The removal of damaged mitochondria and other organelles by autophagy is an essential housekeeping function in all tissues. However, there is increasing evidence that the accumulation of damaged mitochondria contributes to the neurovascular complications associated with diabetes. Our preliminary data show that autophagy proteins are expressed at high levels in healthy retina but are dramatically reduced within the neurovascular diabetic retina, suggesting that the age-related decrease in autophagic proteolysis observed in tissues will be intensified in the diabetic with increasing duration of disease. Moreover, there is a disturbed circadian rhythm in diabetic humans and animals. However, the impact of circadian dysfunction on autophagy in the neurovascular retina is unknown. Our preliminary investigations in mice show that the autophagy proteins Atg9 and LC3 are highest in the retina at around 8:15 am and 8:15 pm, in contrast, Beclin expression was highest at midnight. These observations suggest that in retinal neurovascular tissue, autophagy shows circadian rhythmicity independent of feeding cycles or light cycle. We also show in vitro that endothelial cells in which the peripheral clock has been synchronized demonstrate a peak in autophagy flux which is lost when the clock protein Bmal1 is knocked down. Based on these novel preliminary findings, we put forward the following hypothesis: Circadian-regulated autophagy plays a critical role in neurovascular cell homeostasis within the retina and that diabetes alters this circadian rhythmicity leading to dysregulated autophagy and diabetic retinopathy. To test our hypothesis we propose the following aims: 1) to confirm that in healthy mice autophagy in neural and vascular cells of the retina is under circadian regulation;2) to determine how autophagy is altered following retinal cell-specific clock gene knockdown in mice and if this is associated with functional defects of the neurovascular retina;3) to determine whether autophagic flux and its circadian pattern is altered in neural and vascular cells of the retina of STZ diabetic mice. The data generated will provide novel insights into the pathogenesis of diabetic retinopathy and may identify autophagic proteins as new therapeutic targets for DR.
This proposal represents a paradigm shift in our understanding and interpretation of events surrounding the pathogenesis of diabetic retinopathy. It will be the first study to demonstrate circadian regulation of autophagy flux in the both neural and vascular cells of the retina. It will provide a direct link between loss of circadian rhythmicity in diabetes and autophagic dysregulation in retinal cells in retinopathy. It will emphasize the need to reconsider the timing of our analyses of retinal tissues as expression levels can be conflicting dependent on the time of day animals are sacrificed. Finally, it will identify novel therapeutic targets to prevent the neurovascular complications associated with diabetes.
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