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Lalit P Singh, Takhellambam S Devi, Mallika Somayajulu; Role of TXNIP in mitochondrial dysfunction and mitophagy in diabetic retinopathy. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5447.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the role of the pro-oxidant thioredoxin-interacting protein (TXNIP) in mitochondrial dysfunction and autophagy/mitophagy in diabetic retinopathy.
Streptozotocin-induced type 1 diabetes rats were maintained for 8 weeks and siRNA targeted to TXNIP was delivered intravitreally ten days before terminating the experiments. Retina were harvested and analyzed for mRNA and protein expression for mitochondrial (MT) function and autophagy/mitophagy by using QPCR, Western blotting (WB) and immunohistochemistry (IHC). To understand further the molecular mechanisms, a rat Müller glial cell line (rMC1) was exposed to high glucose (HG, 25 mM vs LG, 5.5 mM) for 5 days in culture and mitochondrial function/dysfunction and autophagy/mitophagy were investigated.
Diabetes increases TXNIP expression in the rat retina, which parallels increases in MT gene expression for aconitase 2, VDAC1, and HO-1 as indicated by QPCR, WB and IHC. In addition, autophagic LC3B punctae are increased in the diabetic rat retina versus non-diabetic rat retinas. Knockdown of TXNIP by intravitreal siRNA delivery blunts LC3B punctae and normalizes several neuronal and glial aberrant gene expressions in the diabetic retina while hyperglycemia is maintained. In vitro studies using rMC1 cultures reveal that HG causes MT ROS generation (MitoSox assay), lowers MT membrane potential ((ΔΨm↓, JC1 probe) and decreases oxygen consumption rates (OCR as measured by SeaHorse FX-24). Under these conditions, TXNIP is significantly upregulated and mitophagy is activated as measured by co-localization of LC3B punctae with Cox-I, a subunit of the MT-ETC complex- IV or LAMP2A, a lysosomal membrane protein, and Cox-I in autophagolysosome. Azaserine (0.5 mM), an inhibitor of the hexosamine pathway and of TXNIP, reduces HG-induced TXNIP expression and mitophagy. HG also decreases Trx2 and autophagy-related gene ATG4B, considered to be targets of TXNIP. ATG4B is critical for pro-LC3B processing to LC3BI for lipidation and also delipidation of LC3BII from autophagosomes for recycling LC3BI. CRISPR-Cas9 gRNA genome editing for TXNIP restores the expression of Trx2, ATG4B and fission proteins Drp1 and fis1. ATG5 and p62/SQSTM1 mRNA levels are not significantly changed.
The results suggest that TXNIP plays an important role in MT redox imbalance, membrane depolarization, bioenergetic stress and mitophagic removal of damaged MT in DR.
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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