Defective mitochondrial function & genomic maintenance with diabetic retinopathy

 描述(申请人提供)：糖尿病视网膜病变是糖尿病影响视网膜的主要神经血管继发并发症。目前尚无公认的药物疗法来预防或治疗糖尿病视网膜病变。糖尿病视网膜病变的病理生理机制尚不清楚，但对视网膜神经缺陷和血-视网膜屏障破坏的研究进展迅速，提示神经解剖学改变和功能障碍先于血管改变。最近的研究发现，糖尿病患者全视网膜和视网膜内皮细胞线粒体功能受损与内皮细胞凋亡有关 以及血-视网膜屏障的破坏，但神经视网膜的线粒体功能障碍尚未得到具体检查。神经元线粒体可能对代谢紊乱特别敏感，而提供局部ATP生产和钙缓冲的突触线粒体群体可能特别容易受到损伤。在此之前，我们和其他人已经证明了糖尿病患者视网膜突触丢失和神经细胞凋亡。我们推测，突触线粒体基因组维持减少和视网膜神经元线粒体功能受损导致突触丢失，最终导致神经元凋亡。在目标1中，我们建议研究1型糖尿病大鼠模型中突触和体细胞视网膜部分线粒体基因组的维持情况，以进行mtDNA拷贝数和异质性(缺失和突变)评估。使用一种新的线粒体DNA拷贝数绝对定量方法和下一代测序来评估异质性和mtDNA突变率，我们将全面确定mtDNA基因组的变化。在目标2中，将通过测量与目标1相同的动物模型中分离的视网膜突触体的耗氧率来研究糖尿病患者视网膜神经线粒体功能的变化。此外，还将进行线粒体膜电位的定性染色作为活性的替代，以便在原位定位线粒体的活性。识别线粒体在基因组维持和功能方面的缺陷将为未来DR相关疾病机制的研究提供靶点，也将为药物开发和进一步了解DR的疾病进展提供靶点。此外，这些拟议的研究将有助于确定视网膜神经元线粒体是糖尿病视网膜病变进展的一个因素。

 DESCRIPTION (provided by applicant): Diabetic retinopathy is a major neurovascular secondary complication of diabetes affecting the retina. There are no approved pharmacological therapies for prevention or treatment of DR. The pathophysiology sequella of DR is still undetermined, however research into neural deficits and blood-retinal barrier breakdown in the retina is advancing rapidly and suggest that neuronal anatomical changes and dysfunction precede vascular changes. Recent studies have identified impaired mitochondrial function in whole retinas and retinal endothelial cells with diabetes correlating to endothelial cell apoptosis and the breakdown of the blood-retinal barrier, but mitochondrial dysfunction of the neural retina has not been examined specifically. Neuronal mitochondria are potentially especially sensitive to metabolic disruption, and synaptic mitochondrial populations that provide local ATP production and calcium buffering may be especially prone to damage. Previously, we and others have demonstrated loss of retinal synapses and neuronal apoptosis with diabetes. We hypothesize that decreased synaptic mitochondrial genomic maintenance and impaired mitochondrial function in retinal neurons contributes to synapse loss and eventually neuronal apoptosis. In Aim 1, we propose to investigate mitochondrial genomic maintenance in a rat model of type-1 diabetes in both synaptic and somatic retinal fractions for mtDNA copy number and heteroplasmy (deletions and mutations) assessment. Using a novel absolute quantitation method of mtDNA copy number and next generation sequencing to assess heteroplasmy and mtDNA mutation rates we will comprehensively determine alterations to the mtDNA genome. In Aim 2, changes in neural retina mitochondrial function with diabetes will be investigated through measuring oxygen consumption rates of isolated retinal synaptosomes from the same animal model from Aim 1. Additionally, qualitative staining of mitochondrial membrane potentials as a surrogate for activity in order to localize mitochondrial activity in situ will be carried out. Identification of mitochondrial defects in genomic maintenance and function will provide targets for future investigations of disease mechanisms involved in DR, as well as targets for drug development and further insight to the DR disease progression. Additionally, these proposed studies will be beneficial in identifying the retinal neuronal mitochondria as a contributing facto to the progression of diabetic retinopathy.