and mutations in this crystallin result in elevated pH. Here we investigated whether phosphorylation of V0 a1 is compromised due to a spontaneous mutation (Nuc1) in the Cryba1 gene (encoding βA3/A1-crystallin), leading to elevated pH in astrocytes.

Methods : The assembly/disassembly of the V-ATPase in wild type (WT) and Nuc1 astrocytes was evaluated by quantitating the presence of V1A and V0d1 subunits of V-ATPase and vinculin by western blotting. The phosphorylation state of the V-ATPase V0 a1 subunit was determined by the electrophoretic mobility of V0a1 from WT and Nuc1 rat astrocytes. The migration of V0 a1 was compared by SDS-PAGE, with or without Phos-tag. V0a1 in the aforementioned experiments was detected by western blotting.

Results : The assembly of the V-ATPase was significantly reduced in Nuc1 compared to WT astrocytes. We have previously shown that the endolysosomal pH in Nuc1 astrocytes is abnormally elevated compared to WT. Here we show that this elevated pH results from reduced V-ATPase assembly. We also observed a substantial decrease in the amount of V0 a1 in Nuc1 compared to WT astrocytes. Furthermore, SDS PAGE with Phos-tag indicated phosphorylated and non-phosphorylated forms of V0 a1 in Nuc1, while only the non-phosphorylated form could be detected in WT astrocytes.

Conclusions : Our results show that the elevated pH in the lysosomes of Nuc1 astrocytes is due to decreased level of V-ATPase assembly, which is affected by the phosphorylation status of V0 a1. This suggests that interaction of βA3/A1-crystallin with V0 a1 subunit of V-ATPase in WT astrocytes is responsible for preventing the V0a1 from being phosphorylated. These studies further corroborate the importance of βA3/A1-crystallin-V0 a1 interaction in maintaining endolysosomal pH, thereby providing a unique opportunity to treat PFV disease by rejuvenating V-ATPase activity in astrocytes.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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