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...

The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS), considered as the pathogenic agent of many diseases and of aging. We have investigated the role of Complex I in superoxide radical production and found by combined use of specific inhibitors of Complex I that the one-electron donor in the Complex to oxygen is a redox center located prior to the sites where three different types of coenzyme Q (CoQ) competitors bind, to be identified with an Fe-S cluster, most probably N2, or possibly an ubisemiquinone intermediate insensitive to all the above inhibitors. Short-chain coenzyme Q analogues enhance superoxide formation, presumably by mediating electron transfer from N2 to oxygen. The clinically used CoQ analogue idebenone is particularly effective, raising doubts about its safety as a drug. The mitochondrial theory of aging considers somatic mutations of mitochondrial DNA induced by ROS as the primary cause of energy decline; in rat liver mitochondria, Complex I appears to be most affected by aging and to become strongly rate limiting for electron transfer. Mitochondrial energetics is also deranged in human platelets upon aging, as demonstrated by the decreased Pasteur effect (enhancement of lactate production by respiratory inhibitors). Cells counteract oxidative stress by antioxidants: CoQ is the only lipophilic antioxidant to be biosynthesized. Exogenous CoQ, however, protects cells from oxidative stress by conversion into its reduced antioxidant form by cellular reductases. The plasma membrane oxidoreductase and DT-diaphorase are two such systems: likewise, they are overexpressed under oxidative stress conditions.

As noted by Warburg, many cancer cells depend on the consumption of glucose. We performed a genetic screen to identify factors responsible for glucose addiction and recovered the two subunits of the xCT antiporter (system xc−), which plays an antioxidant role by exporting glutamate for cystine. Disruption of the xCT antiporter greatly improves cell viability after glucose withdrawal, because conservation of glutamate enables cells to maintain mitochondrial respiration. In some breast cancer cells, xCT antiporter expression is upregulated through the antioxidant transcription factor Nrf2 and contributes to their requirement for glucose as a carbon source. In cells carrying patient-derived mitochondrial DNA mutations, the xCT antiporter is upregulated and its inhibition improves mitochondrial function and cell viability. Therefore, although upregulation of the xCT antiporter promotes antioxidant defence, it antagonizes glutamine metabolism and restricts nutrient flexibility. In cells with mitochondrial dysfunction, the potential utility of xCT antiporter inhibition should be further tested.

Cardiolipin is a mitochondrion-specific phospholipid that stabilizes the assembly of respiratory chain complexes, favoring full-yield operation. It also mediates key steps in apoptosis. In Barth syndrome, an X chromosome-linked cardiomyopathy caused by tafazzin mutations, cardiolipins display acyl chain modifications and are present at abnormally low concentrations, whereas monolysocardiolipin accumulates. Using immortalized lymphoblasts from Barth syndrome patients, we showed that the production of abnormal cardiolipin led to mitochondrial alterations. Indeed, the lack of normal cardiolipin led to changes in electron transport chain stability, resulting in cellular defects. We found a destabilization of the supercomplex (respirasome) I+III2+IVn but also decreased amounts of individual complexes I and IV and supercomplexes I+III and III+IV. No changes were observed in the amounts of individual complex III and complex II. We also found decreased levels of complex V. This complex is not part of the supercomplex suggesting that cardiolipin is required not only for the association/stabilization of the complexes into supercomplexes but also for the modulation of the amount of individual respiratory chain complexes. However, these alterations were compensated by an increase in mitochondrial mass, as demonstrated by electron microscopy and measurements of citrate synthase activity. We suggest that this compensatory increase in mitochondrial content prevents a decrease in mitochondrial respiration and ATP synthesis in the cells. We also show, by extensive flow cytometry analysis, that the type II apoptosis pathway was blocked at the mitochondrial level and that the mitochondria of patients with Barth syndrome cannot bind active caspase-8. Signal transduction is thus blocked before any mitochondrial event can occur. Remarkably, basal levels of superoxide anion production were slightly higher in patients' cells than in control cells as previously evidenced via an increased protein carbonylation in the taz1Δ mutant in the yeast. This may be deleterious to cells in the long term. The consequences of mitochondrial dysfunction and alterations to apoptosis signal transduction are considered in light of the potential for the development of future treatments.

Using molecular, biochemical, and untargeted stable isotope tracing approaches, we identify a previously unappreciated glutamine-derived α-ketoglutarate (αKG) energy-generating anaplerotic flux to be critical in mitochondrial DNA (mtDNA) mutant cells that harbor human disease-associated oxidative phosphorylation defects. Stimulating this flux with αKG supplementation enables the survival of diverse mtDNA mutant cells under otherwise lethal obligatory oxidative conditions. Strikingly, we demonstrate that when residual mitochondrial respiration in mtDNA mutant cells exceeds 45% of control levels, αKG oxidative flux prevails over reductive carboxylation. Furthermore, in a mouse model of mitochondrial myopathy, we show that increased oxidative αKG flux in muscle arises from enhanced alanine synthesis and release into blood, concomitant with accelerated amino acid catabolism from protein breakdown. Importantly, in this mouse model of mitochondriopathy, muscle amino acid imbalance is normalized by αKG supplementation. Taken together, our findings provide a rationale for αKG supplementation as a therapeutic strategy for mitochondrial myopathies.

Mitochondria are strongly involved in the production of reactive oxygen species, considered as the pathogenic agent of many diseases and of aging. The mitochondrial theory of aging considers somatic mutations of mitochondrial DNA induced by oxygen radicals as the primary cause of energy decline; experimentally, complex I appears to be mostly affected and to become strongly rate limiting for electron transfer. Mitochondrial bioenergetics is also deranged in human platelets upon aging, as shown by the decreased Pasteur effect (enhancement of lactate production by respiratory chain inhibition). Cells counteract oxidative stress by antioxidants; among lipophilic antioxidants, coenzyme Q is the only one of endogenous biosynthesis. Exogenous coenzyme Q, however, protects cells from oxidative stress by conversion into its reduced antioxidant form by cellular reductases.

In the mammalian mitochondrial electron transfer system, the majority of electrons enter at complex I, go through complexes III and IV, and are finally delivered to oxygen. Previously we generated several mouse cell lines with suppressed expression of the nuclearly encoded subunit 4 of complex IV. This led to a loss of assembly of complex IV and its defective function. Interestingly, we found that the level of assembled complex I and its activity were also significantly reduced, whereas levels and activity of complex III were normal or up-regulated. The structural and functional dependence of complex I on complex IV was verified using a human cell line carrying a nonsense mutation in the mitochondrially encoded complex IV subunit 1 gene. Our work documents that, although there is no direct electron transfer between them, an assembled complex IV helps to maintain complex I in mammalian cells.

Huntington's disease (HD) is a neurodegenerative disorder caused by an abnormal expansion of a CAG repeat encoding a polyglutamine tract in the huntingtin (Htt) protein. The mutation leads to neuronal death through mechanisms which are still unknown. One hypothesis is that mitochondrial defects may play a key role. In support of this, the activity of mitochondrial complex II (C-II) is preferentially reduced in the striatum of HD patients. Here, we studied C-II expression in different genetic models of HD expressing N-terminal fragments of mutant Htt (mHtt). Western blot analysis showed that the expression of the 30 kDa Iron–Sulfur (Ip) subunit of C-II was significantly reduced in the striatum of the R6/1 transgenic mice, while the levels of the FAD containing catalytic 70 kDa subunit (Fp) were not significantly changed. Blue native gel analysis showed that the assembly of C-II in mitochondria was altered early in N171-82Q transgenic mice. Early loco-regional reduction in C-II activity and Ip protein expression was also demonstrated in a rat model of HD using intrastriatal injection of lentiviral vectors encoding mHtt. Infection of the rat striatum with a lentiviral vector coding the C-II Ip or Fp subunits induced a significant overexpression of these proteins that led to significant neuroprotection of striatal neurons against mHtt neurotoxicity. These results obtained in vivo support the hypothesis that structural and functional alterations of C-II induced by mHtt may play a critical role in the degeneration of striatal neurons in HD and that mitochondrial-targeted therapies may be useful in its treatment.