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Lalit P Singh, Takhellambam Singh Devi, Mallika Somayajulu; High glucose induces mitochondrial dysfunction and mitophagy in retinal Müller cells: Role of TXNIP. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5202.
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© ARVO (1962-2015); The Authors (2016-present)
Thioredoxin-interacting protein (TXNIP) is involved in oxidative stress and apoptosis in diabetic retinopathy (DR). However, the role of TXNIP in the removal of damaged mitochondria (MT) via mitophagy, a process of macroautophagy, remains unexplored. Here, we investigate the associated cellular and molecular mechanisms underlying mitophagy in retinal cells under sustained hyperglycemia.
We maintained a rat Müller cell line (rMC1) under high glucose (25 mM, HG) or low glucose (5.5 mM, LG) condition for 5 days. MitoSox and JC1 assays were used to detect mitochondrial ROS and membrane depolarization, respectively. QPCR and western blotting measured mRNA and protein levels while confocal microscopy (immunofluorescence analysis) determined protein co-localization in mitochondria. Unpaired two-tailed t-test or one-way ANOVA and Bonferroni post-hoc test determine differences among means+/-sem in multiple sets of experiments. A p-value of <0.05 was considered to be statistically significant.
HG upregulates TXNIP in the cytosol as well as in mitochondria. Moreover, mitochondrial ROS stress and membrane depolarization occur under prolonged hyperglycemia leading to fragmentation. These damaged MT are targeted to lysosome for mitophagic degradation as is evident by co-localization of mitochondrial protein COXIV with autophagosome marker LC3BII and the lysosomal membrane protein LAMP2A. In addition, under HG there is accumulation of dynamin-related fission protein Drp1 and E3 ubiquitin ligase Parkin in damaged MT suggesting their roles in mitochondrial fragmentation and ubiquitination, respectively, which is absent in LG. Subsequently, ubiquitin receptors, optineurin (OPTN) and p62/sequestrome 1, bind to the damaged MT and target them to LC3BII autophagosomes, which then fuse with lysosomes via LAMP2A for degradation. Conversely, TXNIP knockout via CRISPR/Cas9 and TXNIP gRNA prevents the HG-induced mitochondrial damage and mitophagy in rMC1.
TXNIP plays an important role in mitophagy induction in Müller glia under diabetic conditions. We proposed that TXNIP is a potential target for preventing ocular complications of diabetes.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
Figure 1. TXNIP regulates mitophagy in retinal Müller cells under sustained hyperglycemia.
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