Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, Texas 77555-1106, USA.
Invest Ophthalmol Vis Sci. 2011 Jun 1;52(6):3521-9. doi: 10.1167/iovs.10-6163.
Mitochondrial DNA (mtDNA) damage may be associated with age-related diseases, such as age-related macular degeneration (AMD). The present study was designed to test whether the frequency of mtDNA damage, heteroplasmic mtDNA mutations, and repair capacity correlate with progression of AMD.
Macular and peripheral RPE cells were isolated and cultured from human donor eyes with and without AMD. The stages of AMD were graded according to the Minnesota Grading System. Confluent primary RPE cells were used to test the frequency of endogenous mtDNA damage by quantitative PCR. Mutation detection kits were used to detect heteroplasmic mtDNA mutation. To test the mtDNA repair capacity, cultured RPE cells were allowed to recover for 3 and 6 hours after exposure to H(2)O(2), and repair was assessed by quantitative PCR. The levels of human OGG1 protein, which is associated with mtDNA repair, were analyzed by Western blot.
This study showed that mtDNA damage increased with aging and that more lesions occurred in RPE cells from the macular region than the periphery. Furthermore, mtDNA repair capacity decreased with aging, with less mtDNA repair capacity in the macular region compared with the periphery in samples from aged subjects. Most interestingly, the mtDNA damage was positively correlated with the grading level of AMD, whereas repair capacity was negatively correlated. In addition, more mitochondrial heteroplasmic mutations were detected in eyes with AMD.
These data show macula-specific increases in mtDNA damage, heteroplasmic mutations, and diminished repair that are associated with aging and AMD severity.
线粒体 DNA（mtDNA）损伤可能与年龄相关性疾病有关，例如年龄相关性黄斑变性（AMD）。本研究旨在测试 mtDNA 损伤、异质 mtDNA 突变和修复能力的频率是否与 AMD 的进展相关。
从有和无 AMD 的人捐献眼中分离和培养黄斑和周边 RPE 细胞。根据明尼苏达州分级系统对 AMD 进行分级。使用定量 PCR 测试衰老相关 mtDNA 损伤的内源性 mtDNA 损伤频率。使用突变检测试剂盒检测异质 mtDNA 突变。为了测试 mtDNA 修复能力，在暴露于 H（2）O（2）后，培养的 RPE 细胞允许恢复 3 和 6 小时，通过定量 PCR 评估修复情况。Western blot 分析与 mtDNA 修复相关的人类 OGG1 蛋白的水平。
本研究表明 mtDNA 损伤随年龄增长而增加，并且黄斑区的 RPE 细胞中发生的病变更多。此外，mtDNA 修复能力随年龄下降，与年龄相关的样本中黄斑区的 mtDNA 修复能力比周边区更差。最有趣的是，mtDNA 损伤与 AMD 的分级水平呈正相关，而修复能力呈负相关。此外，在 AMD 眼中检测到更多的线粒体异质突变。
这些数据表明，mtDNA 损伤、异质突变和修复能力下降在黄斑区特异性增加，与衰老和 AMD 严重程度相关。
