Diurnal Rhythmicity of Autophagy Is Impaired in the Diabetic Retina.

Xiaoping Qi,Michael E Boulton,Yuanqing Yan,Julia V Busik,Sayak K Mitter,Maria B Grant

doi:10.3390/cells9040905

Abstract

Retinal homeostasis is under both diurnal and circadian regulation. We sought to investigate the diurnal expression of autophagy proteins in normal rodent retina and to determine if this is impaired in diabetic retinopathy. C57BL/6J mice and Bio-Breeding Zucker (BBZ) rats were maintained under a 12h/12h light/dark cycle and eyes, enucleated over a 24 h period. Eyes were also collected from diabetic mice with two or nine-months duration of type 1 diabetes (T1D) and Bio-Breeding Zucker diabetic rat (BBZDR/wor rats with 4-months duration of type 2 diabetes (T2D). Immunohistochemistry was performed for the autophagy proteins Atg7, Atg9, LC3 and Beclin1. These autophagy proteins (Atgs) were abundantly expressed in neural retina and endothelial cells in both mice and rats. A differential staining pattern was observed across the retinas which demonstrated a distinctive diurnal rhythmicity. All Atgs showed localization to retinal blood vessels with Atg7 being the most highly expressed. Analysis of the immunostaining demonstrated distinctive diurnal rhythmicity, of which Atg9 and LC3 shared a biphasic expression cycle with the highest level at 8:15 am and 8:15 pm. In contrast, Beclin1 revealed a 24-h cycle with the highest level observed at midnight. Atg7 was also on a 24-h cycle with peak expression at 8:15 am, coinciding with the first peak expression of Atg9 and LC3. In diabetic animals, there was a dramatic reduction in all four Atgs and the distinctive diurnal rhythmicity of these autophagy proteins was significantly impaired and phase shifted in both T1D and T2D animals. Restoration of diurnal rhythmicity and facilitation of autophagy protein expression may provide new treatment strategies for diabetic retinopathy.

Highlights

Macroautophagy is an evolutionarily conserved cellular catabolic mechanism that facilitates the degradation of damaged cellular organelles and proteins, by targeting them to the lysosomes and recycling the macromolecules for the rebuilding of cellular machinery [1,2,3]
We demonstrate that the spatial distribution and temporal expression of autophagy proteins show a diurnal rhythm and that this is depressed and phase shifted in the diabetic retina
When we examined the tissue at two-hour intervals over a 24 h period, these proteins exhibited a distinct diurnal rhythm, but remarkably showed distinct staining patterns across the layers of the neural retina in mice (Figures 1 and 2)

Summary

Introduction

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved cellular catabolic mechanism that facilitates the degradation of damaged cellular organelles and proteins, by targeting them to the lysosomes and recycling the macromolecules for the rebuilding of cellular machinery [1,2,3]. Autophagy undergoes rhythmic variation in accordance with circadian patterns of rest/activity and feeding in adult mammals [4]. Dysregulated autophagy has been implicated in several neurodegenerative disorders, hepatitis, cancer, aging associated diseases and in the general aging process [5,6,7,8,9]. A growing body of evidence indicates that dysregulated autophagy is linked to diabetes [10,11,12,13]. The disruption of circadian rhythm has a profound negative impact on health and is associated with elevated risk for several diseases [14].

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Conclusion