Abstract
Thioredoxin-interacting protein (TXNIP) is involved in oxidative stress and apoptosis in diabetic retinopathy. 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 diabetic conditions. For this, 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. Our data reveal that HG upregulates TXNIP in the cytosol as well as in the MT. Moreover, mitochondrial oxidative 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, a subunit of cytochrome c oxidase, with autophagosome marker LC3BII and the lysosomal membrane protein LAMP2A. In addition, under HG conditions, there is an 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 conditions. Subsequently, ubiquitin receptors, optineurin and p62/sequestrome 1, bind to the damaged MT and target them to LC3BII autophagosomes. Conversely, TXNIP knockout via CRISPR/Cas9 and TXNIP gRNA prevents the HG-induced mitochondrial damage and mitophagy in rMC1. Last, TXNIP level is also significantly upregulated in the diabetic rat retina in vivo and induces radial glial fibrillary acidic protein expression, a marker for Müller glia activation, and the formation of LC3BII puncta, which are prevented by intravitreal injection of TXNIP siRNA. Therefore, TXNIP represents a potential target for preventing ocular complications of diabetes.
Highlights
Thioredoxin-interacting protein (TXNIP) has been defined as a pro-oxidative stress, pro-inflammatory and pro-apoptotic protein that is strongly induced by diabetes and high glucose (HG) in most tissues examined, including pancreatic beta and retinal cells.[1,2] TXNIP binds to thioredoxin (Trx) and inhibits its thiol-reducing and oxidant-scavenging activity, thereby triggering cellular oxidative stress and apoptosis.[3]
We hypothesize that hyperglycemia-induced TXNIP expression and oxidative stress are involved in mitochondrial dysfunction, and mitophagy in diabetic retinopathy (DR) and retinal cells in vitro
We show that when rat retinal Müller cells are subjected to HG (25 mM glucose, 5 days), there is a significant increase in TXNIP expression both at mRNA and protein levels (Figures 1a–c)
Summary
Thioredoxin-interacting protein (TXNIP) has been defined as a pro-oxidative stress, pro-inflammatory and pro-apoptotic protein that is strongly induced by diabetes and high glucose (HG) in most tissues examined, including pancreatic beta and retinal cells.[1,2] TXNIP binds to thioredoxin (Trx) and inhibits its thiol-reducing and oxidant-scavenging activity, thereby triggering cellular oxidative stress and apoptosis.[3]. The ETC generates superoxide radicals, which can damage mitochondrial proteins, DNA and membrane lipids.[9,10,11] To counter these reactive oxygen species (ROS), several anti-oxidant systems are present in the MT, including glutathione, Trx[2], MnSOD and others In spite of these protective mechanisms, mitochondrial membrane damage and depolarization occur in physiological and pathological conditions, including diabetes, and the damaged MTare segregated by fission.[12] Mito-fission involves the cytosolic dynamin-related protein 1 (Drp1), which is a GTPase, and mitochondrial membrane-bound fission proteins, such as Fis[1], which dock Drp[1] onto the outer mitochondrial membrane.[13,14] In contrast, PINK1, which is an inner mitochondrial membrane kinase, accumulates at the outer membrane of depolarized MT and recruits the E3 ubiquitin ligase Parkin, which ubiquitinates outer membrane proteins, such as voltage-dependent anion-selective channel 1 (VDAC1) and Mfn[2], as a mark for degradation of the damaged MT by mitophagy via the lysosomal degradation.[15,16]. HG induces mitochondrial dysfunction and mitophagy in retinal Müller cells in in vitro
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