TXNIP mediates high glucose-induced mitophagic flux and lysosome enlargement in human retinal pigment epithelial cells

Abstract

Thioredoxin-interacting protein (TXNIP) plays a critical role in oxidative stress, inflammation, apoptosis and the pathogenesis of diabetic retinopathy (DR). However, the role of TXNIP in high glucose-induced retinal pigment epithelium (RPE) dysfunction is still unknown. Here, we show that high glucose (HG; 25 mM,) significantly increases TXNIP expression at both the mRNA and protein levels when compared to low glucose (LG; 5.5 mM) in a human RPE cell line (ARPE-19) and primary human RPE (HRPE) cells. TXNIP upregulation is associated with mitochondrial membrane depolarization, fragmentation and mitophagic flux to lysosomes. We used confocal live-cell imaging of RPE cells expressing mt-Keima, a coral protein that emits green light in mitochondria (alkaline or neutral pH) and red light in the acidic lysosome, to measure mitophagic flux. We observed an elongated mitochondrial network of green mt-Keima under LG, which is fragmented in HG. Red mt-Keima accumulates in lysosomes as small punctate aggregations under LG in both ARPE-19 and HRPE cells, whereas they are significantly enlarged (two- to threefold) under HG. Lysosomal enlargement under HG is further illustrated by lysosomal membrane protein LAMP1-mCherry expression in both ARPE-19 and HRPE cells. Furthermore, HG causes lysosomal cathepsin L inactivation and pro-inflammatory caspase-1 activation in ARPE-19 cells. TXNIP knockdown by shRNA prevents mitochondrial fragmentation, mitophagic flux and lysosome enlargement under HG. In addition, antioxidant N-acetylcysteine (NAC) and Amlexanox (Amlx), an inhibitor of protein kinase TBK1 and of the mitophagic adaptors Optineurin (Optn) and Sequestosome 1 (p62/SQSTM1), prevent mitophagic flux and lysosome enlargement. These results suggest that TXNIP mediates several deleterious effects of high glucose on RPE, which may be implicated in the development of DR.

Faulty homocysteine recycling in diabetic retinopathy

“…Homocysteine was also shown to induce mitochondrial dysfunction, and in retinal ganglion cells, it was implicated in the dysregulation of mitochondrial dynamics [12]. Experimental models of diabetic retinopathy have clearly documented the role of mitochondrial homeostasis in the development of diabetic retinopathy; retinal mitochondria were damaged in diabetes, their copy numbers were decreased, and while the mitochondrial fusion marker, mitofusin 2 (Mfn2), was downregulated, mitophagy markers were upregulated, and capillary cell apoptosis was accelerated [3,4,13,14].…”

Diabetic Retinopathy: The Role of Mitochondria in the Neural Retina and Microvascular Disease

“…This phenomenon is thought to occur in part as a consequence of hyperglycemia-induced expression of thioredoxin-interacting protein (TXNIP), which binds to and inhibits the antioxidants thioredoxin 1 and thioredoxin 2. Hyperglycemia-induced expression of TXNIP is observed in the vasculature, pericytes, and the RPE [ 130 , 185 ], suggesting a conserved mechanism across cell types. Knockdown of TXNIP reduces oxidative stress, improves ATP synthesis, and restores mitophagic flux [ 119 ].…”

Auranofin Mediates Mitochondrial Dysregulation and Inflammatory Cell Death in Human Retinal Pigment Epithelial Cells: Implications of Retinal Neurodegenerative Diseases

“…The cleaved N-terminus portion of gasdermin D is then inserted into the plasma membrane forming pores resulting in plasma membrane permeabilization. Therefore, removal of the damaged mitochondrial by mitophagy, an autophagic process of degrading damaged mitochondria by lysosomes, is critical for maintaining mitochondria health, bioenergetics, and cell survival (Devi et al, 2019). In addition, the RPE phagocytes the damaged photoreceptor outer segment daily and recycle visual pigment retinol for photoreceptor function.…”