Quantitative effect of 4977 bp deletion of mitochondrial DNA on the susceptibility of human cells to UV-induced apoptosis

In this study, we used a series of human cytoplasmic hybrids (cybrids) harboring different proportions of 4977 bp-deleted mtDNA to investigate the quantitative effect of a pathogenic mutation of mtDNA on apoptosis. We found that the sensitivity of human cells to apoptosis triggered by UV irradiation increases with the proportion of 4977 bp-deleted mtDNA. Moreover, UV-induced activation of caspase 3 was preceded by the activation of caspases 8 and 9. Most importantly, we observed that UV-induced cytochrome c release from mitochondria occurred much earlier and was much more pronounced in the cybrids harboring higher proportions of 4977 bp-deleted mtDNA. We suggest that 4977 bp-deleted mtDNA increases the susceptibility of human cells to UV-induced apoptosis in a quantitative manner through cytochrome c release from mitochondria and caspase 3 activation.     

Heteroplasmic mutation of mitochondrial DNA D-loop and 4977-bp deletion in human cancer cells during mitochondrial DNA depletion

Somatic mutations in mitochondrial DNA (mtDNA) have been demonstrated in various human cancers. Many cancers have high frequently of mtDNA with homoplasmic point mutations, and carry less frequently of mtDNA with large-scale deletions as compared with corresponding non-cancerous tissue. Moreover, most cancers harbor a decreased copy number of mtDNA than their corresponding non-cancerous tissue. However, it is unclear whether the process of decreasing in mtDNA content would be involved in an increase in the heteroplasmic level of somatic mtDNA point mutation, and/or involved in a decrease in the proportion of mtDNA with large-scale deletion in cancer cells. In this study, we provided evidence that the heteroplasmic levels of variations in cytidine number in np 303-309 poly C tract of mtDNA in three colon cancer cells were not changed during an ethidium bromide-induced mtDNA depleting process. In the mtDNA depleting process, the proportions of mtDNA with 4977-bp deletion in cybrid cells were not significantly altered. These results suggest that the decreasing process of mtDNA copy number per se may neither contribute to the shift of homoplasmic/heteroplasmic state of point mutation in mtDNA nor to the decrease in proportion of mtDNA with large-scale deletions in cancer cells. Mitochondrial genome instability and reduced mtDNA copy number may independently occur in human cancer.     

Differential accumulations of 4,977 bp deletion in mitochondrial DNA of various tissues in human ageing

Several types of deletions in mitochondrial DNA (mtDNA) have been recetly identified in various tissues of old humans. In order to determine whether there are differences in the incidence and proportion of deleted mtDNAs in different tissues during human ageing, we examined tha 4,977 bp deletion in mtDNA of various tissues from subjects of different ages. Total DNA was extracted from each of the biopsied tissues and was serially diluted by two-fold with distilled water. A 533 bp DNA fragment was amplified by PCR from total mtDNA using a pair of primers L3304-3323 and H3817-3836, and another 524 bp PCR product was amplified from 4,977 bp deleted mtDNA by identical conditions using another pair of primers L8150-8166 and H13631-13650. The maximum dilution fold of each sample that still allowed the ethidium bromide-stained PCR product (533 bp or 524 bp) in the agarose gel to be visible under UV light illumination was taken as the relative abundance of the mtDNA (wild-type or mutant) in the original sample. By this method, we were able to determine the proportion of deleted mtDNA in human tissues. We found that the 4,977 bp deletion started to appear in the second and third decades of life in human muscle and liver tissues. But the deletion was not detectable in the testis until the age of 60 years. Moreover, the proportion of deleted mtDNA varied greatly in different tissues. Among the tissues examined, muscle was found to harbor higher proportin of deleted mtDNA than the other tissues. The average proportion of the 4,977 bp depleted mtDNA of the muscle from subjects over 70 years old was approximately 0.06%, and that of the liver and the testis was 0.0076% and 0.05%, respectively. These findings suggest that the frequency and proportion of the deleted mtDNA in human tissues increase with age and that the mtDNA deletions occur more frequently and abundantly in high energy-demanding tissues during the ageing process of the human.     

Increases in mitochondrial DNA content and 4977-bp deletion upon ATM/Chk2 checkpoint activation in HeLa cells

Activation of the Mec1/Rad53 damage checkpoint pathway influences mitochondrial DNA (mtDNA) content and point mutagenesis in Saccharomyces cerevisiae. The effects of this conserved checkpoint pathway on mitochondrial genomes in human cells remain largely unknown. Here, we report that knockdown of the human DNA helicase RRM3 enhances phosphorylation of the cell cycle arrest kinase Chk2, indicating activation of the checkpoint via the ATM/Chk2 pathway, and increases mtDNA content independently of TFAM, a regulator of mtDNA copy number. Cell-cycle arrest did not have a consistent effect on mtDNA level: knockdown of cell cycle regulators PLK1 (polo-like kinase), MCM2, or MCM3 gave rise, respectively, to decreased, increased, or almost unchanged mtDNA levels. Therefore, we concluded that the mtDNA content increase upon RRM3 knockdown is not a response to delay of cell cycle progression. Also, we observed that RRM3 knockdown increased the levels of reactive oxygen species (ROS); two ROS scavengers, N-acetyl cysteine and vitamin C, suppressed the mtDNA content increase. On the other hand, in RRM3 knockdown cells, we detected an increase in the frequency of the common 4977-bp mtDNA deletion, a major mtDNA deletion that can be induced by abnormal ROS generation, and is associated with a decline in mitochondrial genome integrity, aging, and various mtDNA-related disorders in humans. These results suggest that increase of the mitochondrial genome by TFAM-independent mtDNA replication is connected, via oxidative stress, with the ATM/Chk2 checkpoint activation in response to DNA damage, and is accompanied by generation of the common 4977-bp deletion.     

Detection of mtDNA with 4977 bp deletion in blood cells and atherosclerotic lesions of patients with coronary artery disease

Recent evidence suggests that somatic mutations in nuclear and mitochondrial DNA accumulated during aging, may significantly contribute to the pathogenesis of chronic-degenerative illness such as coronary artery disease (CAD). Mitochondrial DNA with 4977 bp deletion mutation (mtDNA4977) is a common type of mtDNA alteration in humans. However, little attempt has been made to detect the presence of mtDNA4977 deletion in cells and tissues of cardiovascular patients. This study investigated the presence of mtDNA4977 in blood samples of 65 cardiovascular patients and 23 atherosclerotic plaques of human coronaries with severe atherosclerosis. Moreover, the presence of the deletion has been investigated in blood cells from 22 healthy age-matched subjects. The detection of mtDNA4977 has been performed by using a nested polymerase chain reaction (PCR) protocol and normalized to wild-type mtDNA. A significant higher incidence of mtDNA4977 was observed in CAD patients with respect to healthy subjects (26.2% versus 4.5%; P=0.03). Furthermore, the relative amount of the deletion was significantly higher in the patients compared to the control group (P=0.02). The mtDNA4977 was detected in 17 of the 65 patients blood samples (26.2%) and deletion levels ranged from 0.18 to 0.46% of the total mtDNA (mean: 0.34+/-0.02%). For what concerns atherosclerotic lesions, 5 patients (21.7%) showed the deletion ranging from 0.13 to 0.45% of the total mtDNA (mean: 0.35+/-0.06%). In both samples from patients, the incidence and the relative amount of mtDNA4977 was not significantly influenced by atherogenic risk factors and clinical parameters. The obtained results may suggest that the increase of oxidative stress in cardiovascular disease may be responsible for the accumulation of mtDNA damage in coronary artery disease patients.     

Studies on polypeptide composition, hydrolytic activity and proton conduction of mitochondrial FoF1 H+ ATPase in regenerating rat liver

A study of the FoF1 ATPase complex of mitochondria isolated from regenerating rat liver following partial (70%) hepatectomy is presented. As we have previously reported, ATPase activity in submitochondrial particles prepared from regenerating rat liver 24 h following partial hepatectomy was depressed by 75% with respect to controls (submitochondrial particles from sham-operated animals). Polyacrylamide gel electrophoresis and immunodecoration using an antibody raised against isolated bovine heart F1 sector of the FoF1 ATPase indicated a substantial decrease in F1 content in the mitochondrial membrane from regenerating rat liver. Proton conduction by the FoF1 ATPase complex was studied by following the anaerobic relaxation of the transmembrane proton gradient (delta mu H+) generated by succinate-driven respiration. In control rat-liver submitochondrial particles containing the FoF1 moiety of the ATPase complex, anaerobic relaxation of delta mu H+ showed biphasic kinetics, whilst the same process in particles derived from regenerating rat liver exhibited monophasic kinetics and was significantly more rapid. Oligomycin and N,N-dicyclohexyl carbodiimide [(cHxN)2C] inhibited proton conductance by the F1-Fo ATPase complex in submitochondrial particles from both control and regenerating rat liver. Binding of [14C](cHxN)2C and immunodecoration using an antibody raised against bovine heart oligomycin-sensitivity-conferring protein (OSCP) indicated no difference in the content of either the (cHxN)2C binding protein or OSCP between control and regenerating rat-liver mitochondrial membranes. The results reported show that the structural and functional integrity of the Fo-F1 ATPase of rat liver is severely perturbed during regeneration.     

Sirt3 mitigates LPS‐induced mitochondrial damage in renal tubular epithelial cells by deacetylating YME1L1

Acute kidney injury (AKI) is often secondary to sepsis. Increasing evidence suggests that mitochondrial dysfunction contributes to the pathological process of AKI. In this study, we aimed to examine the regulatory roles of Sirt3 in Lipopolysaccharide (LPS)‐induced mitochondrial damage in renal tubular epithelial cells (TECs). Sirt3 knockout mice were intraperitoneally injected with LPS, and cultured TECs were stimulated with LPS to evaluate the effects of Sirt3 on mitochondrial structure and function in TECs. Electron microscopy was used to assess mitochondrial morphology. Immunofluorescence staining was performed to detect protein expression and examine mitochondrial morphology. Western blotting was used to quantify protein expression. We observed that LPS increased apoptosis, induced disturbances in mitochondrial function and dynamics, and downregulated Sirt3 expression in a sepsis‐induced AKI mouse model and human proximal tubular (HK‐2) cells in vitro. Sirt3 deficiency further exacerbated LPS‐induced renal pathological damage, apoptosis and disturbances in mitochondrial function and dynamics. On the contrary, Sirt3 overexpression in HK‐2 cells alleviated these lesions. Functional studies revealed that Sirt3 overexpression alleviated LPS‐induced mitochondrial damage and apoptosis in TECs by promoting OPA1‐mediated mitochondrial fusion through the deacetylation of i‐AAA protease (YME1L1), an upstream regulatory molecule of OPA1. Our study has identified Sirt3 as a vital factor that protects against LPS‐induced mitochondrial damage and apoptosis in TECs via the YME1L1‐OPA1 signaling pathway.

In a physiological state, Sirt3 has a certain deacetylation effect on YME1L1, and YME1L1 deacetylation can promote OPA1‐mediated mitochondrial fusion, so that mitochondrial fusion and fission are in a balanced state. When sepsis‐induced AKI occurs, the expression of Sirt3 is reduced, resulting in a decrease in the deacetylation level of YME1L1 and a decrease in the expression of L‐OPA, which in turn reduces mitochondrial fusion and increases fission, ultimately leading to mitochondrial dysfunction and apoptosis.     

Mitochondrial DNA 4977 bp deletion and OH8dG levels correlate in the brain of aged subjects but not Alzheimer's disease patients.

The levels of mitochondrial DNA 4977 bp deletion (mtDNA4977) and mitochondrial DNA 8'-hydroxy-2'-deoxyguanosine (OH8dG) were determined in the same samples from two brain areas of healthy subjects and Alzheimer's disease (AD) patients. A positive correlation between the age-related increases of mtDNA4977 and of OH8dG levels was found in the brain of healthy individuals. On the contrary, in both brain areas of AD patients, mtDNA4977 levels were very low in the presence of high OH8dG amounts. These results might be explained assuming that the increase of OH8dG above a threshold level, as in AD patients, implies consequences for mtDNA replication and neuronal cell survival.     

Cytoskeletal structure in cells harboring two mutations: R133C in NOTCH3 and 5650G>A in mitochondrial DNA

We have previously described a patient with cerebral autosomal-dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) caused by R133C mutation in NOTCH3 and with a concomitant myopathy caused by a G to A point mutation at base pair 5650 (5650G>A) in the gene encoding tRNA(Ala) in mitochondrial DNA (mtDNA). In the present study, we have examined the morphology of the cytoskeletal components in fibroblasts and myoblasts of this patient. Immunolabeling revealed that tubulin network was sparse and formed asters in these cells, whereas no changes were found in actin and vimentin networks in comparison to the control cell lines. Furthermore, mitochondria were less abundant and the branches of the mitochondrial network were reduced in number. Muscle histochemical analysis showed ragged red fibres (RRFs) and cytochrome c oxidase (COX)-negative fibres. The mean proportion of mtDNA with 5650G>A was lower in histologically normal muscle fibres than in the COX-negative fibres and in the RRFs. These findings suggest that 5650G>A is a pathogenic mtDNA mutation. However, the changes in tubulin network and mitochondrial distribution in patient fibroblasts and myoblasts cannot solely be explained by this mutation.     

Defective dimerization of FoF1‐ATP synthase secondary to glycation favors mitochondrial energy deficiency in cardiomyocytes during aging

Aged cardiomyocytes develop a mismatch between energy demand and supply, the severity of which determines the onset of heart failure, and become prone to undergo cell death. The FoF1‐ATP synthase is the molecular machine that provides >90% of the ATP consumed by healthy cardiomyocytes and is proposed to form the mitochondrial permeability transition pore (mPTP), an energy‐dissipating channel involved in cell death. We investigated whether aging alters FoF1‐ATP synthase self‐assembly, a fundamental biological process involved in mitochondrial cristae morphology and energy efficiency, and the functional consequences this may have. Purified heart mitochondria and cardiomyocytes from aging mice displayed an impaired dimerization of FoF1‐ATP synthase (blue native and proximity ligation assay), associated with abnormal mitochondrial cristae tip curvature (TEM). Defective dimerization did not modify the in vitro hydrolase activity of FoF1‐ATP synthase but reduced the efficiency of oxidative phosphorylation in intact mitochondria (in which membrane architecture plays a fundamental role) and increased cardiomyocytes’ susceptibility to undergo energy collapse by mPTP. High throughput proteomics and fluorescence immunolabeling identified glycation of 5 subunits of FoF1‐ATP synthase as the causative mechanism of the altered dimerization. In vitro induction of FoF1‐ATP synthase glycation in H9c2 myoblasts recapitulated the age‐related defective FoF1‐ATP synthase assembly, reduced the relative contribution of oxidative phosphorylation to cell energy metabolism, and increased mPTP susceptibility. These results identify altered dimerization of FoF1‐ATP synthase secondary to enzyme glycation as a novel pathophysiological mechanism involved in mitochondrial cristae remodeling, energy deficiency, and increased vulnerability of cardiomyocytes to undergo mitochondrial failure during aging.     

Sirt6 deficiency contributes to mitochondrial fission and oxidative damage in podocytes via ROCK1‐Drp1 signalling pathway

ObjectivesIncreasing evidence suggests that mitochondrial dysfunction is the key driver of angiotensin II (Ang II)‐induced kidney injury. This study was designed to investigate whether Sirtuin 6 (Sirt6) could affect Ang II‐induced mitochondrial damage and the potential mechanisms.Materials and MethodsPodocyte‐specific Sirt6 knockout mice were infused with Ang II and cultured podocytes were stimulated with Ang II to evaluate the effects of Sirt6 on mitochondrial structure and function in podocytes. Immunofluorescence staining was used to detect protein expression and mitochondrial morphology in vitro. Electron microscopy was used to assess mitochondrial morphology in mice. Western blotting was used to quantify protein expression.ResultsMitochondrial fission and decreased Sirt6 expression were observed in podocytes from Ang II‐infused mice. In Sirt6‐deficient mice, Ang II infusion induced increased apoptosis and mitochondrial fragmentation in podocytes than that in Ang II‐infused wild‐type mice. In cultured human podocytes, Sirt6 knockdown exacerbated Ang II‐induced mitochondrial fission, whereas Sirt6 overexpression ameliorated the Ang II‐induced changes in the balance between mitochondrial fusion and fission. Functional studies revealed that Sirt6 deficiency exacerbated mitochondrial fission by promoting dynamin‐related protein 1 (Drp1) phosphorylation. Furthermore, Sirt6 mediated Drp1 phosphorylation by promoting Rho‐associated coiled coil‐containing protein kinase 1 (ROCK1) expression.ConclusionOur study has identified Sirt6 as a vital factor that protects against Ang II‐induced mitochondrial fission and apoptosis in podocytes via the ROCK1‐Drp1 signalling pathway.

Schematic of the molecular action proposed in this study. Schematic depicting that Ang II‐induced sirt6 expression decline promotes mitochondrial fission and podocyte injury through ROCK1‐Drp1 signalling. SIRT6 reduction leads to increased levels of ROCK1, thereby enhancing Drp1 phosphorylation at the Ser637 site. The Drp1 phosphorylation finally results in podocyte injury by inducing mitochondrial fission and apoptosis.     

Regulation of energy metabolism in human cells in aging and diabetes: FoF(1), mtDNA, UCP, and ROS

Recent advances in bioenergetics consist of discoveries related to rotational coupling in ATP synthase (FoF(1)), uncoupling proteins (UCP), reactive oxygen species (ROS) and mitochondrial DNA (mtDNA). As shown in cloned sheep, mammalian genomes are composed of both nuclear DNA (nDNA) and maternal mtDNA. Oxidative phosphorylation (oxphos) varies greatly depending on cellular activities, and is regulated by both gene expression and the electrochemical potential difference of H(+) (Delta muH(+)). The expression of both mtDNA (by mtTFA) and nDNA for oxphos and UCP (by NRFs, etc.) is coordinated by a factor called PGC-1. The Delta muH(+) rotates an axis in FoF(1) that is regulated by inhibitors and ATP-sensitive K(+)-channels. We cultured human rho(o) cells (cells without mtDNA) in synthetic media and elucidated relationships among mtDNA, nDNA, Delta muH(+), UCPs, ROS, and apoptosis. These cells lack oxphos-dependent ROS formation and survive under conditions of high O(2). Cells cultured in the absence of ROS scavengers have proliferated for 40 years. UCPs lower Delta muH(+) and prevent ROS formation and resulting apoptosis. These results were applied to diabetology and gerontology. The pancreatic rho(o) cells did not secrete insulin, and mtDNA mutations caused diabetes, owing to the deficient Delta muH(+). Insulin resistance was closely related to UCPs and other energy regulators. The resulting high-glucose environment caused glycation of proteins and ROS-mediated apoptosis in vascular cells involved in diabetic complications. Telomeres, oxphos, and ROS are determinants in cellular aging. Cell division and ROS shortened telomeres and accelerated aging. In aged cells, Delta muH(+) was reduced by the slow respiration, and this change induced apoptosis. Cybrids made from aged cytoplasts and rho(o) cells showed that both decreased expression of nDNA, and somatic mutations of mtDNA are involved in the slowing of respiration in aged cells.     

ATPase activity of human ABCG1 is stimulated by cholesterol and sphingomyelin

ATP-binding cassette protein G1 (ABCG1) is important for the formation of HDL. However, the biochemical properties of ABCG1 have not been reported, and the mechanism of how ABCG1 is involved in HDL formation remains unclear. We established a procedure to express and purify human ABCG1 using the suspension-adapted human cell FreeStyle293-F. ABCG1, fused at the C terminus with green fluorescent protein and Flag-peptide, was solubilized with n-dodecyl-β-D-maltoside and purified via a single round of Flag-M2 antibody affinity chromatography. The purified ABCG1 was reconstituted in liposome of various lipid compositions, and the ATPase activity was analyzed. ABCG1 reconstituted in egg lecithin showed ATPase activity (150 nmol/min/mg), which was inhibited by beryllium fluoride. The ATPase activity of ABCG1, reconstituted in phosphatidylserine liposome, was stimulated by cholesterol and choline phospholipids (especially sphingomyelin), and the affinity for cholesterol was increased by the addition of sphingomyelin. These results suggest that ABCG1 is an active lipid transporter and possesses different binding sites for cholesterol and sphingomyelin, which may be synergistically coupled.     

Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells

Recent evidence suggests that coupled leading and lagging strand DNA synthesis operates in mammalian mitochondrial DNA (mtDNA) replication, but the factors involved in lagging strand synthesis are largely uncharacterised. We investigated the effect of knockdown of the candidate proteins in cultured human cells under conditions where mtDNA appears to replicate chiefly via coupled leading and lagging strand DNA synthesis to restore the copy number of mtDNA to normal levels after transient mtDNA depletion. DNA ligase III knockdown attenuated the recovery of mtDNA copy number and appeared to cause single strand nicks in replicating mtDNA molecules, suggesting the involvement of DNA ligase III in Okazaki fragment ligation in human mitochondria. Knockdown of ribonuclease (RNase) H1 completely prevented the mtDNA copy number restoration, and replication intermediates with increased single strand nicks were readily observed. On the other hand, knockdown of neither flap endonuclease 1 (FEN1) nor DNA2 affected mtDNA replication. These findings imply that RNase H1 is indispensable for the progression of mtDNA synthesis through removing RNA primers from Okazaki fragments. In the nucleus, Okazaki fragments are ligated by DNA ligase I, and the RNase H2 is involved in Okazaki fragment processing. This study thus proposes that the mitochondrial replication system utilises distinct proteins, DNA ligase III and RNase H1, for Okazaki fragment maturation.