Mitochondrial dysfunction in glaucomatous optic neuropathy

描述(申请人提供)：青光眼的特征是视神经(ON)轴突丢失和视网膜神经节细胞(RGC)变性，并与眼内压(IOP)升高直接相关。虽然有证据表明青光眼患者存在线粒体功能障碍，但没有直接证据表明线粒体功能障碍与青光眼的发病机制有关。然而，在青光眼患者和青光眼实验动物模型的视神经头(ONH)中观察到线粒体的聚集。我们最近在青光眼小鼠模型中发现了高眼压引起的线粒体损伤，包括RGCs、SoMAS和ONH轴突内线粒体结构和功能的改变。该项目的总体目标是描述这些效应的特征，并确定新的线粒体相关治疗靶点，以防止青光眼和其他神经退行性疾病中的神经元死亡和轴突损伤。有三个具体目的：(1)确定体外升高的静水压力或体内升高的眼压是否会引起RGCs、SoMAS和ONH轴突线粒体网络的破坏、线粒体DNA的改变和冠状突超微结构的改变。我们将研究体外RGC培养系统和体内青光眼小鼠模型，以表征高眼压引起的线粒体变化与眼压史和mtDNA完整性和分布的关系。(2)确定体外升高的静水压力或体内升高的眼压如何改变线粒体融合/分裂介质，从而降解线粒体生物能并诱导RGC死亡。这些研究将扩大我们在体外和体内压力诱导的视网膜节细胞中OPA1和DRp1变化的初步观察。我们将在体外和体内青光眼小鼠模型中，将这些OPA1和DRp1的变化与视网膜神经节细胞线粒体融合/分裂、细胞ATP耗竭、呼吸缺陷、活性氧产生和细胞凋亡的变化相关联。(3)确定在体外高眼压或体内高眼压后，DRp1表达降低或OPA1表达增加是否能阻止RGC丢失和轴突变性。我们将AAV2-OPA1和AAV2-DrpK38A构建的AAV2-OPA1和AAV2-DrpK38A转基因到体外或体内的青光眼小鼠模型中，然后使用成像和分子生物学技术评估RGC的存活率，以及轴突和线粒体的保存情况。此外，我们还将评估对照组和OPA1突变小鼠的RGC存活率。这些研究将确定与线粒体相关的新治疗策略，以保护RGC死亡和青光眼变性。公共卫生相关性：在包括青光眼在内的多种神经退行性疾病中已发现线粒体功能障碍的证据。然而，没有证据表明线粒体功能障碍与青光眼的发病机制有关。该项目将解决这一问题，并可能确定新的治疗靶点，以防止青光眼和其他神经退行性疾病中的神经元死亡和轴突损伤。

DESCRIPTION (provided by applicant): Glaucoma is characterized by optic nerve (ON) axon loss and retinal ganglion cell (RGC) degeneration and is directly linked with elevated intraocular pressure (IOP). Although evidence of mitochondrial dysfunction was reported in patients with glaucoma, there is no direct evidence that mitochondrial dysfunction contributes to the pathogenesis of glaucoma. However, accumulation of mitochondria has been observed in the optic nerve head (ONH) of patients with glaucoma and of experimental animal models of glaucoma. We have recently identified elevated IOP-induced mitochondrial damage including alterations of mitochondrial structure and function within RGCs somas and the ONH axons in mouse models of glaucoma. The overall goal of this project is to characterize these effects and to identify new mitochondria-associated therapeutic targets that could protect against neuronal death and axon damage in glaucoma and other neurodegenerative diseases. There are three specific aims: (1) To determine whether in vitro elevated hydrostatic pressure or in vivo elevated IOP triggers breakdown of mitochondrial network, alterations of mitochondrial DNA and ultrastructural changes of cristae in RGCs somas and the ONH axons. We will examine an in vitro RGC culture system and in vivo mouse models of glaucoma to characterize the relationship of elevated IOP-induced mitochondrial changes to IOP history and mtDNA integrity and distribution. (2) To determine how in vitro elevated hydrostatic pressure or in vivo elevated IOP alters mitochondrial fusion/fission mediators that degrade mitochondrial bioenergetics and induce RGC death. These studies will extend our preliminary observation of pressure-induced alterations of OPA1 and Drp1 in RGCs in vitro and in vivo. We will correlate these OPA1 and Drp1 changes with alterations of mitochondrial fusion/fission, cellular ATP depletion, respiration deficiency, reactive oxygen species production, and apoptotic cell death in RGCs in vitro and in vivo mouse models of glaucoma. (3) To determine whether reduced Drp1 or increased OPA1 expression will block RGC loss and axon degeneration following in vitro elevated hydrostatic pressure or in vivo elevated IOP. We will transfect AAV2-OPA1 and AAV2-DrpK38A constructs into RGCs in vitro or mouse models of glaucoma in vivo and then assess RGC survival, and preservation of axons and mitochondria using imaging and molecular biological techniques as described in Aims 1 and 2. Further, we will assess RGC survival in control and OPA1 mutant mice treated with laser photocoagulation. These investigations will identify mitochondria-related new therapeutic strategies that protect RGC death and ON degeneration in glaucoma. PUBLIC HEALTH RELEVANCE: Evidence of mitochondrial dysfunction has been identified in a wide variety of neurodegenerative diseases including glaucoma. However, there is no evidence that mitochondrial dysfunction contributes to the pathogenesis of glaucoma. This project will address this issue and may identify new therapeutic targets that could protect against neuronal death and axon damage in glaucoma and other neurodegenerative diseases.