Circadian Rhythmicity in the Expression of Autophagy Proteins in Normal and Diabetic Retinas

Abstract

Purpose: : Little is known about the role of autophagy in the normal retina, whose homeostasis is maintained by circadian rhythms, and if this is impaired in diabetes. We investigated the distribution of autophagy proteins in the normal and diabetic rodent retina and determined if they were under circadian control.

Methods: : Eyes from C57BL/6 mice and rats maintained under a 6am/6pm light/dark cycle were enucleated every 2 hours over a 24 hour period. Eyes were also collected from BBZDR/wor type 2 diabetic rats, STZ diabetic mice over selected timepoints. Eyes were either a) fixed in 4% paraformaldehyde and immunostained for Atg7, Atg9, LC3 and Beclin or b) the retina was isolated and expression of Atg7, Atg9, LC3 and Beclin was determined by RT-PCR. Immunostaining was independently assessed and localization of autophagy proteins within the retinal vasculature was confirmed by dual staining with the endothelial cell marker, TRITC-agglutinin.

Results: : Autophagy proteins in both mice and rats demonstrated a differential staining pattern across the retina which showed circadian rhythmicity. Atg9 and LC3 were present in the ganglion cell layer, outer nuclear layer, and some cells of the inner nuclear layer while Beclin was distributed throughout the retina. Atg7 was predominantly localized to the outer segment of the rods and cones and RPE. Atg9, LC3 and Atg7 all showed some association with retinal vessels. Atg7 and Atg9 were primarily observed at the inner plexus of the retinal vessels while Atg7 was strongly expressed in the larger vessels at the retinal surface. Careful analysis of the immunostaining demonstrated circadian rhythmicity in the expression of the autophagy proteins. Atg9 and LC3 expression revealed a biphasic circadian cycle being highest at around 10:15 am and 10:15 pm. Meanwhile, Beclin expression was on a 24-hour cycle with the highest staining observed around noon. Atg7 had no apparent circadian rhythm in normal animals. In diabetic animals, immunohistochemistry and RT-PCR showed a decrease in the expression of Atg7, Atg9, beclin and LC3 with the exception that Atg7 showed an increase in the RPE. Furthermore, the strong circadian rhythmicity for the expression of these autophagy genes was reduced and phase shifted in diabetic animals.

Conclusions: : Autophagy proteins show both spatial and circadian-dependent expression in the retina which is both reduced in diabetes and phase shifted.

Keywords: microscopy: light/fluorescence/immunohistochemistry • diabetic retinopathy • circadian rhythms