Role of mitochondrial permeability transition pores in mitochondrial autophagy

During autophagy, cells rid themselves of damaged and superfluous mitochondria, as well as other organelles. This activation of mitochondrial turnover could be the result of changes in the physiological state of mitochondria. Confocal microscopy and fluorescence techniques indicate that onset of mitochondrial permeability transition is one such change. The mitochondrial permeability transition is a reversible phenomenon whereby the mitochondrial inner membrane becomes freely permeable to solutes of less than 1500 Da. At onset of the mitochondrial permeability transition, mitochondria depolarize, uncouple, and undergo large amplitude swelling due to opening of permeability transition pores, which may form by aggregation of damaged, misfolded membrane proteins. When injurious cellular stresses occur, cells may protect themselves using autophagy to remove damaged mitochondria and mutated mitochondrial DNA. Ca²⁺ overloading, reactive oxygen and nitrogen species, decreased mitochondrial membrane potential, and oxidation of pyridine nucleotides and glutathione all promote mitochondrial damage and onset of the mitochondrial permeability transition. The mitochondrial permeability transition is also associated with necrosis and apoptosis after a variety of stimuli. This review emphasizes the role of the mitochondrial permeability transition as a key event in mitochondrial autophagy.     

A mutation in a mitochondrial ABC transporter results in mitochondrial dysfunction through oxidative damage of mitochondrial DNA

We have isolated a Saccharomyces cerevisiae mutant that shows an increased tendency to form cytoplasmic petites (respiration-deficient ρ⁻ or ρ⁰ mutants) in response to treatment of cells growing on a solid medium with the DNA-damaging agent methyl methanesulfonate or ultraviolet light. The mutation in this strain, atm1-1, was found to cause a single amino acid substitution in ATM1, a nuclear gene that encodes the mitochondrial ATP-binding cassette (ABC) transporter. When the mutant cells were grown in liquid glucose medium, they accumulated free iron within the mitochondria and at the same time gave rise to spontaneous cytoplasmic petite mutants, as seen previously in cells carrying a mutation in a gene homologous to the human gene responsible for Friedreich's ataxia. Analysis of the effects of free iron and malonic acid (an inhibitor of oxidative respiration in mitochondria) on the incidence of petites among the mutant cells indicated that spontaneous induction of petites was a consequence of oxidative stress rather than a direct effect of either a defect in the ATM1 gene or the accumulation of free iron. We observed an increase in the incidence of strand breaks in the mitochondrial DNA of the atm1-1 mutant cells. Furthermore, we found that rates of induction of petites and accumulation of strand breaks in mitochondrial DNA were enhanced in the atm1-1 mutant by the introduction of another mutation, mhr1-1, which results in a deficiency in mitochondrial DNA repair. These observations indicate that spontaneous induction of petites in the atm1-1 mutant is a consequence of oxidative damage to mitochondrial DNA mediated by enhanced accumulation of mitochondrial iron. Received: 26 March 1999 / Accepted: 29 June 1999     

Increased intrinsic mitochondrial function in humans with mitochondrial haplogroup H

It has been suggested that human mitochondrial variants influence maximal oxygen uptake (VO_2max). Whether mitochondrial respiratory capacity per mitochondrion (intrinsic activity) in human skeletal muscle is affected by differences in mitochondrial variants is not known. We recruited 54 males and determined their mitochondrial haplogroup, mitochondrial oxidative phosphorylation capacity (OXPHOS), mitochondrial content (citrate synthase (CS)) and VO_2max. Intrinsic mitochondrial function is calculated as mitochondrial OXPHOS capacity divided by mitochondrial content (CS). Haplogroup H showed a 30% higher intrinsic mitochondrial function compared with the other haplo group U. There was no relationship between haplogroups and VO_2max. In skeletal muscle from men with mitochondrial haplogroup H, an increased intrinsic mitochondrial function is present.     

Sumo1 conjugates mitochondrial substrates and participates in mitochondrial fission

Mitochondrial fission requires the evolutionarily conserved dynamin related protein (DRP1), which is recruited from the cytosol to the mitochondrial outer membrane to coordinate membrane scission. Currently, the mechanism of recruitment and assembly of DRP1 on the mitochondria is unclear. Here, we identify Ubc9 and Sumo1 as specific DRP1-interacting proteins and demonstrate that DRP1 is a Sumo1 substrate. In addition, a surprising number of Sumo1 conjugates were observed in the mitochondrial fractions, suggesting that sumoylation is a common mitochondrial modification. Video microscopy demonstrates that YFP:Sumo1 is often found at the site of mitochondrial fission and remains tightly associated to the tips of fragmented mitochondria. Consistent with this, fluorescence microscopy revealed that a portion of total cytosolic YFP:Sumo1 colocalizes with endogenous mitochondrial DRP1. Finally, transient transfection of Sumo1 dramatically increases the level of mitochondrial fragmentation. Analysis of endogenous DRP1 levels indicates that overexpression of Sumo1 specifically protects DRP1 from degradation, resulting in a more stable, active pool of DRP1, which at least partially accounts for the excess fragmentation. Together, these data are the first to identify a function for Sumo1 on the mitochondria and suggest a novel role for the participation of Sumo1 in mitochondrial fission.     

Changes in mitochondrial shape in wheat root cells exposed to mitochondrial poisons

It is found that mitochondrial poisons not only modify the functional activity of the organelles but also change the shape of some mitochondria. The ring-shaped organelles were found to appear in root cells of 5- to 6-day-old seedlings of spring wheat (Triticum aestivum L., cvs. Moskovskaya 35 and Lyuba). Using a technique of serial sections, we have shown that the circular profiles of mitochondria within the cell correspond in most cases to bowl-shaped organelles. Spatial reorganization of mitochondria did not depend directly on the respiration rate, duration of exposure to inhibitors, and inhibitor specificity. These observations indicate the reversibility of spatial rearrangements and general nonspecific nature of the detected morphological changes.     

Screening of common mitochondrial mutations in Chinese patients with mitochondrial encephalomyopathies

To investigate the spectrum of common mitochondrial mutations in Northern China during the years of 2000-2005, 552 patients of mitochondrial encephalomyopathies clinically diagnosed as MELAS, MERRF or Leigh's syndrome, 14 cases of LHON and 46 cases of aminoglycoside induced deafness along with their family members, accepted routine point mutation tests at nucleotide positions 3243, 8344, 8993, 11778 or 1555 in mitochondrial genome. PCR-RFLP analysis, site-specific PCR and PCR-sequencing methods were used to identify the mutations. Fifty-seven cases with A3243G mutation, 4 cases with A8344G, 2 cases with T8993C and 1 case with T8993G were identified from the 552 encephalomyopathy patients. In addition, one case with G11778A was found from the 14 cases of LHON, and 5 cases with A1555G from the 46 cases of aminoglycoside ototoxicity patients. Additional screening for T8356G and T3271C merely had limited significance for the diagnosis of MERRF and MELAS. Differential diagnosis among mitochondrial encephalomyopathies was often complicated due to many similar clinical manifestations. For A3243G mutation, the proportion of mutant mtDNA was not related to severity of the disease but to the age of onset.     

Automatic sequencing of mitochondrial tRNA genes in patients with mitochondrial encephalomyopathy

We have investigated nine children with infantile onset of mitochondrial myopathy and two adults with myoclonus epilepsy and ragged-red fibers (MERRF) and chronic progressive external ophthalmoplegia (CPEO), respectively. These patients lacked any of the previously known pathogenic tRNA mutations. Southern blot analysis of muscle mtDNA revealed no deletions. The tRNA genes of muscle mtDNA were sequenced. Restriction enxyme analysis of PCR fragments was performed to verify the presence of the mutations identified by automatic sequencing. Several tRNA mutations were found, but they were all homoplasmic. Furthermore, the mutations were either present in controls or did not change nucleotides conserved between species. This strongly suggests that none of the tRNA mutations identified in the 11 patients with mitochondrial encephalomyopathy was pathogenic. It can thus be concluded that mitochondrial tRNA mutations and mtDNA deletions probably are an infrequent cause of mitochondrial disorders in infants. Patients with MERRF and CPEO may lack both pathogenic point mutations of tRNA genes and deletions of mtDNA.     

Marchantia polymorpha mitochondrial orf identifies transcribed sequence in angiosperm mitochondrial genome

Heterologous hybridizations performed using nine Marchantia polymorpha mitochondrial orfs and the sdh4 gene against angiosperm mtDNA suggested that three of them and the sdh4 gene have been conserved in the mitochondrial genome of different angiosperm species. Solanum tuberosum mtDNA fragments, which hybridized to M. polymorpha orf207 and sdh4 gene, were cloned, sequenced, and their expressions evaluated by Northern and RT-PCR. Hybridizing fragments to sdh4 gene and orf207 from potato mtDNA were shown to be transcribed, but only in the case of sdh4 gene was there homology between the protein encoded by the DNA sequence from M. polymorpha and the potato mitochondrial genome. M. polymorpha orf207 showed little similarity to an open reading frame from potato mtDNA, named here orf78. The putative proteins encoded by both orf207 and orf78 were not related, indicating that these orfs do not constitute homologous sequences.     

Exome sequencing identifies mitochondrial alanyl-tRNA synthetase mutations in infantile mitochondrial cardiomyopathy

Infantile cardiomyopathies are devastating fatal disorders of the neonatal period or the first year of life. Mitochondrial dysfunction is a common cause of this group of diseases, but the underlying gene defects have been characterized in only a minority of cases, because tissue specificity of the manifestation hampers functional cloning and the heterogeneity of causative factors hinders collection of informative family materials. We sequenced the exome of a patient who died at the age of 10 months of hypertrophic mitochondrial cardiomyopathy with combined cardiac respiratory chain complex I and IV deficiency. Rigorous data analysis allowed us to identify a homozygous missense mutation in AARS2, which we showed to encode the mitochondrial alanyl-tRNA synthetase (mtAlaRS). Two siblings from another family, both of whom died perinatally of hypertrophic cardiomyopathy, had the same mutation, compound heterozygous with another missense mutation. Protein structure modeling of mtAlaRS suggested that one of the mutations affected a unique tRNA recognition site in the editing domain, leading to incorrect tRNA aminoacylation, whereas the second mutation severely disturbed the catalytic function, preventing tRNA aminoacylation. We show here that mutations in AARS2 cause perinatal or infantile cardiomyopathy with near-total combined mitochondrial respiratory chain deficiency in the heart. Our results indicate that exome sequencing is a powerful tool for identifying mutations in single patients and allows recognition of the genetic background in single-gene disorders of variable clinical manifestation and tissue-specific disease. Furthermore, we show that mitochondrial disorders extend to prenatal life and are an important cause of early infantile cardiac failure.     

Akt mediates mitochondrial protection in cardiomyocytes through phosphorylation of mitochondrial hexokinase-II

Akt activation supports survival of cardiomyocytes against ischemia/reperfusion, which induces cell death through opening of the mitochondrial permeability transition pore (PT-pore). Mitochondrial depolarization induced by treatment of cardiomyocytes with H(2)O(2) is prevented by activation of Akt with leukemia inhibitory factor (LIF). This protective effect is observed even when cardiomyocytes treated with LIF are permeabilized and mitochondrial depolarization is elicited by elevating Ca(2+). Cell fractionation studies demonstrate that LIF treatment increases both total and phosphorylated Akt in the mitochondrial fraction. Furthermore, the association of Akt with HK-II is increased by LIF. HK-II contains consensus sequences for phosphorylation by Akt and LIF treatment induces PI3K- and Akt-dependent HK-II phosphorylation. Addition of recombinant kinase-active Akt to isolated adult mouse heart mitochondria stimulates phosphorylation of HK-II and concomitantly inhibits the ability of Ca(2+) to induce cytochrome c release. This protection is prevented when HK-II is dissociated from mitochondria by incubation with glucose 6-phosphate or HK-II-dissociating peptide. Finally LIF increases HK-II association with mitochondria and dissociation of HK-II from mitochondria attenuates the protective effect of LIF on H(2)O(2)-induced mitochondrial depolarization in cardiomyocytes. We conclude that Akt has a direct effect at the level of the mitochondrion, which is mediated via phosphorylation of HK-II and results in protection of mitochondria against oxidant or Ca(2+)-stimulated PT-pore opening.     

The mitochondrial genome and human mitochondrial diseases

To date, more than 100 point mutations and several hundreds of structural rearrangements of mitochondrial DNA (mtDNA) are known too be connected with characteristic neuromuscular and other mitochondrial syndromes varying form those causing death at the neonatal stage to diseases with late ages of onset. The immediate cause of mitochondrial disorders is a defective oxidative phosphorylation. Wide phenotypic variation and the heteroplasmy phenomenon, which some authors include in mutation load, are characteristic of human mitochondrial diseases. As the numbers of cases identified and pedigrees described increase, data on the genotype--phenotype interaction and the structure and frequency of pathogenic and conditionally pathogenic mtDNA mutations in human populations are rapidly accumulated. The data on the genetics and epidemiology of mitochondrial diseases are not only important for differential diagnosis and genetic counseling. Since both neutral and mildly pathogenic mutations of mtDNA are progressively accumulated in maternal phyletic lines, molecular analysis of these mutations permits not only reconstruction of the genealogical tree of modern humans, but also estimation of the role that these mutations play in natural selection.     

Cardiolipin and mitochondrial phosphatidylethanolamine have overlapping functions in mitochondrial fusion in Saccharomyces cerevisiae

The two non-bilayer forming mitochondrial phospholipids cardiolipin (CL) and phosphatidylethanolamine (PE) play crucial roles in maintaining mitochondrial morphology. We have shown previously that CL and PE have overlapping functions, and the loss of both is synthetically lethal. Because the lack of CL does not lead to defects in the mitochondrial network in Saccharomyces cerevisiae, we hypothesized that PE may compensate for CL in the maintenance of mitochondrial tubular morphology and fusion. To test this hypothesis, we constructed a conditional mutant crd1Δpsd1Δ containing null alleles of CRD1 (CL synthase) and PSD1 (mitochondrial phosphatidylserine decarboxylase), in which the wild type CRD1 gene is expressed on a plasmid under control of the TET(OFF) promoter. In the presence of tetracycline, the mutant exhibited highly fragmented mitochondria, loss of mitochondrial DNA, and reduced membrane potential, characteristic of fusion mutants. Deletion of DNM1, required for mitochondrial fission, restored the tubular mitochondrial morphology. Loss of CL and mitochondrial PE led to reduced levels of small and large isoforms of the fusion protein Mgm1p, possibly accounting for the fusion defect. Taken together, these data demonstrate for the first time in vivo that CL and mitochondrial PE are required to maintain tubular mitochondrial morphology and have overlapping functions in mitochondrial fusion.     

Preventing mitochondrial fission impairs mitochondrial function and leads to loss of mitochondrial DNA

Mitochondria form a highly dynamic tubular network, the morphology of which is regulated by frequent fission and fusion events. However, the role of mitochondrial fission in homeostasis of the organelle is still unknown. Here we report that preventing mitochondrial fission, by down-regulating expression of Drp1 in mammalian cells leads to a loss of mitochondrial DNA and a decrease of mitochondrial respiration coupled to an increase in the levels of cellular reactive oxygen species (ROS). At the cellular level, mitochondrial dysfunction resulting from the lack of fission leads to a drop in the levels of cellular ATP, an inhibition of cell proliferation and an increase in autophagy. In conclusion, we propose that mitochondrial fission is required for preservation of mitochondrial function and thereby for maintenance of cellular homeostasis.