Accumulation Of mtDNA Deletions Research Articles

Role of p53, Mitochondrial DNA Deletions, and Paternal Age in Autism: A Case-Control Study.

The tumor suppressor p53 responds to a variety of environmental stressors by regulating cell cycle arrest, apoptosis, senescence, DNA repair, bioenergetics and mitochondrial DNA (mtDNA) copy number maintenance. Developmental abnormalities have been reported in p53-deficient mice, and altered p53 and p53-associated pathways in autism (AU). Furthermore, via the Pten-p53 crosstalk, Pten haploinsufficient-mice have autisticlike behavior accompanied by brain mitochondrial dysfunction with accumulation of mtDNA deletions. mtDNA copy number and deletions, and p53 gene copy ratios were evaluated in peripheral blood monocytic cells from children aged 2-5 years with AU (n = 66), race-, gender-, and age-matched typically neurodeveloping children (n = 46), and both parents from each diagnostic group, recruited by the Childhood Autism Risk from Genes and Environment study at the University of California, Davis. mtDNA deletions and higher p53 gene copy ratios were more common in children with AU and their fathers. The incidence of mtDNA deletions in fathers of children with AU was increased 1.9-fold over fathers of typically neurodeveloping children, suggesting a role for deficient DNA repair capacity not driven by paternal age. Deletions in mtDNA and altered p53 gene copy ratios seem to result from genetics (children with severity scores ≥8) and/or act in concert with environmental factors (children with 6-7 severity scores). Given pro- and antioxidant activities of p53, and associations of genomic instability with disorders other than AU, our study suggests a link between DNA repair capacity, genomic instability in the 17p13.1 region influenced by environmental triggers, and AU diagnosis.

Defective mitochondrial disulfide relay system, altered mitochondrial morphology and function in Huntington's disease

A number of studies have been conducted that link mitochondrial dysfunction (MD) to Huntington's disease (HD); however, contradicting results had resulted in a lack of a clear mechanism that links expression of mutant Huntingtin protein and MD. Mouse homozygous (HM) and heterozygous (HT) mutant striatal cells with two or one allele encoding for a mutant huntingtin protein with 111 polyGln repeats showed a significant impairment of the mitochondrial disulfide relay system (MDRS). This system (consisting of two proteins, Gfer and Mia40) is involved in the mitochondrial import of Cys-rich proteins. The Gfer-to-Mia40 ratio was significantly altered in HM cells compared with controls, along with the expression of mitochondrial proteins considered substrates of the MDRS. In progenitors and differentiated neuron-like HM cells, impairment of MDRS were accompanied by deficient oxidative phosphorylation, Complex I, IV and V activities, decreased mtDNA copy number and transcripts, accumulation of mtDNA deletions and changes in mitochondrial morphology, consistent with other MDRS-deficient biological models, thus providing a framework for the energy deficits observed in this HD model. The majority (>90%) of the mitochondrial outcomes exhibited a gene-dose dependency with the expression of mutant Htt. Finally, decreases in the mtDNA copy number, along with the accumulation of mtDNA deletions, provide a mechanism for the progressive neurodegeneration observed in HD patients.

Mitochondrial Dysfunction in Pten Haplo-Insufficient Mice with Social Deficits and Repetitive Behavior: Interplay between Pten and p53

Etiology of aberrant social behavior consistently points to a strong polygenetic component involved in fundamental developmental pathways, with the potential of being enhanced by defects in bioenergetics. To this end, the occurrence of social deficits and mitochondrial outcomes were evaluated in conditional Pten (Phosphatase and tensin homolog) haplo-insufficient mice, in which only one allele was selectively knocked-out in neural tissues. Pten mutations have been linked to Alzheimer's disease and syndromic autism spectrum disorders, among others. By 4–6 weeks of age, Pten insufficiency resulted in the increase of several mitochondrial Complex activities (II–III, IV and V) not accompanied by increases in mitochondrial mass, consistent with an activation of the PI3K/Akt pathway, of which Pten is a negative modulator. At 8–13 weeks of age, Pten haplo-insufficient mice did not show significant behavioral abnormalities or changes in mitochondrial outcomes, but by 20–29 weeks, they displayed aberrant social behavior (social avoidance, failure to recognize familiar mouse, and repetitive self-grooming), macrocephaly, increased oxidative stress, decreased cytochrome c oxidase (CCO) activity (50%) and increased mtDNA deletions in cerebellum and hippocampus. Mitochondrial dysfunction was the result of a downregulation of p53-signaling pathway evaluated by lower protein expression of p21 (65% of controls) and the CCO chaperone SCO2 (47% of controls), two p53-downstream targets. This mechanism was confirmed in Pten-deficient striatal neurons and, HCT 116 cells with different p53 gene dosage. These results suggest a unique pathogenic mechanism of the Pten-p53 axis in mice with aberrant social behavior: loss of Pten (via p53) impairs mitochondrial function elicited by an early defective assembly of CCO and later enhanced by the accumulation of mtDNA deletions. Consistent with our results, (i) SCO2 deficiency and/or CCO activity defects have been reported in patients with learning disabilities including autism and (ii) mutated proteins in ASD have been found associated with p53-signaling pathways.

Mitochondrial biogenesis fails in secondary biliary cirrhosis in rats leading to mitochondrial DNA depletion and deletions

Chronic cholestasis is characterized by mitochondrial dysfunction, associated with loss of mitochondrial membrane potential, decreased activities of respiratory chain complexes, and ATP production. Our aim was to determine the molecular mechanisms that link long-term cholestasis to mitochondrial dysfunction. We studied a model of chronic cholestasis induced by bile duct ligation in rats. Key sensors and regulators of the energetic state and mitochondrial biogenesis, mitochondrial DNA (mtDNA)-to-nuclear DNA (nDNA) ratio (mtDNA/nDNA) relative copy number, mtDNA deletions, and indexes of apoptosis (BAX, BCL-2, and cleaved caspase 3) and cell proliferation (PCNA) were evaluated. Our results show that long-term cholestasis is associated with absence of activation of key sensors of the energetic state, evidenced by decreased SIRT1 and pyruvate dehydrogenase kinase levels and lack of AMPK activation. Key mitochondrial biogenesis regulators (PGC-1α and GABP-α) decreased and NRF-1 was not transcriptionally active. Mitochondrial transcription factor A (TFAM) protein levels increased transiently in liver mitochondria at 2 wk after bile duct ligation, but they dramatically decreased at 4 wk. Reduced TFAM levels at this stage were mirrored by a marked decrease (65%) in mtDNA/nDNA relative copy number. The blockade of mitochondrial biogenesis should not be ascribed to activation of apoptosis or inhibition of cell proliferation. Impaired mitochondrial turnover and loss of the DNA stabilizing effect of TFAM are likely the causative event involved in the genetic instability evidenced by accumulation of mtDNA deletions. In conclusion, the lack of stimulation of mitochondrial biogenesis leads to mtDNA severe depletion and deletions in long-term cholestasis. Hence, long-term cholestasis should be considered a secondary mitochondrial hepatopathy.

The potential benefits of creatine and conjugated linoleic acid as adjuncts to resistance training in older adults

Human aging is associated with a significant reduction in muscle mass (sarcopenia) resulting in muscle weakness and functional limitations in the elderly. Sarcopenia has been associated with mitochondrial dysfunction and the accumulation of mtDNA deletions. Resistance training increases muscle strength and size and can increase mitochondrial capacity and decrease oxidative stress in older adults. Creatine monohydrate (CrM) and conjugated linoleic acid (CLA) have biological effects that could enhance some of the beneficial effects of resistance training in older adults (i.e., up arrow fat-free mass, down arrow total body fat). We have completed two resistance-training studies with CrM alone and CrM+CLA supplementation in older adults to evaluate the independent effects of exercise and dietary supplements, as well as their interactive effects. Our studies, and several others, have found that CrM enhanced the resistance exercise mediated gains in fat-free mass and strength. More recently, we found that the addition of CLA also lead to a significant reduction of body fat after six months of resistance training in older adults. Older adults have fewer wild-type mtDNA copies and higher amounts of mtDNA deletions as compared with younger adults in mature skeletal muscle; however, these deletions are not seen in the satellite cell-derived myoblast cultures. These findings, and the fact that mtDNA deletions are lower and wild-type mtDNA copy number is higher after resistance training in older adults, suggests that activation of satellite cells secondary to resistance exercise-induced muscle damage can dilute or "shift" the proportion of mtDNA genotype towards that of a younger adult. Recent evidence suggests that CrM supplementation in combination with strength training can enhance satellite cell activation and total myonuclei number per muscle fiber in young men. Future studies are required to determine whether the mitochondrial adaptations to resistance exercise in older adults are further enhanced with CrM supplementation and whether this is due to increased recruitment of satellite cells. It will also be important to determine whether these changes are maintained over a longer time period.

Mitochondrial Dysfunction as the Molecular Basis of Bipolar Disorder

Multiple lines of evidence, such as impaired energy metabolism in the brain detected by magnetic resonance spectroscopy, a possible role of maternal inheritance, co-morbidity with mitochondrial diseases, the effects of mood stabilisers on mitochondria, increased mitochondrial DNA (mtDNA) deletion in the brain, and association with mtDNA mutations/polymorphisms or nuclear-encoded mitochondrial genes, suggest that mitochondrial dysfunction is an important component of bipolar disorder. Global reduction of mitochondria-related gene expression in the postmortem brains of patients with bipolar disorder may also be an indicator, but such findings are affected by sample pH and thus need to be interpreted with caution. A recently developed animal model carrying mtDNA deletion in neurons suggested that accumulation of mtDNA deletions causes bipolar disorder-like phenotypes. The next step in the study of mitochondrial dysfunction in bipolar disorder should be clarification of how mitochondrial dysfunction, a nonspecific risk factor, can cause specific symptoms of bipolar disorder. Two hypothetical mechanisms are mtDNA neuroplasticity and nonvisual photoreception impairment. Further study of mitochondrial dysfunction in bipolar disorder is expected to be useful for the development of new mood stabilisers.

Mitochondrial DNA deletions in rhesus macaque oocytes and embryos

Mitochondria are the most abundant organelles in mammalian oocytes and early embryos. Mitochondrial DNA (mtDNA) mutations, including the common deletion, have been found in skeletal muscle fibres from aged rhesus macaques. The specific aims of this study were to determine whether the mitochondrial common deletion is present in rhesus oocytes after hormonal stimulation and in embryos generated by in vitro production, or whether this deletion is already present in the immature oocyte. Using a nested primer PCR strategy, we found a significant increase in the proportion of mtDNA deletions in stimulated oocytes and embryos from rhesus macaques, compared with mtDNA deletions in immature, unstimulated oocytes derived from necropsied ovaries of age-matched monkeys. The common deletion is larger in the rhesus (5704 bp) than in humans (4977 bp). Accumulation of mtDNA deletions in oocytes may contribute to mitochondrial dysfunction and impaired ATP production. We propose the rhesus to be an excellent model to assess the quality of gametes and embryos and their developmental competence in primates, including humans.

Deleted Mitochondrial DNA in Human Luteinized Granulosa Cells

The rearrangement of mitochondrial DNA in luteinized granulosa cells was determined in order to evaluate the fertilization capacity of oocytes and the development of embryos. Multiple deletions of mtDNA were found in luteinized granulosa cells from in vitro fertilization (IVF) patients. The 4977-base pair (bp) deletion was the most frequent deletion found in human granulosa cells. No significant difference was noted between mtDNA deletions of granulosa cells based on the fertilization capacity of oocytes and the development of embryos. To determine the relationship of proportions of mtDNA rearrangements with the aging process, granulosa cells were grouped into three different cohorts according to maternal age: younger than 32 years, between 32 and 37 years, and older than 37 years. No statistical correlation was noted between patient age and the frequency of occurrence of multiple mtDNA deletions. However, an increase in granulosa cell apoptosis was associated with an increase in mtDNA deletions. Accumulation of mtDNA deletions may contribute to mitochondrial dysfunction and impaired ATP production. We concluded that the accumulation of rearranged mtDNA in granulosa cells might not interfere with fertilization of human oocytes and further embryonic development; it was, however, associated with apoptosis processes.

128 MITOCHONDRIAL DNA DELETIONS IN RHESUS MACAQUE OOCYTES, EMBRYOS, AND ADULT AND EMBRYONIC STEM CELLS

Mitochondria are the most abundant organelles in mammalian oocytes and early embryos. Previous data have shown that mitochondrial DNA (mtDNA) deletions are present both in human oocytes and in embryos from in vitro fertilization (IVF) patients and suggest that accumulation of these deletions may contribute to mitochondrial dysfunction and impaired ATP production. In addition, high levels of mitochondrial mutations are present in skeletal muscle fibers from aged rhesus macaques. The specific aims of this study were to determine whether the mitochondrial common deletion is present in non-human primate oocytes and embryos generated by IVF and to determine whether mtDNA mutations are already present in immature oocytes from rhesus ovaries. Using a nested primer polymerase chain reaction (PCR) strategy, we determined the frequency of the rhesus common deletion in immature oocytes compared with stimulated oocytes and embryos. There was a low incidence (21%) of the rhesus common deletion present in immature, unstimulated oocytes derived from necropsied ovaries of 2 to 10-yr-old rhesus macaques. However, there was >3-fold increase (71.4%) in the frequency of deleted mtDNA in stimulated oocytes and IVF embryos from age-matched fertile monkeys. We postulated that, in addition to skeletal muscle, a similar time-dependent accumulation of mtDNA deletions occurs in fertile rhesus macaque oocytes and embryos. We are now investigating the effects of culture and passage number on mtDNA deletions in primate adult and embryonic stem cells. We propose the rhesus monkey to be an excellent model to assess the quality of gametes and embryos, as well as stem cells, and their developmental competence in human and non-human primates. This study was supported by National Institutes of Health grants RR15395 and HD045966.

Mitochondrial DNA 4,977‐bp Deletion in Paired Oral Cancer and Precancerous Lesions Revealed by Laser Microdissection and Real‐Time Quantitative PCR

Abstract: Oral cancer is the fourth leading cause of cancer deaths among men in Taiwan and is closely associated with areca quid chewing habits. Recent studies showed that mitochondrial DNA (mtDNA) mutations occur in various tumors, including oral cancers, and that the accumulation of mtDNA deletions could be an important contributor to carcinogenesis. Using laser microdissection, we have analyzed mtDNA deletions by pairwise comparisons in oral cancer, precancerous cells, and their adjacent submucosal stoma tissues in 12 patients with areca quid chewing history. Real‐time quantitative polymerase chain reaction (RTQPCR) was performed to detect and quantify mtDNA with the 4,977‐bp deletion in the histologically defined specified cell groups. Quantitative analysis of 60 samples by RTQPCR revealed that the average proportions of 4,977‐bp deleted mtDNA over total mtDNA were 0.137%, 0.367%, and 0.001% in cancer, precancer cells, and lymphocytes of lymph node biopsies, respectively. Pairwise analysis of the proportion of mtDNA deletion in cancer, precancer, and their stroma tissues revealed a consistent trend among these patients. All of the patients (12/12) presented a higher proportion of mtDNA with 4,977‐bp deletion in the lesions than in the lymphocytes, with average increases of 198‐fold in cancer and 546‐fold in precancer cells. A decrease in the proportion of deleted mtDNA was observed in 8 of 12 patients when the disease progressed from precancer to cancer lesions. Interestingly, 7 of 12 cancer tissues and 8 of 12 precancer lesions exhibited an average of 6.3‐fold and 17.4‐fold increases in the proportion of 4,977‐bp deleted mtDNA in the stromal cells than in the lesion cells, respectively. The observation that the proportion of 4,977‐bp deleted mtDNA in all oral lesions was higher than normal and consistently decreased during cancer progression from precancer to primary cancer suggests that accumulation and subsequent cytoplasmic segregation of the mutant mtDNA during cell division may play an important role in oral carcinogenesis. This study also demonstrates that laser microdissection combined with RTQPCR is an efficient and sensitive tool to gain insight into the role that mtDNA mutation may play in carcinogenesis.

Mitochondrial DNA 4,977-bp deletion in paired oral cancer and precancerous lesions revealed by laser microdissection and real-time quantitative PCR.

Oral cancer is the fourth leading cause of cancer deaths among men in Taiwan and is closely associated with areca quid chewing habits. Recent studies showed that mitochondrial DNA (mtDNA) mutations occur in various tumors, including oral cancers, and that the accumulation of mtDNA deletions could be an important contributor to carcinogenesis. Using laser microdissection, we have analyzed mtDNA deletions by pairwise comparisons in oral cancer, precancerous cells, and their adjacent submucosal stoma tissues in 12 patients with areca quid chewing history. Real-time quantitative polymerase chain reaction (RTQPCR) was performed to detect and quantify mtDNA with the 4,977-bp deletion in the histologically defined specified cell groups. Quantitative analysis of 60 samples by RTQPCR revealed that the average proportions of 4,977-bp deleted mtDNA over total mtDNA were 0.137%, 0.367%, and 0.001% in cancer, precancer cells, and lymphocytes of lymph node biopsies, respectively. Pairwise analysis of the proportion of mtDNA deletion in cancer, precancer, and their stroma tissues revealed a consistent trend among these patients. All of the patients (12/12) presented a higher proportion of mtDNA with 4,977-bp deletion in the lesions than in the lymphocytes, with average increases of 198-fold in cancer and 546-fold in precancer cells. A decrease in the proportion of deleted mtDNA was observed in 8 of 12 patients when the disease progressed from precancer to cancer lesions. Interestingly, 7 of 12 cancer tissues and 8 of 12 precancer lesions exhibited an average of 6.3-fold and 17.4-fold increases in the proportion of 4,977-bp deleted mtDNA in the stromal cells than in the lesion cells, respectively. The observation that the proportion of 4,977-bp deleted mtDNA in all oral lesions was higher than normal and consistently decreased during cancer progression from precancer to primary cancer suggests that accumulation and subsequent cytoplasmic segregation of the mutant mtDNA during cell division may play an important role in oral carcinogenesis. This study also demonstrates that laser microdissection combined with RTQPCR is an efficient and sensitive tool to gain insight into the role that mtDNA mutation may play in carcinogenesis.

Multiple rearrangements of mitochondrial DNA in unfertilized human oocytes

Objective: To determine the rearrangement of mitochondrial DNA (mtDNA) in unfertilized human oocytes and compromised embryos to evaluate the fertilization capacity of oocytes. Design: Prospective laboratory research. Setting: IVF laboratory in a university hospital. Patient(s): One hundred twenty-four unfertilized oocytes, 98 arrested embryos, and 45 tripronucleate (3PN) embryos from 65 female patients undergoing in vitro fertilization (IVF). Intervention(s): Unfertilized oocytes and poor quality embryos were collected 48 hours after IVF. Main Outcome Measure(s): Comparison of the frequency of mtDNA deletions and fertilization rates of oocytes. Result(s): Multiple deletions of mtDNA were found in unfertilized oocytes and arrested embryos obtained from IVF patients. A 4977-bp deletion was the most frequent deletion in human oocytes and embryos. About 66.1% of the unfertilized oocytes, 34.8% of the arrested or fragmented embryos, and 21.1% of the 3PN embryos harbored the 4977-bp deletion of mtDNA. There was a significant increase in the proportion of deleted mtDNA in unfertilized oocytes. Conclusion(s): Accumulation of mtDNA deletions may contribute to mitochondrial dysfunction and impaired ATP production. We conclude that the accumulation of rearranged mtDNA may interfere with fertilization of human oocytes and further embryonic development.

Accumulation of mitochondrial DNA deletions in human oral tissues — effects of betel quid chewing and oral cancer

Accumulation of mitochondrial DNA (mtDNA) mutations in human tissues has been associated with intrinsic aging and environmental insult. Recently, mtDNA mutations have been detected in various tumors, including head and neck tumors. However, the factors affecting the occurrence and accumulation of mtDNA deletions in tumor tissues are poorly understood. In Taiwan, betel quid chewing is a major risk factor for oral cancer. Using polymerase chain reaction (PCR) techniques, we examined large-scale deletions of mtDNA in 53 pairs of tumor and non-tumor oral tissues from the patients with or without betel quid chewing history. The results revealed that irrespective of the history of betel quid chewing, the incidences of the 4977 bp deletion and other deletions of mtDNA were lower in the tumor portion as compared with the non-tumor portion. The average proportions of the 4977 bp deleted mtDNA in the tumor tissues of the betel quid chewers and non-betel quid chewers were 13- and 5-fold, respectively, lower than those in the corresponding non-tumor tissues. Moreover, the average proportion of 4977 bp deleted mtDNA was significantly higher ( P<0.05) in the non-tumor oral tissues of the patients with betel quid chewing history than that of the patients without the history of betel quid chewing. These results suggest that betel quid chewing may increase mtDNA mutation in human oral tissues and that accumulation of mtDNA deletions and subsequent cytoplasmic segregation of these mutations during cell division could be an important contributor to the early phase of oral carcinogenesis.

Human Skin Mitochondrial DNA Deletions Associated with Light Exposure

By polymerase chain reaction technique and DNA sequencing, we demonstrated the existence of multiple deletions in the mitochondrial DNA (mtDNA) of human skin tissues obtained from different body sites of an 86-year-old male farmer. We examined four types of skin tissues of different physiologic conditions, including sun-exposed, nonexposed, precancerous, and cancerous. The results showed that the common age-related 4977-base-pair (bp)-deleted mtDNA was present in different proportions in all the tissues examined. Quantitative PCR revealed that the amount of the 4977-bp-deleted mtDNA was associated with the physiologic conditions of the skin tissues. The sun-exposed skin tissues harbored higher level of the 4977-bp-deleted mtDNA (12.5%) than the nonexposed normal aged skin tissues (0.4%). On the other hand, in all the exposed skin tissues examined, the slower growing aged skin tissues harbored higher level of the 4977-bp-deleted mtDNA than the faster growing skin tissues in cancerous and precancerous skin tissues. In addition, we found at least four new deletions in the aforementioned different body sites with sizes of 7031, 7150, 7288, and 7485 bp, respectively. These findings provide an evidence of photooxidation-induced mtDNA mutations, since the deletions deposit in different patterns and proportions in different skin tissues of the patient. These findings suggest that, besides the intrinsic formation and accumulation of mtDNA deletions in human tissues during normal aging process, physiological and environmental factors may also play important roles in eliciting the deposit of deleted mtDNA molecules in the tissue cells.

Detection of the 4977 Base Pair Mitochondrial DNA Deletion in Paraffin-Embedded Heart Tissue Using the Polymerase Chain Reaction—A New Method to Probe Sudden Cardiac Death Molecular Mechanisms?

Detection of mitochondrial DNA deletions is performed in fresh or frozen material. At our institute, however, heart samples from subjects referred for autopsy are systematically processed for histologic examination (that is, paraffin-embedded). We were interested to know if mtDNA deletions can be detected in such material. Our data indicate that: 1) the most frequently observed deletion--the 4977 base pair deletion--can easily be detected in paraffin-embedded heart tissue; 2) this assay is sufficiently sensitive, since very low levels of the deletion can be found in normal heart tissue from young adults; and 3) buffered formalin appears to be the fixative of choice. Recent literature shows that repeated episodes of ischemia result in the accumulation of mtDNA deletions in myocardial cells. Because ischemic heart disease is a major cause of sudden cardiac death, a sensitive method for the detection of mtDNA damage in myocardial cells will be an important tool to facilitate understanding of unexpected cardiac arrest mechanisms.