Circadian-dependent autophagy in retinal maintenance and diabetes

昼夜节律依赖性自噬在视网膜维护和糖尿病中的作用

基本信息

批准号：
8698848
负责人：
Michael Edwin Boulton
金额：
$ 12.61万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-10 至 2014-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8698848
关键词：
Address Age-Months Animal Model Animals Autophagocytosis Blood Vessels C57BL/6 Mouse Cells Circadian Rhythms Clinical Clock protein Conflict (Psychology)Data Defect Dependovirus Diabetes Mellitus Diabetic Retinopathy Diabetic mouse Endothelial Cells Event Excision Feedback Functional disorder Gene Expression Generations Genes Homeostasis Hour Housekeeping Human Immunohistochemistry In Vitro Injection of therapeutic agent Investigation Knowledge Light Link Maintenance Mitochondria Mus Organelles Pathogenesis Pathology Pattern Periodicity Peripheral Play Proteins Proteolysis Regulation Reporting Retina Retinal Retinal Diseases Reverse Transcriptase Polymerase Chain Reaction Rhodopsin Role Signal Pathway Small Interfering RNA Staining method Stains Streptozocin Testing Time Tissues Vascular Endothelial Cell Western Blotting Wild Type Mouse age related base cadherin 5 circadian pacemaker diabetic feeding illness length in vivo insight intravitreal injection killings knock-down new therapeutic target non-diabetic novel prevent promoter relating to nervous system research study transcription factor type I diabetic

项目摘要

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.

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重要的管家功能。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高水平表达，但在神经血管糖尿病视网膜内显着减少，这表明在组织中观察到的自噬蛋白水解与年龄相关的减少在糖尿病患者中随着病程的延长而加剧。此外，患有糖尿病的人类和动物的昼夜节律受到干扰。然而，昼夜节律功能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，相比之下，Beclin 表达在午夜最高。这些观察结果表明，在视网膜神经血管组织中，自噬表现出与进食周期或光周期无关的昼夜节律。我们还在体外证明，外周时钟已同步的内皮细胞表现出自噬通量峰值，当时钟蛋白 Bmal1 被敲低时，自噬通量就会消失。基于这些新颖的初步发现，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发挥着关键作用，糖尿病改变了这种昼夜节律，导致自噬失调和糖尿病视网膜病变。为了检验我们的假设，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处于昼夜节律调节之下； 2) 确定小鼠视网膜细胞特异性时钟基因敲低后自噬如何改变，以及这是否与神经血管视网膜的功能缺陷有关； 3) 确定STZ糖尿病小鼠视网膜神经细胞和血管细胞的自噬通量及其昼夜节律模式是否发生改变。生成的数据将为糖尿病视网膜病变的发病机制提供新的见解，并可能将自噬蛋白确定为 DR 的新治疗靶点。