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的基本管家功能。然而，越来越多的证据表明，受损线粒体的积累导致了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中显著减少，这表明组织中观察到的与年龄相关的自噬蛋白分解减少在糖尿病患者中将随着病程的延长而加剧。此外，糖尿病人和动物的昼夜节律受到干扰。然而，昼夜节律紊乱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左右和晚上8：15左右在视网膜中的表达最高，相比之下，Beclin的表达在午夜最高。这些观察表明，在视网膜神经血管组织中，自噬表现出与摄食周期或光周期无关的昼夜节律性。我们还在体外表明，在外周时钟已经同步的内皮细胞中，自噬流量出现一个峰值，当时钟蛋白BMal1被击倒时，自噬流量就会丢失。基于这些新的初步发现，我们提出了以下假设：昼夜节律调节的自噬在视网膜内神经血管细胞的稳态中起着关键作用，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。为了验证我们的假说，我们提出了以下目标：1)确认健康小鼠视网膜神经和血管细胞的自噬是否受昼夜节律的调节；2)确定在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)确定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模式是否发生了改变。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。 公共卫生相关性：这一建议代表了我们对糖尿病视网膜病变发病机制的理解和解释的范式转变。这将是第一个证明视网膜神经和血管细胞中自噬流量的昼夜调节的研究。它将在糖尿病患者的昼夜节律性丧失和视网膜病变的视网膜细胞自噬失调之间提供直接的联系。它将强调重新考虑我们对视网膜组织分析的时机的必要性，因为表达水平可能是相互冲突的，取决于动物被牺牲的时间。最后，它将确定新的治疗目标，以预防与糖尿病相关的神经血管并发症。

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. PUBLIC HEALTH RELEVANCE: 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.