September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Cellular effects of lysosomal dysfunction in retinal pigment epithelial cells
Author Affiliations & Notes
  • Mallika Valapala
    Optometry, Indiana University, Bloomington, Indiana, United States
  • Abdulla Alamri
    Optometry, Indiana University, Bloomington, Indiana, United States
  • Rhonda Grebe
    UVEA, Johns Hopkins Univ Sch of Med, Baltimore, Maryland, United States
  • J Samuel Zigler
    UVEA, Johns Hopkins Univ Sch of Med, Baltimore, Maryland, United States
  • James T Handa
    UVEA, Johns Hopkins Univ Sch of Med, Baltimore, Maryland, United States
  • Debasish Sinha
    UVEA, Johns Hopkins Univ Sch of Med, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Mallika Valapala, None; Abdulla Alamri, None; Rhonda Grebe, None; J Zigler, None; James Handa, None; Debasish Sinha, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 6048. doi:
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    • Get Citation

      Mallika Valapala, Abdulla Alamri, Rhonda Grebe, J Samuel Zigler, James T Handa, Debasish Sinha; Cellular effects of lysosomal dysfunction in retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6048.

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

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Abstract

Purpose : The retinal pigment epithelial (RPE) is not only one of the most active phagocytic cell types in the body but also has a high rate of autophagy. Lysosomes are involved in the terminal stages of phagocytosis and autophagy, hence dysfunctional lysosomes lead to impaired cellular clearance in the RPE. The objective of this study was to analyze if the transcriptional program regulating lysosomal biogenesis and function can be induced in the RPE following lysosomal disruption by treatment with ammonium chloride. We also examined the cellular effects of lysosomal dysfunction in the RPE using Cryba1 (gene encoding βA3/A1-crystallin) knockout mice.

Methods : Confluent monolayers of ARPE-19 cells were treated with 1 mM ammonium chloride (to block endolysosomal acidification) for 18 hrs. Ammonium chloride was later removed and the cells were subjected to a 6 hr recovery period. Quantitative real time PCR (qPCR) analysis was performed to study the expression of the following genes involved in lysosomal biogenesis and function: Lysosome associated membrane protein 1(LAMP-1), Cathepsin D (CTSD), Cathepsin A (CTSA), Cathepsin F (CTSF), hexosaminidase A, and ATPase, H+ transporting, lysosomal V0 subunit a1 (ATP6V0A1) encoding for a subunit of V-ATPase. Immunofluorescence and immunoblotting was used to determine the expression of the lysosomal marker, LAMP-1. Cellular ultrastructure of the Cryba1 KO RPE was studied by transmission electron microscopy (TEM).

Results : qPCR analysis of ARPE-19 cells treated with ammonium chloride and subjected to a 6 hr recovery period revealed significant induction of genes regulating both lysosomal biogenesis (LAMP-1,HexA) and function (CTSD, CTSA,CTSF and ATP6V0A1) compared to untreated cells. Immunoblotting and immunofluorescence studies showed increased expression of LAMP-1 in cells recovered from ammonium chloride treatment compared to untreated cells. TEM of 25 month old Cryba1 KO mice revealed many vacuole-like structures containing undigested cellular material and photoreceptor outer segments.

Conclusions : Our studies suggest that perturbation of lysosomal function by pre-treatment with ammonium chloride followed by a recovery period results in a rapid induction of the transcriptional program that induces lysosomal biogenesis and function in ARPE-19 cells. Impaired lysosomal function seen in the Cryba1 KO mice leads to RPE cellular abnormalities and eventual degeneration

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

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