Thioredoxin interacting protein is critical for development of early diabetic retinopathy

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1. Scientific Abstract: The retinal being a part of the central nervous system is made up of fully differentiated and non-dividing cells - such as neurons, Muller cells (MC) and pigmented epithelium (RPE) – which may not be renewed if death occurs. Retinal cells consume large amounts of glucose and oxygen to generate ATP in mitochondria for their visual function. During ATP production via the mitochondrial electron transport chain (ETC), electrons leak which is captured by molecular oxygen producing reactive oxygen species (ROS) that damage mitochondrial membrane, proteins and DNA. Therefore, removal of damaged mitochondria via lysosomal degradation by mitophagy, a specific process of autophagy, and synthesis of new mitochondria (mitogenesis) are needed for mitochondrial homeostasis. We have published that thioredoxin-interacting protein (TXNIP) mediates cellular oxidative stress and mitophagic flux in retinal MC under high glucose conditions in vitro and in vivo in diabetic retinas. Furthermore, retinal neurons may undergo cell death by ferroptosis, a newly identified non-apoptotic mechanism of cell demise due to membrane lipid peroxidation by inhibition of glutathione peroxidase 4 (GPX4) and iron accumulation. Cell death by ferroptosis in diabetic retinopathy (DR) is still unknown. We hypothesize that the TXNIP-PARKIN-TFEB pathway is critical for mitophagy and biogenesis of mitochondrion and lysosome in retinal cells in DR. Parkin is an E3 ubiquitin ligase, which is recruited to damaged mitochondria by Pink1 (a mitochondrial protein kinase) for tagging ubiquitin for degradation by mitophagy. Parkin also promotes mitogenesis via ubiquitin-mediated degradation of PARIS (Parkin Interacting Substrate), which blocks PGC1α nuclear translocation, a critical step in mitogenesis. Similarly, Transcription factor EB (TFEB) is a lysosome- associated protein that mediates lysosomal biogenesis, mitogenesis (via PGC1α transcriptional activation) and expression of autophagy-related genes (ATGs) via a CLEAR gene network (CLEAR - Coordinated Lysosomal Expression And Regulation). We will investigate our hypothesis in 3 specific aims that: (i) Parkin is required for mitophagy and mitogenesis in DR; (ii) TFEB is critical for lysosome biogenesis and mitogenesis in DR; and (iii) TXNIP promoter-mediated gene therapy prevents early retinal abnormalities in DR. The proposal is significant in that the study targets the mitophagy-lysosome axis dysregulation in stress, a common feature of age-related chronic neurodegenerative diseases including DR. The role of Parkin and TFEB in mitochondrial quality control in DR has not been addressed before. The proposed gene therapy approach is innovative because the TXNIP promoter linked therapeutic gene (an anti-oxidant gene or a neurotrophic factor) with be induced under high glucose environment, such as seen in diabetes, while mostly unresponsive under physiological glucose levels.