Potential Combination Drug Therapy to Prevent Redox Stress and Mitophagy Dysregulation in Retinal Müller Cells under High Glucose Conditions: Implications for Diabetic Retinopathy.

Lalit Pukhrambam Singh,Takhellambam S Devi

doi:10.3390/diseases9040091

Abstract

Chronic hyperglycemia-induced thioredoxin-interacting protein (TXNIP) expression, associated oxidative/nitrosative stress (ROS/RNS), and mitochondrial dysfunction play critical roles in the etiology of diabetic retinopathy (DR). However, there is no effective drug treatment to prevent or slow down the progression of DR. The purpose of this study is to examine if a combination drug treatment targeting TXNIP and the mitochondria-lysosome pathway prevents high glucose-induced mitochondrial stress and mitophagic flux in retinal Müller glial cells in culture, relevant to DR. We show that diabetes induces TXNIP expression, redox stress, and Müller glia activation (gliosis) in rat retinas when compared to non-diabetic rat retinas. Furthermore, high glucose (HG, 25 mM versus low glucose, LG 5.5 mM) also induces TXNIP expression and mitochondrial stress in a rat retinal Müller cell line, rMC1, in in vitro cultures. Additionally, we develop a mitochondria-targeted mCherry and EGFP probe tagged with two tandem COX8a mitochondrial target sequences (adenovirus-CMV-2×mt8a-CG) to examine mitophagic flux in rMC1. A triple drug combination treatment was applied using TXNIP-IN1 (which inhibits TXNIP interaction with thioredoxin), Mito-Tempo (mitochondrial anti-oxidant), and ML-SA1 (lysosome targeted activator of transient calcium channel MCOLN1/TRPML1 and of transcription factor TFEB) to study the mitochondrial–lysosomal axis dysregulation. We found that HG induces TXNIP expression, redox stress, and mitophagic flux in rMC1 versus LG. Treatment with the triple drug combination prevents mitophagic flux and restores transcription factor TFEB and PGC1α nuclear localization under HG, which is critical for lysosome biosynthesis and mitogenesis, respectively. Our results demonstrate that 2×mt8a-CG is a suitable probe for monitoring mitophagic flux, both in live and fixed cells in in vitro experiments, which may also be applicable to in vivo animal studies, and that the triple drug combination treatment has the potential for preventing retinal injury and disease progression in diabetes.

Highlights

Diabetic retinopathy (DR) is a devastating complication of the eyes affecting millions of people around the world
We have utilized the Ad-CMV-mt-Keima probe in our studies [20]. Using these two mitophagic probes, we examined the mitophagic flux in a retinal Müller cell line under high glucose conditions and the effect of treatment with triple drug combination, targeting thioredoxin-interacting protein (TXNIP) and the mitochondrial–lysosomal axis
Our results showed that high glucose induces TXNIP expression, redox stress, and mitophagic flux in rMC1, which are prevented by treatment with the triple drug combination, which may have potential in DR therapy

Summary

Introduction

Diabetic retinopathy (DR) is a devastating complication of the eyes affecting millions of people around the world. No effective therapy to prevent or treat the disease is available so far. There is a need for developing new and innovative drug therapies to prevent or slow down the progression of DR. The retina is a complex tissue responsible for visual perception and a window to the outside environment [1,2,3,4]. It is composed of three neuronal layers consisting of rod and cone photoreceptors (PRs) in the outer nuclear layer (ONL), bipolar neurons in the inner nuclear layer (INL), and retinal ganglion layer (RGC) that is connected by the outer and inner plexiform layers (OPL and IPL, respectively) (Figure 1).

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