July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Txnip Mediates Mitophagic Flux and Lysosomal Destablization in Human Retinal Pigment Epithelial cells under Diabetic Conditions
Author Affiliations & Notes
  • Lalit P Singh
    Wayne State Univ Sch of Med, West Bloomfield, Michigan, United States
  • Thangal Yumnamcha
    Wayne State Univ Sch of Med, West Bloomfield, Michigan, United States
  • Fayi Yao
    Wayne State Univ Sch of Med, West Bloomfield, Michigan, United States
  • Mallika Somayajulu
    Wayne State Univ Sch of Med, West Bloomfield, Michigan, United States
  • Takhellambam S Devi
    Wayne State Univ Sch of Med, West Bloomfield, Michigan, United States
  • Footnotes
    Commercial Relationships   Lalit Singh, Wayne State University (E); Thangal Yumnamcha, None; Fayi Yao, None; Mallika Somayajulu, None; Takhellambam Devi, None
  • Footnotes
    Support  NIH/NEI: R01 EY023992
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3552. doi:
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      Lalit P Singh, Thangal Yumnamcha, Fayi Yao, Mallika Somayajulu, Takhellambam S Devi; Txnip Mediates Mitophagic Flux and Lysosomal Destablization in Human Retinal Pigment Epithelial cells under Diabetic Conditions. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3552.

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

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Abstract

Purpose : Retinal pigment epithelium (RPE) plays an important role in maintaining outer retinal blood barrier and photoreceptor function. Here we examine if TXNIP upregulation in RPE by high glucose play a role in mitophagy dysregulation and lysosome destabilization.

Methods : We maintained an RPE cell line ARPE-19 or primary human RPE (HRPE) under high glucose (25 mM, HG) or low glucose (5.5 mM, LG) condition for 5 days. Mitochondrial ROS, membrane depolarization and bioenergetics were detected by MitoSox, JC1 and ATP assays . QPCR and western blotting measured mRNA and protein levels while immunofluorescence confocal microscopy detected protein localization in mitochondria and lysosomes. A mitophagy probe (Mt-keima) was employed to examine mitophagic flux in live cells while LAMP1-mCherry detected lysosome sizes. Unpaired t-test or one-way ANOVA with Bonferroni post-hoc test determine differences among means+/-sem in multiple sets of experiments with p<0.05 as statistically significant.

Results : HG significantly increases TXNIP mRNA and protein levels compared to LG both in ARPE-19 and HRPE cells. TXNIP upregulation is associated with mitochondrial membrane depolarization, fragmentation and mitophagic flux to lysosomes. Confocal live cell imaging of RPE cells expressing Mt-keima, a coral protein that emits green in mitochondria (alkaline or neutral pH) and red in acidic lysosome, show mitochondrial network of green Mt-keima under LG, which is fragmented in HG. Red Mt-keima after mitophagic flux to lysosomes accumulate as small punctates under LG both in HRPE and ARPE-19 cells while they are enlarged (2-3 folds, p<0.05) in HG. Lysosomal enlargement under HG is further illustrated by LAMP1-mCherry expression and cathepsin L-FITC co-localization. Furthermore, HG causes cathepsin L inactivation and caspase-1 activation in ARPE-19 cells. TXNIP knockdown by shRNA prevents mitochondrial fragmentation, mitophagic flux and lysosome enlargement under HG. In addition, anti-oxidant N-acetylcysteine and amlexanox, an inhibitor of protein kinase TBK1 and of mitophagic adaptors Optineurin and p62, prevent mitophagic flux and lysosome enlargement.

Conclusions : These results suggest that, under high glucose, TXNIP mediates mitophagic flux and lysososmal destabilization causing inflammasome/caspase-1 activation implicating RPE dysfunction in the development of diabetic retinopathy.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

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