Decrease of MtDNA copy number affects mitochondrial function and involves in the pathological consequences of ischaemic stroke

The mtDNA copy number can affect the function of mitochondria and play an important role in the development of diseases. However, there are few studies on the mechanism of mtDNA copy number variation and its effects in IS. The specific mechanism of mtDNA copy number variation is still unclear. In this study, mtDNA copy number of 101 IS patients and 101 normal controls were detected by qRT‐PCR, the effect of D‐loop variation on mtDNA copy number of IS patients was explored. Then, a TFAM gene KD‐OE PC12 cell model was constructed to explore the effect of mtDNA copy number variation on mitochondrial function. The results showed that the mtDNA copy number level of the IS group was significantly lower than that of the normal control group (p < 0.05). The relative expression of TFAM gene mRNA in the cells of the OGD/R treatment group was significantly lower than that of the control group (p < 0.05). In addition, after TFAM gene knockdown and over‐expression plasmids were transfected into HEK 293T cells, mtDNA copy number and ATP production level of Sh‐TFAM transfection group was significantly decreased (p < 0.05), while mtDNA copy number and ATP production level of OE‐TFAM transfected group were significantly higher than that of blank control group and OE‐ctrl negative control group (p < 0.01). Our study demonstrated that mitochondrial D‐loop mutation and TFAM gene dysfunction can cause the decrease of mtDNA copy number, thus affecting the mitochondrial metabolism and function of nerve cells, participating in the pathological damage mechanism of IS.     

Human prohibitin 1 maintains the organization and stability of the mitochondrial nucleoids

Mitochondrial prohibitin (PHB) proteins have diverse functions, such as the regulation of apoptosis and the maintenance of mitochondrial morphology. In this study, we clarified a novel mitochondrial function of PHB1 that regulates the organization and maintenance of mitochondrial DNA (mtDNA). In PHB1-knockdown cells, we found that mtDNA is not stained by fluorescent dyes, such as ethidium bromide and PicoGreen, although the mitochondrial membrane potential still maintains. We also demonstrated that mtDNA, which is predominantly found in the NP-40-insoluble fraction when isolated from normal mitochondria, is partially released into the soluble fraction when isolated from PHB1-knockdown cells, indicating that the organization of the mitochondrial nucleoids has been altered. Furthermore, we found that PHB1 regulates copy number of mtDNA by stabilizing TFAM protein, a known protein component of the mitochondrial nucleoids. However, TFAM does not affect the organization of mtDNA as observed in PHB1-knockdown cells. Taken together, these results demonstrate that PHB1 maintains the organization and copy number of the mtDNA through both TFAM-independent and -dependent pathways.     

Involvement of autophagy and mitochondrial dynamics in determining the fate and effects of irreparable mitochondrial DNA damage

Mitochondrial DNA (mtDNA) is different in many ways from nuclear DNA. A key difference is that certain types of DNA damage are not repaired in the mitochondrial genome. What, then, is the fate of such damage? What are the effects? Both questions are important from a health perspective because irreparable mtDNA damage is caused by many common environmental stressors including ultraviolet C radiation (UVC). We found that UVC-induced mtDNA damage is removed slowly in the nematode Caenorhabditis elegans via a mechanism dependent on mitochondrial fusion, fission, and autophagy. However, knockdown or knockout of genes involved in these processes—many of which have homologs involved in human mitochondrial diseases—had very different effects on the organismal response to UVC. Reduced mitochondrial fission and autophagy caused no or small effects, while reduced mitochondrial fusion had dramatic effects.     

Enhanced phosphorylation of AMPK by lutein and oxidised lutein that lead to mitochondrial biogenesis in hyperglycemic HepG2 cells

The stimulation of adenosine monophosphate-activated protein kinase (AMPK) is a prime target to decrease the hyperglycemic condition, hence it is a lutein (L) and oxidised lutein (OXL) is a target molecule for the treatment of type II diabetes. In the current study, a plausible interaction of L and OXL with AMPK was investigated by molecular docking. In addition, the effect of L and OXL for the activation of AMPK that triggers the downstream regulator peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), TFAM expression, mitochondrial DNA (mtDNA), mitochondrial biogenesis and superoxide dismutase 2 (SOD2) in high glucose treated HepG2 cells were investigated by quantitative polymerase chain reaction and Western blot analysis. Molecular docking reveals higher binding affinity of L (ΔG = −6.3 kcal/mol) and OXL (ΔG = −15.5 kcal/mol) with AMPK, compared with metformin (ΔG = −5.0 kcal/mol). The phosphorylation of AMPK increased by 1.3- and 1.5-fold with L and OXL treatment, respectively, in high glucose induced HepG2 cells. The activation of PGC-1α is significant (P < 0.05) in OXL group than L. Similarly, TFAM expression is increased with L and OXL compared with the high glucose group. Further increase in SOD2 and mtDNA, confirms the efficacy of L and OXL in restoring the mitochondrial biogenesis in high glucose induced cells through AMPK, PGC-1α, and TFAM.     

The implications of mitochondrial DNA copy number regulation during embryogenesis

Mutations of mitochondrial DNA (mtDNA) cause a wide array of multisystem disorders, particularly affecting organs with high energy demands. Typically only a proportion of the total mtDNA content is mutated (heteroplasmy), and high percentage levels of mutant mtDNA are associated with a more severe clinical phenotype. MtDNA is inherited maternally and the heteroplasmy level in each one of the offspring is often very different to that found in the mother. The mitochondrial genetic bottleneck hypothesis was first proposed as the explanation for these observations over 20 years ago. Although the precise bottleneck mechanism is still hotly debated, the regulation of cellular mtDNA content is a key issue. Here we review current understanding of the factors regulating the amount of mtDNA within cells and discuss the relevance of these findings to our understanding of the inheritance of mtDNA heteroplasmy.     

Mitochondrial quality control: Cell-type-dependent responses to pathological mutant mitochondrial DNA

Pathological mutations in the mitochondrial DNA (mtDNA) produce a diverse range of tissue-specific diseases and the proportion of mutant mitochondrial DNA can increase or decrease with time via segregation, dependent on the cell or tissue type. Previously we found that adenocarcinoma (A549.B2) cells favored wild-type (WT) mtDNA, whereas rhabdomyosarcoma (RD.Myo) cells favored mutant (m3243G) mtDNA. Mitochondrial quality control (mtQC) can purge the cells of dysfunctional mitochondria via mitochondrial dynamics and mitophagy and appears to offer the perfect solution to the human diseases caused by mutant mtDNA. In A549.B2 and RD.Myo cybrids, with various mutant mtDNA levels, mtQC was explored together with macroautophagy/autophagy and bioenergetic profile. The 2 types of tumor-derived cell lines differed in bioenergetic profile and mitophagy, but not in autophagy. A549.B2 cybrids displayed upregulation of mitophagy, increased mtDNA removal, mitochondrial fragmentation and mitochondrial depolarization on incubation with oligomycin, parameters that correlated with mutant load. Conversely, heteroplasmic RD.Myo lines had lower mitophagic markers that negatively correlated with mutant load, combined with a fully polarized and highly fused mitochondrial network. These findings indicate that pathological mutant mitochondrial DNA can modulate mitochondrial dynamics and mitophagy in a cell-type dependent manner and thereby offer an explanation for the persistence and accumulation of deleterious variants.     

Mmm2p, a mitochondrial outer membrane protein required for yeast mitochondrial shape and maintenance of mtDNA nucleoids

The mitochondrial outer membrane protein, Mmm1p, is required for normal mitochondrial shape in yeast. To identify new morphology proteins, we isolated mutations incompatible with the mmm1-1 mutant. One of these mutants, mmm2-1, is defective in a novel outer membrane protein. Lack of Mmm2p causes a defect in mitochondrial shape and loss of mitochondrial DNA (mtDNA) nucleoids. Like the Mmm1 protein (Aiken Hobbs, A.E., M. Srinivasan, J.M. McCaffery, and R.E. Jensen. 2001. J. Cell Biol. 152:401-410.), Mmm2p is located in dot-like particles on the mitochondrial surface, many of which are adjacent to mtDNA nucleoids. While some of the Mmm2p-containing spots colocalize with those containing Mmm1p, at least some of Mmm2p is separate from Mmm1p. Moreover, while Mmm2p and Mmm1p both appear to be part of large complexes, we find that Mmm2p and Mmm1p do not stably interact and appear to be members of two different structures. We speculate that Mmm2p and Mmm1p are components of independent machinery, whose dynamic interactions are required to maintain mitochondrial shape and mtDNA structure.     

Depletion of mitochondrial DNA by down-regulation of deoxyguanosine kinase expression in non-proliferating HeLa cells

Purine deoxyribonucleotides required for mitochondrial DNA replication are either imported from the cytosol or derived from phosphorylation of deoxyadenosine or deoxyguanosine catalyzed by mitochondrial deoxyguanosine kinase (DGUOK). DGUOK deficiency has been linked to mitochondrial DNA depletion syndromes suggesting an important role for this enzyme in dNTP supply. We have generated HeLa cell lines with 20-30% decreased levels of DGUOK mRNA by the expression of small interfering RNAs directed towards the DGUOK mRNA. The cells with decreased expression of the enzyme showed similar levels of mtDNA as control cells when grown exponentially in culture. However, mtDNA levels rapidly decreased in the cells when cell cycle arrest was induced by serum starvation. DNA incorporation of 9-beta-d-arabino-furanosylguanine (araG) was lower in the cells with decreased deoxyguanosine kinase expression, but the total rate of araG phosphorylation was increased in the cells. The increase in araG phosphorylation was shown to be due to increased expression of deoxycytidine kinase. In summary, our findings show that DGUOK is required for mitochondrial DNA replication in resting cells and that small changes in expression of this enzyme may cause mitochondrial DNA depletion. Our data also suggest that alterations in the expression level of DGUOK may induce compensatory changes in the expression of other nucleoside kinases.