A growing body of evidence suggests that impaired mitochondrial energy production and increased oxidative radical damage to the mitochondria could be causally involved in motor neuron death in amyotrophic lateral sclerosis (ALS) and in familial ALS associated with mutations of Cu,Zn superoxide dismutase (SOD1). For example, morphologically abnormal mitochondria and impaired mitochondrial histoenzymatic respiratory chain activities have been described in motor neurons of patients with sporadic ALS. To investigate further the role of mitochondrial alterations in the pathogenesis of ALS, we studied mitochondria from transgenic mice expressing wild type and G93A mutated hSOD1. We found that a significant proportion of enzymatically active SOD1 was localized in the intermembrane space of mitochondria. Mitochondrial respiration, electron transfer chain, and ATP synthesis were severely defective in G93A mice at the time of onset of the disease. We also found evidence of oxidative damage to mitochondrial proteins and lipids. On the other hand, presymptomatic G93A transgenic mice and mice expressing the wild type form of hSOD1 did not show significant mitochondrial abnormalities. Our findings suggest that G93A-mutated hSOD1 in mitochondria may cause mitochondrial defects, which contribute to precipitating the neurodegenerative process in motor neurons.Amyotrophic lateral sclerosis (ALS) 1 is a devastating neurodegenerative disease affecting spinal cord and cortical motor neurons. The onset of the disease is generally in the 4th and 5th decade, and it progresses over an average of 5 years leading to progressive paralysis and premature death (1). Although the majority of the cases are sporadic and due to unknown causes, about 5-10% are familial (FALS), of which ϳ25% are associated with mutations in the Cu,Zn superoxide dismutase gene (SOD1) (2-6). The symptoms and pathology of FALS patients resemble those of patients with the sporadic form of ALS, suggesting that the mechanisms of neurodegeneration share common pathways. Since the initial report (7) of mutant SOD1, more than 90 different mutations of the SOD1 gene have been found in FALS patients. Because these mutations do not always affect the dismutase activity (8, 9), a toxic gain of function of the mutated protein has been postulated, possibly causing enhanced reactive oxygen species generation (10).In the motor neurons of transgenic mice expressing the G93A-mutated SOD1 (11), among other pathological features is the presence of membrane-bound vacuoles deriving from mitochondrial degeneration. In these mice, the onset of the paralysis is immediately preceded by an increase in degenerating mitochondria (12), suggesting that mitochondrial alterations might represent a triggering factor in precipitating the degeneration of motor neurons. A decrease of mitochondrial membrane potential and disturbed mitochondrial calcium homeostasis have also been reported (13) in cultured primary motor neurons from G93A mice. Reduced respiratory chain activities were found in the spinal c...

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Mitochondrial DNA (mtDNA) mutations cause heterogeneous disorders in humans. MtDNA exists in multiple copies per cell, and mutations need to accumulate beyond a critical threshold to cause disease, because coexisting wild-type mtDNA can complement the genetic defect. A better understanding of the molecular determinants of functional complementation among mtDNA molecules could help us shedding some light on the mechanisms modulating the phenotypic expression of mtDNA mutations in mitochondrial diseases. We studied mtDNA complementation in human cells by fusing two cell lines, one containing a homoplasmic mutation in a subunit of respiratory chain complex IV, COX I, and the other a distinct homoplasmic mutation in a subunit of complex III, cytochrome b. Upon cell fusion, respiration is recovered in hybrids cells, indicating that mitochondria fuse and exchange genetic and protein materials. Mitochondrial functional complementation occurs frequently, but with variable efficiency. We have investigated by native gel electrophoresis the molecular organization of the mitochondrial respiratory chain in complementing hybrid cells. We show that the recovery of mitochondrial respiration correlates with the presence of supramolecular structures (supercomplexes) containing complexes I, III and IV. We suggest that critical amounts of complexes III or IV are required in order for supercomplexes to form and provide mitochondrial functional complementation. From these findings, supercomplex assembly emerges as a necessary step for respiration, and its defect sets the threshold for respiratory impairment in mtDNA mutant cells.

The MTG1 gene of Saccharomyces cerevisiae, corresponding to ORF YMR097c on chromosome XIII, codes for a mitochondrial protein essential for respiratory competence. A human homologue of Mtg1p capable of partially rescuing the respiratory deficiency of a yeast mtg1 mutant has also been localized in mitochondria. Mtg1p is a member of a family of GTPases with largely unknown functions. The respiratory deficiency of mtg1 mutants stems from a defect in mitochondrial protein synthesis. Mutations in the 21S rRNA locus are able to suppress the translation defect of mtg1 null mutants. This points to the 21S rRNA or the large ribosomal subunit as the most likely target of Mtg1p action. The presence of mature size 15S and 21S mitochondrial rRNAs in mtg1 mutants excludes Mtg1p from being involved in transcription or processing of these RNAs. More likely, Mtg1p functions in assembly of the large ribosomal subunit. This is consistent with the peripheral localization of Mtg1p on the matrix side of the inner membrane and the results of in vivo mitochondrial translation assays in a temperature-sensitive mtg1 mutant.

Inter-molecular heterologous mitochondrial DNA (mtDNA) recombination is known to occur in yeast and plants. Nevertheless, its occurrence in human cells is still controversial. To address this issue we have fused two human cytoplasmic hybrid cell lines, each containing a distinct pathogenic mtDNA mutation and specific sets of genetic markers. In this hybrid model, we found direct evidence of recombination between these two mtDNA haplotypes. Recombinant mtDNA molecules in the hybrid cells were identified using three independent experimental approaches. First, recombinant molecules containing genetic markers from both parental alleles were demonstrated with restriction fragment length polymorphism of polymerase chain reaction products, by measuring the relative frequencies of each marker. Second, fragments of recombinant mtDNA were cloned and sequenced to identify the regions involved in the recombination events. Finally, recombinant molecules were demonstrated directly by Southern blot using appropriate combinations of polymorphic restriction sites and probes. This combined approach confirmed the existence of heterogeneous species of recombinant mtDNA molecules in the hybrid cells. These findings have important implications for mtDNA-related diseases, the interpretation of human evolution and population genetics and forensic analyses based on mtDNA genotyping.

The mechanisms that regulate oxidative phosphorylation in mammalian cells are largely unknown. To address this issue, cybrids were generated by fusing osteosarcoma cells devoid of mitochondrial DNA (mtDNA) with platelets from a patient with a stop-codon mutation in cytochrome c oxidase subunit I (COX I). The molecular and biochemical characteristics of cybrids harboring varying levels of mutated mitochondrial DNA were studied. We found a direct correlation between the levels of mutated COX I DNA and mutated COX I mRNA, whereas the levels of COX I total mRNA were unchanged. COX I polypeptide synthesis and steady-state levels were inversely proportional to mutation levels. Cytochrome c oxidase subunit II was reduced proportionally to COX I, indicating impairment in complex assembly. COX enzymatic activity was inversely proportional to the levels of mutated mtDNA. However, both cell respiration and ATP synthesis were preserved in cells with lower proportions of mutated genomes, with a threshold at ϳ40%, and decreased linearly with increasing mutated mtDNA. These results indicate that COX levels in mutated cells were not regulated at the transcriptional, translational, and post-translational levels. Because of a small excess of COX capacity, the levels of expression of COX subunits exerted a relatively tight control on oxidative phosphorylation.

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Mutations in mitochondrial DNA (mtDNA) cause impairment of ATP synthesis. It was hypothesized that high-energy compounds, such as ATP, are compartmentalized within cells and that different cell functions are sustained by different pools of ATP, some deriving from mitochondrial oxidative phosphorylation (OXPHOS) and others from glycolysis. Therefore, an OXPHOS dysfunction may affect different cell compartments to different extents. To address this issue, we have used recombinant forms of the ATP reporter luciferase localized in different cell compartmentsthe cytosol, the subplasma membrane region, the mitochondrial matrix, and the nucleus-of cells containing either wild-type or mutant mtDNA. We found that with glycolytic substrates, both wild-type and mutant cells were able to maintain adequate ATP supplies in all compartments. Conversely, with the OXPHOS substrate pyruvate ATP levels collapsed in all cell compartments of mutant cells. In wild-type cells normal levels of ATP were maintained with pyruvate in the cytosol and in the subplasma membrane region, but, surprisingly, they were reduced in the mitochondria and, to a greater extent, in the nucleus. The severe decrease in nuclear ATP content under "OXPHOS-only" conditions implies that depletion of nuclear ATP plays an important, and hitherto unappreciated, role in patients with mitochondrial dysfunction. INTRODUCTIONThe concept that high-energy compounds are compartmentalized in cells was proposed more than 20 years ago (Erickson-Viitanen et al., 1982a, 1982bSaks et al., 1994). For example, it was suggested that in normal smooth muscle cells the contractile functions are supported by mitochondrial ATP derived from the respiratory chain and oxidative phosphorylation (OXPHOS), whereas the plasma membrane proton pumps are supported by ATP from anaerobic glycolysis (Ishida et al., 1994). In addition, recent studies showed that the import of histones into the nuclei of neonatal cardiomyocytes is strictly dependent on a concerted interaction between mitochondrial ATP synthesis and the trafficking of high-energy phosphoryls (Dzeja et al., 2002).In a technical advance, targeted luciferase has been used as an ATP sensor to investigate the kinetics of the variation of ATP concentration beneath the plasma membrane, in the mitochondria, and in the cytosol of pancreatic ␤-cells in response to glucose stimulation (Kennedy et al., 1999). These experiments demonstrated that in response to the administration of glucose and potassium, ATP levels increased in the plasma membrane of ␤-cells in concert with that in mitochondria, whereas cytosolic ATP showed only a transient increase. On the other hand, studies using the ATP-dependent potassium channel as an ATP sensor showed that in Xenopus oocytes and in cultured mammalian cells there was no gradient between bulk cytosolic ATP and subplasma membrane ATP, suggesting that ATP diffuses freely between these two cell compartments (Gribble et al., 2000).Despite a growing body of evidence that high-energy molecules such as ATP and phos...