Circadian-dependent autophagy in retinal maintenance and diabetes

描述（由申请人提供）：通过自噬去除受损的线粒体和其他细胞器是所有组织中必不可少的清洁功能。然而，越来越多的证据表明，受损线粒体的积累会导致与糖尿病相关的神经血管并发症。我们的初步数据显示，自噬蛋白在健康视网膜中表达水平较高，但在神经血管性糖尿病视网膜中表达水平显著降低，这表明随着糖尿病病程的延长，组织中观察到的自噬蛋白水解的年龄相关性降低将会加剧。此外，糖尿病人和动物的昼夜节律紊乱。然而，昼夜节律障碍对神经血管视网膜自噬的影响尚不清楚。我们对小鼠的初步研究表明，自噬蛋白Atg9和LC3在上午8:15和晚上8:15左右在视网膜中表达最高，而Beclin在午夜表达最高。这些观察结果表明，在视网膜神经血管组织中，自噬表现出独立于摄食周期或光周期的昼夜节律性。我们还在体外实验中表明，外周时钟同步的内皮细胞显示出自噬通量的峰值，当时钟蛋白Bmal1被敲低时，自噬通量就会消失。基于这些新的初步发现，我们提出以下假设：昼夜节律调节的自噬在视网膜内神经血管细胞稳态中起关键作用，糖尿病改变了这种昼夜节律性，导致自噬失调和糖尿病视网膜病变。为了验证我们的假设，我们提出以下目标：1)确认健康小鼠视网膜神经和血管细胞的自噬是受昼夜节律调节的；2)确定小鼠视网膜细胞特异性时钟基因敲除后自噬是如何改变的，以及这是否与神经血管视网膜的功能缺陷有关；3)观察STZ糖尿病小鼠视网膜神经和血管细胞的自噬通量及其昼夜节律模式是否发生改变。所产生的数据将为糖尿病视网膜病变的发病机制提供新的见解，并可能确定自噬蛋白作为DR的新治疗靶点。

DESCRIPTION (provided by applicant): 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.