Qi Xiaoping, Lewin Alfred S, Hauswirth William W, Guy John
Department of Ophthalmology, Center for Vision Science, University of Florida, College of Medicine, Gainesville, Florida32610-0284, USA.
Invest Ophthalmol Vis Sci. 2003 Mar;44(3):1088-96. doi: 10.1167/iovs.02-0864.
Reactive oxygen species (ROS) are suspected to play a pivotal role in the pathogenesis of Leber hereditary optic neuropathy (LHON), caused by mutated complex I subunit genes. It seems surprising that optic neuropathy has not been described in animals with a knockout of genes encoding critical anti-ROS defenses. If ROS have a role in the optic nerve injury of LHON, then increasing mitochondrial levels of ROS should induce optic neuropathy.
To develop an animal model system for study of oxidative injury to the optic nerve, mitochondrial defenses were decreased against ROS by designing hammerhead ribozymes to degrade SOD2 mRNA. Several potential ribozymes were analyzed in vitro. The one with the best kinetic characteristics was cloned into a recombinant adeno-associated virus (rAAV) vector for delivery and testing in cells and animals. The effects of the AAV-expressing ribozyme on murine cell growth, SOD2 mRNA and protein, cellular ROS levels, and apoptosis were evaluated by RNase protection assay, immunoblot analysis, and ROS- and apoptosis-activated fluorescent probes. The rAAV-ribozyme was then injected into the eyes of DBA/1J mice, and the effect on the optic nerve was evaluated by ocular histopathologic examination.
The AAV-expressing ribozyme decreased SOD2 mRNA and protein levels by as much as 85%, increased cellular superoxide, reduced mitochondrial membrane potential, and culminated in the death of infected cell lines by apoptosis without significantly altering complex I and III activity, somewhat spared in the most common LHON mutation (G11778A), although adenosine triphosphate (ATP) synthesis is markedly reduced. When inoculated into the eyes of mice, the AAV-expressing ribozyme led to loss of axons and myelin in the optic nerve and ganglion cells in the retina, the hallmarks of optic nerves examined at autopsy of patients with LHON.
The striking similarity of the optic neuropathy to the histopathology of LHON is powerful evidence supporting ROS as a key factor in the pathogenesis of LHON.
活性氧（ROS）被怀疑在由线粒体复合体I亚基基因突变引起的Leber遗传性视神经病变（LHON）的发病机制中起关键作用。令人惊讶的是，在编码关键抗ROS防御的基因敲除动物中尚未描述视神经病变。如果ROS在LHON的视神经损伤中起作用，那么增加线粒体ROS水平应该会诱发视神经病变。
为了开发一个用于研究视神经氧化损伤的动物模型系统，通过设计锤头状核酶降解SOD2 mRNA来降低线粒体对ROS的防御能力。在体外分析了几种潜在的核酶。将具有最佳动力学特性的核酶克隆到重组腺相关病毒（rAAV）载体中，用于在细胞和动物中进行递送和测试。通过核糖核酸酶保护试验、免疫印迹分析以及ROS和凋亡激活荧光探针评估表达核酶的AAV对小鼠细胞生长、SOD2 mRNA和蛋白质、细胞ROS水平以及凋亡的影响。然后将rAAV-核酶注射到DBA/1J小鼠的眼睛中，并通过眼部组织病理学检查评估对视神经的影响。
表达核酶的AAV使SOD2 mRNA和蛋白质水平降低多达85%，增加细胞超氧化物，降低线粒体膜电位，并最终导致感染的细胞系通过凋亡死亡，而不会显著改变复合体I和III的活性，在最常见的LHON突变（G11778A）中有所保留，尽管三磷酸腺苷（ATP）合成明显减少。当接种到小鼠眼中时，表达核酶的AAV导致视神经和视网膜神经节细胞中的轴突和髓鞘丢失，这是LHON患者尸检时视神经的特征。
视神经病变与LHON组织病理学的显著相似性是支持ROS作为LHON发病机制关键因素的有力证据。
