Acute glutathione depletion restricts mitochondrial ATP export in cerebellar granule neurons

Decreases in GSH pools detected during ischemia sensitize neurons to excitotoxic damage. Thermodynamic analysis predicts that partial GSH depletion will cause an oxidative shift in the thiol redox potential. To investigate the acute bioenergetic consequences, neurons were exposed to monochlorobimane (mBCl), which depletes GSH by forming a fluorescent conjugate. Neurons transfected with redox-sensitive green fluorescent protein showed a positive shift in thiol redox potential synchronous with the formation of the conjugate. Mitochondria within neurons treated with mBCl for 1 h failed to hyperpolarize upon addition of oligomycin to inhibit their ATP synthesis. A decreased ATP turnover was confirmed by monitoring neuronal oxygen consumption in parallel with mitochondrial membrane potential (Deltapsi(m)) and GSH-mBCl formation. mBCl progressively decreased cell respiration, with no effect on mitochondrial proton leak or maximal respiratory capacity, suggesting adequate glycolysis and a functional electron transport chain. This approach to "state 4" could be mimicked by the adenine nucleotide translocator inhibitor bongkrekic acid, which did not further decrease respiration when administered after mBCl. The cellular ATP/ADP ratio was decreased by mBCl, and consistent with mitochondrial ATP export failure, respiration could not respond to an increased cytoplasmic ATP demand by plasma membrane Na(+) cycling; instead, mitochondria depolarized. More prolonged mBCl exposure induced mitochondrial failure, with Deltapsi(m) collapse followed by cytoplasmic Ca(2+) deregulation. The initial bioenergetic consequence of neuronal GSH depletion in this model is thus an inhibition of ATP export, which precedes other forms of mitochondrial dysfunction.     

Ubiquitination and proteasomal degradation of ATG12 regulates its proapoptotic activity

During macroautophagy, conjugation of ATG12 to ATG5 is essential for LC3 lipidation and autophagosome formation. Additionally, ATG12 has ATG5-independent functions in diverse processes including mitochondrial fusion and mitochondrial-dependent apoptosis. In this study, we investigated the regulation of free ATG12. In stark contrast to the stable ATG12–ATG5 conjugate, we find that free ATG12 is highly unstable and rapidly degraded in a proteasome-dependent manner. Surprisingly, ATG12, itself a ubiquitin-like protein, is directly ubiquitinated and this promotes its proteasomal degradation. As a functional consequence of its turnover, accumulation of free ATG12 contributes to proteasome inhibitor-mediated apoptosis, a finding that may be clinically important given the use of proteasome inhibitors as anticancer agents. Collectively, our results reveal a novel interconnection between autophagy, proteasome activity, and cell death mediated by the ubiquitin-like properties of ATG12.     

Metabolic and Antioxidant System Alterations in an Astrocytoma Cell Line Challenged with Mitochondrial DNA Deletion

Oxidative stress can induce mitochondrial dysfunction, mitochondrial DNA (mtDNA) depletion, and neurodegeneration, although the underlying mechanisms are poorly understood. The major mitochondrial antioxidant system that protects cells consists of manganese superoxide dismutase (MnSOD), glutathione peroxidase (GPx) and glutathione (GSH). To investigate the putative adaptive changes in antioxidant enzyme protein expression and targeting to mitochondria as mtDNA depletion occurs, we progressively depleted U87 astrocytoma cells of mtDNA by chronic treatment with ethidium bromide (EB, 50 ng/ml). Cellular MnSOD protein expression was markedly increased in a time-related manner while that of GPx showed time-related decreases. The mtDNA depletion also altered targeting or subcellular distribution of GPx, suggesting the importance of intact mtDNA in mitochondrial genome-nuclear genome signaling/communication. Cellular NADP⁺-ICDH activity also showed marked, time-related increases while their GSH content decreased. Thus, our findings suggest that interventions to elevate MnSOD, GPx, NADP⁺-ICDH, and GSH levels may protect brain cells from oxidative stress.     

Ubiquitination and proteasomal degradation of ATG12 regulates its proapoptotic activity

During macroautophagy, conjugation of ATG12 to ATG5 is essential for LC3 lipidation and autophagosome formation. Additionally, ATG12 has ATG5-independent functions in diverse processes including mitochondrial fusion and mitochondrial-dependent apoptosis. In this study, we investigated the regulation of free ATG12. In stark contrast to the stable ATG12–ATG5 conjugate, we find that free ATG12 is highly unstable and rapidly degraded in a proteasome-dependent manner. Surprisingly, ATG12, itself a ubiquitin-like protein, is directly ubiquitinated and this promotes its proteasomal degradation. As a functional consequence of its turnover, accumulation of free ATG12 contributes to proteasome inhibitor-mediated apoptosis, a finding that may be clinically important given the use of proteasome inhibitors as anticancer agents. Collectively, our results reveal a novel interconnection between autophagy, proteasome activity, and cell death mediated by the ubiquitin-like properties of ATG12.     

mtDNA replicative potential remains constant during ageing: polymerase gamma activity does not correlate with age related cytochrome oxidase activity decline in platelets.

Progressive age-related oxidative phosphorylation (OxPhos) decline is well known in human tissues. Depletion of mitochondrial DNA (mtDNA) causes OxPhos defects in patients with myopathic syndromes and deficient mtDNA replication has been observed in cells cultured from patients with mitochondrial disease. Patients undergoing treatment for AIDS develop OxPhos defects via mtDNA depletion resulting from inhibition of mtDNA polymerase gamma (Polgamma) by 2'-deoxy 3'-azido thymidine. These findings by others give rise to a possible link between mtDNA replication and bioenergetic decline in disease and during ageing. We have designed an in vitro assay for Polgamma function in small tissue samples to explore this possible link. Platelet homogenate Polgamma showed an activity with a K m of 150 microM (dTTP), a V max of 11.8 pmol/min/mg, inhibited (41% inhibition; 50 microM) by ethidium bromide. Determination of several storage characteristics showed that platelets were a convenient source of Polgamma for assay. Polgamma activity in 45 subjects did not coincide with significant age-related decline (P<0.002; P) observed in cytochrome oxidase (CytOx) activity or with citrate synthase activity. Of the activities studied, the only significant age-wise variation was a 24% CytOx deficiency in elderly (>50; n = 19) compared to young (<51; n = 24) individuals (P<0.01; t). These results suggest a maintenance of total cellular mtDNA Polgamma processive levels during ageing, largely independent of total cellular bioenergetic status or mitochondrial number/density. The processive component of Polgamma is therefore unlikely to make a major contribution to age-related bioenergetic activity decline. This does not, however, preclude the possibility that transient periods of inhibition at crucial points of the cell cycle or development may augment existing intracellular deficiencies. The assay described here greatly facilitates study of Polgamma activity in patients with conditions involving mtDNA depletion or rearrangement.     

Distinct nuclear gene expression profiles in cells with mtDNA depletion and homoplasmic A3243G mutation

The pathobiochemical pathways determining the wide variability in phenotypic expression of mitochondrial DNA (mtDNA) mutations are not well understood. Most pathogenic mtDNA mutations induce a general defect in mitochondrial respiration and thereby ATP synthesis. Yet phenotypic expression of the different mtDNA mutations shows large variations that are difficult to reconcile with ATP depletion as sole pathogenic factor, implying that additional mechanisms contribute to the phenotype. Here, we use DNA microarrays to identify changes in nuclear gene expression resulting from the presence of the A3243G diabetogenic mutation and from a depletion of mtDNA (rho0 cells). We find that cells respond mildly to these mitochondrial states with both general and specific changes in nuclear gene expression. This observation indicates that cells can sense the status of mtDNA. A number of genes show divergence in expression in rho0 cells compared to cells with the A3243G mutation, such as genes involved in oxidative phosphorylation. As a common response in A3243G and rho0 cells, mRNA levels for extracellular matrix genes are up-regulated, while the mRNA levels of genes involved in ubiquitin-mediated protein degradation and in ribosomal protein synthesis is down-regulated. This reduced expression is reflected at the level of cytosolic protein synthesis in both A3243G and rho0 cells. Our finding that mitochondrial dysfunction caused by different mutations affects nuclear gene expression in partially distinct ways suggests that multiple pathways link mitochondrial function to nuclear gene expression and contribute to the development of the different phenotypes in mitochondrial disease.     

Depletion of mitochondrial DNA in the skeletal muscle of two cirrhotic patients with severe asthenia

Qualitative and quantitative alterations of mitochondrial DNA (mtDNA) in the skeletal muscle from two patients with cirrhosis and severe asthenia have been studied. The 4977 bp (mtDNA(4977)) and the 7436 bp (mtDNA(7436)) mtDNA deletions, as well as other mtDNA deletions, revealed by long extension PCR (LX-PCR), were found in the two patients, whereas the 10,422 bp (mtDNA(10,422)) mtDNA deletion was absent. Altogether, the qualitative alterations of mtDNA in cirrhotic patients with severe asthenia were comparable to those of age-matched healthy individuals. The mtDNA content, on the contrary, was substantially decreased in both patients with respect to control. Such mtDNA depletion might be explained by an increased, disease-related, oxidative damage to mtDNA, which probably affects the replication of the mitochondrial genome as already suggested in other oxidative stress-associated diseases.     

Mitochondrial DNA mutations in diseases of energy metabolism

A variety of degenerative diseases involving deficiencies in mitochondrial bioenergetics have been associated with mitochondrial DNA (mtDNA) mutations. Maternally inherited mtDNA nucleotide substitutions range from neutral polymorphisms to lethal mutations. Neutral polymorphisms are ancient, having accumulated along mtDNA lineages, and thus correlate with ethnic and geographic origin. Mildly deleterious base substitutions have also occurred along mtDNA lineages and have been associated with familial deafness and some cases of Alzheimer's Disease and Parkinson's Disease. Moderately deleterious nucleotide substitutions are more recent and cause maternally-inherited diseases such as Leber's Hereditary Optic Neuropathy (LHON) and Myoclonic Epilepsy and Ragged-Red Fiber Disease (MERRF). Severe nucleotide substitutions are generally new mutations that cause pediatric diseases such as Leigh's Syndrome and dystonia. MtDNA rearrangements also cause a variety of phenotypes. The milder rearrangements generally involve duplications and can cause maternally-inherited adult-onset diabetes and deafness. More severe rearrangements frequently involving detetions have been associated with adult-onset Chronic Progressive External Ophthalmoplegia (CPEO) and Kearns-Sayre Syndrome (KSS) or the lethal childhood disorder, Pearson's Marrow/Pancreas Syndrome. Defects in nuclear-cytoplasmic interaction have also been observed, and include an autosomal dominant mutation causing multiple muscle mtDNA deletions and a genetically complex disease resulting in the tissue depletion of mtDNAs. MtDNA nucleotide substitution and rearrangement mutations also accumulate with age in quiescent tissues. These somatic mutations appear to degrade cellular bioenergetic capacity, exacerbate inherited mitochondrial defects and contribute to tissue senescence. Thus, bioenergetic defects resulting from mtDNA mutations may be a common cause of human degenerative disease.     

TOM40 Mediates Mitochondrial Dysfunction Induced by α-Synuclein Accumulation in Parkinson’s Disease

Alpha-synuclein (α-Syn) accumulation/aggregation and mitochondrial dysfunction play prominent roles in the pathology of Parkinson’s disease. We have previously shown that postmortem human dopaminergic neurons from PD brains accumulate high levels of mitochondrial DNA (mtDNA) deletions. We now addressed the question, whether alterations in a component of the mitochondrial import machinery -TOM40- might contribute to the mitochondrial dysfunction and damage in PD. For this purpose, we studied levels of TOM40, mtDNA deletions, oxidative damage, energy production, and complexes of the respiratory chain in brain homogenates as well as in single neurons, using laser-capture-microdissection in transgenic mice overexpressing human wildtype α-Syn. Additionally, we used lentivirus-mediated stereotactic delivery of a component of this import machinery into mouse brain as a novel therapeutic strategy. We report here that TOM40 is significantly reduced in the brain of PD patients and in α-Syn transgenic mice. TOM40 deficits were associated with increased mtDNA deletions and oxidative DNA damage, and with decreased energy production and altered levels of complex I proteins in α-Syn transgenic mice. Lentiviral-mediated overexpression of Tom40 in α-Syn-transgenic mice brains ameliorated energy deficits as well as oxidative burden. Our results suggest that alterations in the mitochondrial protein transport machinery might contribute to mitochondrial impairment in α-Synucleinopathies.     

Mitochondrial DNA deletions and differential mitochondrial DNA content in Rhesus monkeys: Implications for aging

The purpose of this study was to determine the relationship between mitochondrial DNA (mtDNA) deletions, mtDNA content and aging in rhesus monkeys. Using 2 sets of specific primers, we amplified an 8 kb mtDNA fragment covering a common 5.7 kb deletion and the entire 16.5 kb mitochondrial genome in the brain and buffy-coats of young and aged monkeys. We studied a total of 66 DNA samples: 39 were prepared from a buffy-coat and 27 were prepared from occipital cortex tissues. The mtDNA data were assessed using a permutation test to identify differences in mtDNA, in the different monkey groups. Using real-time RT-PCR strategy, we also assessed both mtDNA and nuclear DNA levels for young, aged and male and female monkeys. We found a 5.7 kb mtDNA deletion in 81.8% (54 of 66) of the total tested samples. In the young group of buffy-coat DNA, we found 5.7 kb deletions in 7 of 17 (41%), and in the aged group, we found 5.7 kb deletions in 12 of 22 (54%), suggesting that the prevalence of mtDNA deletions is related to age. We found decreased mRNA levels of mtDNA in aged monkeys relative to young monkeys. The increases in mtDNA deletions and mtDNA levels in aged rhesus monkeys suggest that damaged DNA accumulates as rhesus monkeys age and these altered mtDNA changes may have physiological relevance to compensate decreased mitochondrial function.     

Mitochondria: Biogenesis and mitophagy balance in segregation and clonal expansion of mitochondrial DNA mutations

Mitochondria are cytoplasmic organelles containing their own multi-copy genome. They are organized in a highly dynamic network, resulting from balance between fission and fusion, which maintains homeostasis of mitochondrial mass through mitochondrial biogenesis and mitophagy. Mitochondrial DNA (mtDNA) mutates much faster than nuclear DNA. In particular, mtDNA point mutations and deletions may occur somatically and accumulate with aging, coexisting with the wild type, a condition known as heteroplasmy. Under specific circumstances, clonal expansion of mutant mtDNA may occur within single cells, causing a wide range of severe human diseases when mutant overcomes wild type. Furthermore, mtDNA deletions accumulate and clonally expand as a consequence of deleterious mutations in nuclear genes involved in mtDNA replication and maintenance, as well as in mitochondrial fusion genes (mitofusin-2 and OPA1), possibly implicating mtDNA nucleoids segregation. We here discuss how the intricacies of mitochondrial homeostasis impinge on the intracellular propagation of mutant mtDNA.This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.     

Mitochondrial DNA deletions and differential mitochondrial DNA content in Rhesus monkeys: implications for aging

The purpose of this study was to determine the relationship between mitochondrial DNA (mtDNA) deletions, mtDNA content and aging in rhesus monkeys. Using 2 sets of specific primers, we amplified an 8 kb mtDNA fragment covering a common 5.7 kb deletion and the entire 16.5 kb mitochondrial genome in the brain and buffy-coats of young and aged monkeys. We studied a total of 66 DNA samples: 39 were prepared from a buffy-coat and 27 were prepared from occipital cortex tissues. The mtDNA data were assessed using a permutation test to identify differences in mtDNA, in the different monkey groups. Using real-time RT-PCR strategy, we also assessed both mtDNA and nuclear DNA levels for young, aged and male and female monkeys. We found a 5.7 kb mtDNA deletion in 81.8% (54 of 66) of the total tested samples. In the young group of buffy-coat DNA, we found 5.7 kb deletions in 7 of 17 (41%), and in the aged group, we found 5.7 kb deletions in 12 of 22 (54%), suggesting that the prevalence of mtDNA deletions is related to age. We found decreased mRNA levels of mtDNA in aged monkeys relative to young monkeys. The increases in mtDNA deletions and mtDNA levels in aged rhesus monkeys suggest that damaged DNA accumulates as rhesus monkeys age and these altered mtDNA changes may have physiological relevance to compensate decreased mitochondrial function.     

Characterization of the C584R variant in the mtDNA depletion syndrome gene FBXL4, reveals a novel role for FBXL4 as a regulator of mitochondrial fusion

Mutations in FBXL4 (F-Box and Leucine rich repeat protein 4), a nuclear-encoded mitochondrial protein with an unknown function, cause mitochondrial DNA depletion syndrome. We report two siblings, from consanguineous parents, harbouring a previously uncharacterized homozygous variant in FBXL4 (c.1750 T > C; p.Cys584Arg). Both patients presented with encephalomyopathy, lactic acidosis and cardiac hypertrophy, which are reported features of FBXL4 impairment. Remarkably, dichloroacetate (DCA) administration to the younger sibling improved metabolic acidosis and reversed cardiac hypertrophy. Characterization of FBXL4 patient fibroblasts revealed severe bioenergetic defects, mtDNA depletion, fragmentation of mitochondrial networks, and abnormalities in mtDNA nucleoids. These phenotypes, observed with other pathogenic FBXL4 variants, confirm the pathogenicity of the p.Cys584Arg variant. Although treating FBXL4 fibroblasts with DCA improved extracellular acidification, in line with reduced lactate levels in patients, DCA treatment did not improve any of the other mitochondrial functions. Nonetheless, we highlight DCA as a potentially effective drug for the management of elevated lactate and cardiomyopathy in patients with pathogenic FBXL4 variants. Finally, as the exact mechanism through which FBXL4 mutations lead to mtDNA depletion was unknown, we tested the hypothesis that FBXL4 promotes mitochondrial fusion. Using a photo-activatable GFP fusion assay, we found reduced mitochondrial fusion rates in cells harbouring a pathogenic FBXL4 variant. Meanwhile, overexpression of wildtype FBXL4, but not the p.Cys584Arg variant, promoted mitochondrial hyperfusion. Thus, we have uncovered a novel function for FBXL4 in promoting mitochondrial fusion, providing important mechanistic insights into the pathogenic mechanism underlying FBXL4 dysfunction.