Damage to Mitochondrial DNA in Aging

工作综述：一种新的机理理论 有丝分裂后组织的老化表明 线粒体DNA的氧化损伤(mt-DNA)导致 线粒体生物发生受损，因此无法维持 细胞能量动态平衡。我们正在测试这其中的几个要素 假设。在这个项目中，我们量化了 8-羟基脱氧鸟苷(8OHdG)，其氧化产物 不同日龄大鼠肝线粒体DNA中的脱氧鸟苷， 高效液相色谱分离核苷和高效毛细管电泳法测定核苷 电化学阵列检测器。我们发现8-OHdG比 在mt-DNA中比在核DNA中普遍存在。此外，还有一个 随着年龄的增长显著增加；从每100000个脱氧鸟苷中增加8个 6个月至14个月的残留物，12个月的残留物和23个月的残留物 年龄。核DNA没有这种与年龄相关的变化。AS 利用GC/MS分离/检测8-OHdG的前期工作 Mt-DNA无随年龄增长而增加的趋势。我们拿了一些 煞费苦心地验证高效液相色谱/电化学法。两种疾病的治疗 从组织和商业小牛胸腺DNA中提取MT-DNA 8-OHdG特异性甲酰胺嘧啶糖基酶(FPG酶) 导致化学计量比的峰被归因于 8-OHdG。光在空气中引起的氧化损伤 亚甲基蓝同样被FPG酶识别，并且 高效液相色谱技术，鼓励我们使用FPG的组合 处理和Southern杂交检测8-OHdG含量 从极小的组织样本中提取线粒体DNA(如T淋巴细胞) 根据FACS技术进行分类，在当前和未来的工作中。这是 对我们来说很重要，因为我们以前的许多工作都涉及 衰老大鼠心脏与单个大鼠心脏mt-DNA的提取 太小了，不能支持高效液相分析。我们还计划延长 这项工作是对另一种氧化产物5-羟胞嘧啶的测定 Mt-DNA产物。我们已经建立了层析和 DR法合成5-羟胞嘧啶的电势特性 丽贝卡·塞尔泽，也来自LMG。在一个相关的项目中 研究人T淋巴细胞的线粒体DNA损伤。 函数的情况下，我们已经证明了 旧细胞中mt-DNA“常见”5-kb缺失(多于 70年)捐赠者，但未能制造出全长的PCR产物 来自这些细胞的线粒体基因组，尽管成功地 用杨氏DNA合成全长产物 30年)捐赠者。尽管有必要做进一步的工作来 描述阻止mt-DNA的损伤的性质 作为模板，这些对人类T淋巴细胞的研究提供了 线粒体DNA损伤程度与 线粒体的功能，在逐个细胞的基础上。

Summary of work: One theory of the mechanism of aging of post-mitotic tissues proposes that an accumulation of oxidative damage to mitochondrial DNA (mt-DNA) leads to impaired biogenesis of mitochondria and thus a failure to maintain cellular energy homeostasis. We are testing several elements of this hypothesis. In this project, we have quantitated the occurrence of 8-OHdeoxyguanosine (8OHdG), an oxidative product of deoxyguanosine, in mt-DNA from liver of rats of various ages, using separation of nucleosides by HPLC and measurement by an electrochemical array detector. We found that 8-OHdG is more prevalent in mt-DNA than in nuclear DNA. Further, there is a significant increase with aging; from 8 per 100000 deoxyguanosine residues at 6 months to 14 at 12 months and 22 at 23 months of age. There was no such age-linked change in nuclear DNA. As previous work using GC/MS for separation/detection of 8-OHdG had shown no increase with aging in mt-DNA. we have taken some pains to validate the HPLC/EC technique. Treatment both of mt-DNA from tissues and of commercial calf thymus DNA with the 8-OHdG-specific formamidopyrimidine glycosylase (fpg enzyme) resulted in stoichiometric removal of the peak attributed to 8-OHdG. Oxidative damage induced by light in the presence of methylene blue was equally recognized by the fpg enzyme and HPLC techniques, encouraging us to use a combination of fpg treatment and Southern blotting to investigate the 8-OHdG content of mt-DNA from very small tissue samples, eg T-lymphocytes sorted by FACS technology, in current and future work. This is important to us since much of our previous work has involved the aging rat heart and the yield of mt-DNA isolated from one rat heart is too small to support the HPLC analysis. We also plan to extend this work to the measurement of 5-OHcytosine as another oxidative product of mt-DNA. We have established the chromatographic and potentiometric characteristics of 5-OHcytosine synthesized by Dr Rebecca Selzer, also of LMG. In a related project which investigates damage to mt-DNA of human T-lymphocytes as a function of donor age, we have demonstrated the existence of the "common" 5-kb deletion of mt-DNA in cells from old (more than 70 years) donors and have failed to make a full-length PCR product from the mitochondrial genome in these cells, despite the successful synthesis of a full-length product using DNA from young (less than 30 years) donors. Although further work is necessary to characterize the nature of the damage which prevents the mt-DNA acting as a template, these studies with human T-lymphocytes offer the potential of correlating extent of damage to mt-DNA with mitochondrial functioning, on a cell-by-cell basis.