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

Takhellambam S Devi,Thangal Yumnamcha,Mallika Somayajulu,Fayi Yao,Lalit P Singh,Renu A Kowluru

doi:10.1242/bio.038521

Takhellambam S Devi, Thangal Yumnamcha + Show 4 more

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https://doi.org/10.1242/bio.038521

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Journal: Biology open	Publication Date: Jan 1, 2019
Citations: 40	License type: CC BY 4.0

Affiliation: Wayne State University

Abstract

ABSTRACTThioredoxin-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.

Highlights

Diabetic retinopathy (DR) is the number one cause of blindness among the working adult population around the globe, including the US
high glucose (HG) levels induce Thioredoxin-interacting protein (TXNIP) expression and mitochondrial dysfunction in retinal pigment epithelium (RPE) cells Treatment of ARPE-19 for 5 days with HG leads to significant increases in TXNIP expression when compared to low glucose (LG) conditions (Fig. 1A,B and Fig. S1)
Cell viability is reduced under HG but this is rescued by the antioxidant N-acetylcysteine (NAC) (Fig. 1E), suggesting that oxidative stress is involved in this process

Summary

Introduction

Diabetic retinopathy (DR) is the number one cause of blindness among the working adult population around the globe, including the US. DR is primarily defined by microvascular complications of retinal blood vessels, including endothelial dysfunction, pericyte dropout, basement membrane thickening, acellular capillary. In DR, photoreceptor dysfunction occurs early, before microvascular damage (Simó et al, 2018; Tonade et al, 2017). The retinal pigment epithelium (RPE) is a single layer of fully differentiated cells that separates the neuroretina from the fenestrated choriocapillaris that help form the outer blood-retinal barrier (Simó et al, 2010)

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