Mitochondrial Genome Mutation in Cell Death and Aging

This article reviews the concept, molecular genetics, and pathology of cell death and agingin relation to mitochondrial genome mutation. Accumulating evidence emphasizes the role ofgenetic factors in the development of naturally occurring cell death and aging. The ATPrequired for a cell's biological activity is almost exclusively produced by mitochondria. Eachmitochondrion possesses its own DNA (mtDNA) that codes essential subunits of themitochondrial energy-transducing system. Recent studies confirm that mtDNA is unexpectedly fragileto hydroxyl radical damage, hence to the oxygen stress. Cellular mtDNA easily fragmentsinto over a hundred-types of deleted mtDNA during the life of an individual. Cumulativeaccumulation of these oxygen damages and deletions in mtDNA results in a defective energytransducing system and in bioenergetic crisis. The crisis leads cells to the collapse ofmitochondrial trans-membrane potential, to the release of the apoptotic protease activating factors intocytosol, to uncontrolled cell death, to tissue degeneration and atrophy, and to aging. Thetotal base sequencing of mtDNA among individuals revealed that germ-line point mutationstransmitted from ancestors accelerate the somatic oxygen damages and mutations in mtDNAleading to phenotypic expression of premature aging and degenerative diseases. A practicalsurvey of point mutations will be useful for genetic diagnosis in predicting the life-span ofan individual.     

PCR-Free Enrichment of Mitochondrial DNA from Human Blood and Cell Lines for High Quality Next-Generation DNA Sequencing

Recent advances in sequencing technology allow for accurate detection of mitochondrial sequence variants, even those in low abundance at heteroplasmic sites. Considerable sequencing cost savings can be achieved by enriching samples for mitochondrial (relative to nuclear) DNA. Reduction in nuclear DNA (nDNA) content can also help to avoid false positive variants resulting from nuclear mitochondrial sequences (numts). We isolate intact mitochondrial organelles from both human cell lines and blood components using two separate methods: a magnetic bead binding protocol and differential centrifugation. DNA is extracted and further enriched for mitochondrial DNA (mtDNA) by an enzyme digest. Only 1 ng of the purified DNA is necessary for library preparation and next generation sequence (NGS) analysis. Enrichment methods are assessed and compared using mtDNA (versus nDNA) content as a metric, measured by using real-time quantitative PCR and NGS read analysis. Among the various strategies examined, the optimal is differential centrifugation isolation followed by exonuclease digest. This strategy yields >35% mtDNA reads in blood and cell lines, which corresponds to hundreds-fold enrichment over baseline. The strategy also avoids false variant calls that, as we show, can be induced by the long-range PCR approaches that are the current standard in enrichment procedures. This optimization procedure allows mtDNA enrichment for efficient and accurate massively parallel sequencing, enabling NGS from samples with small amounts of starting material. This will decrease costs by increasing the number of samples that may be multiplexed, ultimately facilitating efforts to better understand mitochondria-related diseases.     

Effects of Chloroplast DNA Content on the Cell Proliferation and Aging in Chlamydomonas reinhardtii

Chlamydomonas is an unicellular green alga that contains one cup-shaped chloroplast with about 60 copies of cpDNA. Chloroplasts (cp) multiply in the cytoplasm of the plant cell by binary division, with multiple copies of cpDNA transmitted and maintained in successive generations. The effect of cpDNA copy number on cell proliferation and aging was investigated using a C. reinhardtii moc mutant, which has an undispersed cp-nucleoid and unequal segregation of cpDNA during cell division. When the mother cell divided into four daughters, one moc daughter cell chloroplast contained about 60 copies of cpDNA, and the chloroplasts in the three other daughter cells contained the 4–7 copies of cpDNA. In liquid medium, the number of moc cells at the period of stationary phase was about one-third that of the wild type. To observe the process of proliferation and aging in the mother cell, we used solid medium. Three out of four moc cell spores were preferentially degenerated 60 days after cell transfer. To confirm this, wild-type and moc mother cells containing four daughter cells were treated with novobiocin to inhibit cpDNA replication. Cell degeneration increased only in the moc strain following novobiocin introduction. In total, our results suggest that cells possessing smaller amounts of cpDNA degenerate and age more rapidly. Received 7 September 2000/ Accepted in revised form 14 February 2001     

线粒体DNA突变与衰老关系的研究进展

衰老的分子机制复杂,目前的研究尚未能对衰老的关键轴心获得一致而明晰的理论认识,各种衰老假说相继被提出,其中具有代表性的为端粒学说。端粒-p53-线粒体衰老轴线自从被提出,已经获得越来越多的实验论证,较多的研究也表明线粒体DNA突变与衰老有着密切的相关性,但突变与衰老之间的因果关系存在较大争议。我们简要综述了近年来线粒体DNA突变与衰老关系的相关研究进展。     

Gynaecography in Premature Ovarian Failure and Ovarian Dysgenesis

Gynaecography—the radiological visualization of the internal female genitalia after pneumoperitoneum—is a safe and simple procedure. When taken in conjunction with the clinical and laboratory findings the results of gynaecography are often a sufficient basis for diagnosis and a plan of management in women presenting with menstrual irregularity or infertility.     

鼠毛及脑线粒体DNA片段缺失与增龄的关系

以聚合酶链反应（ＰＣＲ）技术检测不同年龄Ｂａｌｂ／ｃ小鼠脑细胞线粒体ＤＮＡ片段缺失与增龄的关系．发现老年鼠脑细胞线粒体３８６７ｂｐ片段缺失率为５０％;而断奶鼠与青年鼠均无此缺失片段出现;用鼠毛为材料进行无损伤检测亦获类似的结果．有人认为线粒体ＤＮＡ片段缺失率可作为生物衰老的一种生物学标志     

Low Expression of Mfn2 Is Associated with Mitochondrial Damage and Apoptosis of Ovarian Tissues in the Premature Ovarian Failure Model

Background

This study aimed to construct a working model for detecting the mitochondrial damage and expression of Mfn2. It furthermore explored the pathogenesis of premature ovarian failure (POF) induced by cisplatin.

Method

Forty young female mice were divided randomly into two groups. The first was the treatment group intraperitoneally administered cisplatin (1.5mg/kg). The untreated control group was likewise injected with physiological saline for 10 days. One month later, we observed the ovarian weight and morphological changes, particularly the development of follicles and concentration of sex hormones. Immunohistochemistry and western blotting were used to measure the two groups. We later evaluated ovarian cell apoptosis with TUNEL and analyzed Bcl-2 and Bax levels. We used transmission electron microscopy in order to observe the ultrastructure of ovarian cells. The phosphomolybdic acid colorimetric method was used to measure the ATP content in the ovarian tissue. Finally, the mitochondrial membrane potential of ovarian cells was detected with JC-1 dye.

Results

The cisplatin resulted in a decline of body weight, reduced ovarian weight significantly, and resulted in disorders of the extrous cycle. The follicles’ number decreased within the tissue’s stromal hyperplasia. Moreover, E₂ levels were reduced, and elevated gonadotropin levels were observed. However, Mfn2 was present in the cell’s cytoplasm in both groups. Nevertheless, the Mfn2 levels and the expression of Bcl-2 were significantly decreased (p<0.05), but the expression of Bax and the apoptosis index (AI) was increased. In addition, the ATP levels (35.2 ±5.7μmol/g) of the control group were significantly higher (13.5 ± 3.8 μmol/g). Lastly, an obvious impairment of mitochondrial function and structure was observed.

Conclusion

The intreperitoneal injection of cisplatin, when administered for 10 days, establishes a POF model. Thus, the above results suggest that lower expression of Mfn2 may be involved in the mechanism of premature ovarian failure by affecting both the mitochondria’s energy metabolism and its apoptosis. This decides the termination of the follicles’ development.     

自由基对线粒体DNA的氧化损伤与衰老

自由基是一类氧化剂,对生物具有多种损害作用．衰老的自由基学说是有关衰老机理的诸多学说之一．线粒体DNA组成结构特殊,易受自由基攻击;目前认为,线粒体DNA的氧化损伤是自由基引起衰老的分子基础．     

Mitochondrial DNA Copy Number in Peripheral Blood and Melanoma Risk

Mitochondrial DNA (mtDNA) copy number in peripheral blood has been suggested as risk modifier in various types of cancer. However, its influence on melanoma risk is unclear. We evaluated the association between mtDNA copy number in peripheral blood and melanoma risk in 500 melanoma cases and 500 healthy controls from an ongoing melanoma study. The mtDNA copy number was measured using real-time polymerase chain reaction. Overall, mean mtDNA copy number was significantly higher in cases than in controls (1.15 vs 0.99, P<0.001). Increased mtDNA copy number was associated with a 1.45-fold increased risk of melanoma (95% confidence interval: 1.12-1.97). Significant joint effects between mtDNA copy number and variables related to pigmentation and history of sunlight exposure were observed. This study supports an association between increased mtDNA copy number and melanoma risk that is independent on the known melanoma risk factors (pigmentation and history of sunlight exposure).     

激素替代周期子宫内膜微创术治疗卵巢早衰的疗效分析

目的:探讨激素替代周期子宫内膜微创术治疗卵巢早衰患者的临床疗效及安全性。方法:选择2013年12月~2015年12月于我院进行治疗的卵巢早衰患者68例,随机分为实验组与对照组,每组34例,实验组患者采用激素替代周期子宫内膜微创术进行治疗,对照组患者服用中药抗衰复巢汤。比较治疗前后两组患者血清卵泡刺激素(FSH)、促黄体生成素(LH)及雌二醇(E2)水平,同时对两组患者的不良反应情况进行统计,治疗结束后比较两组患者的临床疗效。结果:与治疗前相比,两组患者血清FSH、LH水平均降低,E2水平均升高;治疗结束后,与对照组相比,实验组患者血清FSH、LH水平较低,E2水平较高(P0.05),不良反应发生率较低(P0.05),临床总有效率较高(P0.05)。结论:应用激素替代周期子宫内膜微创术可以减少卵巢早衰患者在治疗后的不良反应,提高临床疗效。     

骨髓间充质干细胞移植对VCD 所致卵巢损伤的修复研究

目的:探讨小鼠骨髓间充质干细胞(MSCs)移植对去氧乙烯基环己烯(VCD)所致卵巢早衰治疗的可行性。方法:采用VCD(160mg kg-1,day-1)连续腹腔注射来诱导小鼠卵巢早衰。每侧卵巢注射转染了绿色荧光基因小鼠骨髓来源的MSCs,于移植后14、28天及45天,取各组血液标本及卵巢组织,同时观察小鼠动情周期的变化;酶联免疫法检测血清FSH、LH水平,显微镜下观察MSC在卵巢的分布。结果:MSCs移植后各组均可见绿色荧光,并且主要分布于卵巢间质区,卵巢泡膜细胞区也可见绿色荧光细胞。MSCs组动情周期较实验对照组缩短,FSH与LH水平较实验对照组低,差异具有显著性。结论:骨髓间充质干细胞可改善卵巢早衰小鼠的卵巢内分泌功能,并且长时间存在于卵巢组织。骨髓间充质干细胞可能成为卵巢早衰治疗的新方法。     

骨髓问充质干细胞移植对VCD所致卵巢损伤的修复研究

目的：探讨小鼠骨髓间充质干细胞（MSCs）移植对去氧乙烯基环己烯（VCD）所致卵巢早衰治疗的可行性。方法：采用VCD（160mgkg^-l,day^-1）连续腹腔注射来诱导小鼠卵巢早衰。每侧卵巢注射转染了绿色荧光基因小鼠骨髓来源的MSCs,于移植后14、28天及45天,取各组血液标本及卵巢组织,同时观察小鼠动情周期的变化;酶联免疫法检测血清FSH、LH水平,显微镜下观察MSC在卵巢的分布。结果：MSCs移植后各组均可见绿色荧光,并且主要分布于卵巢间质区,卵巢泡膜细胞区也可见绿色荧光细胞。MSCs组动情周期较实验对照组缩短,FSH与LH水平较实验对照组低,差异具有显著性。结论：骨髓间充质干细胞可改善卵巢早衰小鼠的卵巢内分泌功能,并且长时间存在于卵巢组织。骨髓间充质干细胞可能成为卵巢早衰治疗的新方法。     

人线粒体DNA缺失肝癌细胞株建立

人线粒体DNA缺失肝癌细胞株(ρ0SK-Hep1)的建立及鉴定。采用溴化乙锭(EB)诱导,PCR、Southern杂交及选择性培养方法进行鉴定。ρ0SK-Hep1细胞在含EB、无尿嘧啶和丙酮酸的选择培养基中从第1天始细胞逐渐悬浮、肿胀,培养第5天以后大量悬浮死亡,且贴壁疏松,10～12天细胞完全悬浮死亡。在选择性培养基中可以正常生长增殖成单层,有少量悬浮。同期非选择培养的SK-Hep1细胞生长正常。PCR结果显示,细胞色素氧化酶I、II(COXI、COXII)及内参G3PDH在SK-Hep1细胞中均可扩增出相应的条带,ρ0SK-Hep1细胞只见内参条带的形成。Southern杂交结果显示,ρ0SK-Hep1细胞未见COXI、COXII杂交条带形成。经EB诱导后成功地获得了ρ0SK-Hep1细胞株。     

CSB-PGBD3 Mutations Cause Premature Ovarian Failure

Premature ovarian failure (POF) is a rare, heterogeneous disorder characterized by cessation of menstruation occurring before the age of 40 years. Genetic etiology is responsible for perhaps 25% of cases, but most cases are sporadic and unexplained. In this study, through whole exome sequencing in a non-consanguineous family having four affected members with POF and Sanger sequencing in 432 sporadic cases, we identified three novel mutations in the fusion gene CSB-PGBD3. Subsequently functional studies suggest that mutated CSB-PGBD3 fusion protein was impaired in response to DNA damage, as indicated by delayed or absent recruitment to damaged sites. Our data provide the first evidence that mutations in the CSB-PGBD3 fusion protein can cause human disease, even in the presence of functional CSB, thus potentially explaining conservation of the fusion protein for 43 My since marmoset. The localization of the CSB-PGBD3 fusion protein to UVA-induced nuclear DNA repair foci further suggests that the CSB-PGBD3 fusion protein, like many other proteins that can cause POF, modulates or participates in DNA repair.