Elena García-Arumí

Enhanced ROS production and antioxidant defenses in cybrids harbouring mutations in mtDNA

It has been suggested that mutations in mitochondrial DNA (mtDNA) can produce an increase in reactive oxygen species (ROS) and that this can play a major role in the pathogenic mechanisms of mitochondrial encephalomyopathies. Many studies exist using electron transport chain (ETC) inhibitors, however there are only a few studies that examine ROS production associated with mutations in the mtDNA. To investigate this issue, we have studied ROS production, antioxidant defences and oxidative damage to lipids and proteins in transmitochondrial cybrids carrying different mtDNA mutations. Here, we report that two different mutant cell lines carrying mutations in their mitochondrial tRNA genes (A3243G in tRNA LeuUUR and A8344G in tRNA Lys) showed an increased ROS production with a parallel increase in the antioxidant enzyme activities, which may protect cells from oxidative damage in our experimental conditions (no overt oxidative damage to lipids and proteins has been observed). In contrast, cytochrome c oxidase (COX) mutant cybrids (carrying the stop-codon mutation G6930A in the COXI gene) showed neither an increase in ROS production nor elevation of antioxidant enzyme activities or oxidative damage. These results suggest that the specific location of mutations in mtDNA has a strong influence on the phenotype of the antioxidant response. Therefore, this issue should be carefully considered when antioxidant therapies are investigated in patients with mitochondrial disorders.

Quantification of mitochondrial DNA copy number: pre-analytical factors.

Mitochondrial DNA (mtDNA) content is important for understanding many cellular processes. Several pre-analytical factors, from sample collection to DNA extraction can affect measurement of mtDNA copy number. In the present study, whole blood samples yielded a higher mtDNA copy number than buffy coat samples. mtDNA content is affected by the cell separation method used and the time between blood withdrawal and cell separation. Thus, reference values must be established with the same type of sample. As to the DNA isolation and purification method, the manual phenol method can give randomly false high values. The QIAamp DNA Mini Kit provided the most highly reproducible mtDNA/nDNA yield.

Oxidative Damage Compromises Energy Metabolism in the Axonal Degeneration Mouse Model of X-Adrenoleukodystrophy

AIMS Chronic metabolic impairment and oxidative stress are associated with the pathogenesis of axonal dysfunction in a growing number of neurodegenerative conditions. To investigate the intertwining of both noxious factors, we have chosen the mouse model of adrenoleukodystrophy (X-ALD), which exhibits axonal degeneration in spinal cords and motor disability. The disease is caused by loss of function of the ABCD1 transporter, involved in the import and degradation of very long-chain fatty acids (VLCFA) in peroxisomes. Oxidative stress due to VLCFA excess appears early in the neurodegenerative cascade. RESULTS In this study, we demonstrate by redox proteomics that oxidative damage to proteins specifically affects five key enzymes of glycolysis and TCA (Tricarboxylic acid) cycle in spinal cords of Abcd1(-) mice and pyruvate kinase in human X-ALD fibroblasts. We also show that NADH and ATP levels are significantly diminished in these samples, together with decrease of pyruvate kinase activities and GSH levels, and increase of NADPH. INNOVATION Treating Abcd1(-) mice with the antioxidants N-acetylcysteine and α-lipoic acid (LA) prevents protein oxidation; preserves NADH, NADPH, ATP, and GSH levels; and normalizes pyruvate kinase activity, which implies that oxidative stress provoked by VLCFA results in bioenergetic dysfunction, at a presymptomatic stage. CONCLUSION Our results provide mechanistic insight into the beneficial effects of antioxidants and enhance the rationale for translation into clinical trials for X-adrenoleukodystrophy.

The Eutherian Armcx genes regulate mitochondrial trafficking in neurons and interact with Miro and Trak2.

Brain function requires neuronal activity-dependent energy consumption. Neuronal energy supply is controlled by molecular mechanisms that regulate mitochondrial dynamics, including Kinesin motors and Mitofusins, Miro1-2 and Trak2 proteins. Here we show a new protein family that localizes to the mitochondria and controls mitochondrial dynamics. This family of proteins is encoded by an array of armadillo (Arm) repeat-containing genes located on the X chromosome. The Armcx cluster is unique to Eutherian mammals and evolved from a single ancestor gene (Armc10). We show that these genes are highly expressed in the developing and adult nervous system. Furthermore, we demonstrate that Armcx3 expression levels regulate mitochondrial dynamics and trafficking in neurons, and that Alex3 interacts with the Kinesin/Miro/Trak2 complex in a Ca(2+)-dependent manner. Our data provide evidence of a new Eutherian-specific family of mitochondrial proteins that controls mitochondrial dynamics and indicate that this key process is differentially regulated in the brain of higher vertebrates.

Mutational analysis of the Cu/Zn superoxide dismutase gene in a Catalan ALS population: Should all sporadic ALS cases also be screened for SOD1?

BACKGROUND SOD1 gene mutations are the most common identified cause of ALS, accounting for approximately 20% of familial ALS cases and around 4% of sporadic ALS cases. However, the prevalence of SOD1 varies in different ethnic groups. No previous epidemiological studies have been carried out in Catalonia. OBJECTIVE To determine the prevalence of SOD1 gene mutations in a Catalan ALS population, and to analyze the genotype-phenotype relationship. MATERIALS AND METHODS 30 different FALS pedigrees and 94 sporadic ALS patients were screened for SOD1 mutations using direct sequence analysis. RESULTS Five of the 30 FALS pedigrees (16.6%) carried a SOD1 mutant. The mutations identified in this group were G37R, D76V, S105L, I112M and N139H. Four SOD1 mutants (4.25%) were found in the sporadic ALS group (SALS). The overall frequency (FALS plus SALS) of SOD1 mutations in our series was 6.45%. In the SALS group, D90A was identified in a patient presenting the typical Scandinavian phenotype. A 53-year-old woman with no family history of ALS carried the N139H mutation. Two unrelated sporadic ALS cases carried the A140A SOD1 mutant. CONCLUSIONS The prevalence of the SOD1 mutation in FALS in Catalonia is similar to levels in other Mediterranean countries, but lower than those in reports studying the Belgian, Japanese, and Scottish populations. The prevalence of the SOD1 mutation was 4.25% in patients with no family history of ALS. These results may have significant repercussions on genetic counseling, and screening for the SOD1 mutation in sporadic ALS cases must therefore be considered.

Accumulation of mitochondrial DNA deletions within dopaminergic neurons triggers neuroprotective mechanisms.

Acquired alterations in mitochondrial DNA are believed to play a pathogenic role in Parkinsons disease. In particular, accumulation of mitochondrial DNA deletions has been observed in substantia nigra pars compacta dopaminergic neurons from patients with Parkinsons disease and aged individuals. Also, mutations in mitochondrial DNA polymerase gamma result in multiple mitochondrial DNA deletions that can be associated with levodopa-responsive parkinsonism and severe substantia nigra pars compacta dopaminergic neurodegeneration. However, whether mitochondrial DNA deletions play a causative role in the demise of dopaminergic neurons remains unknown. Here we assessed the potential pathogenic effects of mitochondrial DNA deletions on the dopaminergic nigrostriatal system by using mutant mice possessing a proofreading-deficient form of mitochondrial DNA polymerase gamma (POLGD257A), which results in a time-dependent accumulation of mitochondrial DNA deletions in several tissues, including the brain. In these animals, we assessed the occurrence of mitochondrial DNA deletions within individual substantia nigra pars compacta dopaminergic neurons, by laser capture microdissection and quantitative real-time polymerase chain reaction, and determined the potential deleterious effects of such mitochondrial DNA alterations on mitochondrial function and dopaminergic neuronal integrity, by cytochrome c oxidase histochemistry and quantitative morphology. Nigral dopaminergic neurons from POLGD257A mice accumulate mitochondrial DNA deletions to a similar extent (∼40-60%) as patients with Parkinsons disease and aged individuals. Despite such high levels of mitochondrial DNA deletions, the majority of substantia nigra pars compacta dopaminergic neurons from these animals did not exhibit mitochondrial dysfunction or degeneration. Only a few individual substantia nigra pars compacta neurons appeared as cytochrome c oxidase-negative, which exhibited higher levels of mitochondrial DNA deletions than cytochrome c oxidase-positive cells (60.38±3.92% versus 45.18±2.83%). Survival of dopaminergic neurons in POLGD257A mice was associated with increased mitochondrial DNA copy number, enhanced mitochondrial cristae network, improved mitochondrial respiration, decreased exacerbation of mitochondria-derived reactive oxygen species, greater striatal dopamine levels and resistance to parkinsonian mitochondrial neurotoxins. These results indicate that primary accumulation of mitochondrial DNA deletions within substantia nigra pars compacta dopaminergic neurons, at an extent similar to that observed in patients with Parkinsons disease, do not kill dopaminergic neurons but trigger neuroprotective compensatory mechanisms at a mitochondrial level that may account for the high pathogenic threshold of mitochondrial DNA deletions in these cells.

Measurements of the Antioxidant Enzyme Activities of Superoxide Dismutase, Catalase, and Glutathione Peroxidase

Publisher Summary This chapter describes the activity assays of the major reactive oxygen species (ROS) defense system enzymes—superoxide dismutases, glutathione peroxidases (GPx), and catalase (CAT)—in cultured cells, tissue homogenates, and mammalian mitochondria. To cope with the damaging actions of ROS, organisms have evolved a sophisticated ROS defense system (RDS), consisting of low-molecular-weight antioxidants, such as glutathione, ascorbic acid, tocopherol, and uric acid, and specialized ROS-detoxifying enzymes, such as SODs, CAT, GPxs, and various thio-, peroxi-, and glutaredoxins. These enzymes represent the primary line of ROS defense. Defense enzymes can remove ROS directly or can repair the damage to other macromolecules caused by ROS. Other enzymes are involved in the renewal of the reducing power of defense enzymes. Failure of RDS to cope with the intracellular ROS production results in oxidative stress, which contributes to the damage and death of cells. Therefore, measuring the activity of RDS enzymes is a valuable diagnostic tool to determine the role of the oxidative stress in the pathology of a particular disease.

Free radicals-mediated damage in transmitochondrial cells harboring the T14487C mutation in the ND6 gene of mtDNA

We have studied the production of reactive oxygen species (ROS) in transmitochondrial cells, harboring homoplasmic levels of the T14487C mtDNA mutation in the ND6 gene of mitochondrial DNA (mtDNA). Previous work has shown that this mutation causes complex I deficiency. Here, we show that this mutation causes an overproduction of ROS leading to an increase in the oxidation of lipids and mtDNA without modification of antioxidant enzyme activities. We suggest that mutations in mtDNA affecting complex I activity may result in oxidative cellular damage, and reinforce the possible role of ROS‐mediated mechanisms participating in some mtDNA‐related disorders.

Mitochondrial DAMPs Induce Endotoxin Tolerance in Human Monocytes: An Observation in Patients with Myocardial Infarction

Monocyte exposure to mitochondrial Danger Associated Molecular Patterns (DAMPs), including mitochondrial DNA (mtDNA), induces a transient state in which these cells are refractory to further endotoxin stimulation. In this context, IRAK-M up-regulation and impaired p65 activity were observed. This phenomenon, termed endotoxin tolerance (ET), is characterized by decreased production of cytokines in response to the pro-inflammatory stimulus. We also show that monocytes isolated from patients with myocardial infarction (MI) exhibited high levels of circulating mtDNA, which correlated with ET status. Moreover, a significant incidence of infection was observed in those patients with a strong tolerant phenotype. The present data extend our current understanding of the implications of endotoxin tolerance. Furthermore, our data suggest that the levels of mitochondrial antigens in plasma, such as plasma mtDNA, should be useful as a marker of increased risk of susceptibility to nosocomial infections in MI and in other pathologies involving tissue damage.

A novel exon 5 mutation (N139H) in the SOD1 gene in a Spanish family associated with incomplete penetrance

BACKGROUND Allelic heterogeneity and phenotype variability-especially in age at onset, penetrance and progression-are reported in ALS1 families. For this reason, SOD1 gene mutation data in ALS1 patients are currently being gathered to better understand the genotype-phenotype relationship in this disorder. Here, we report the clinical and molecular characteristics of a Spanish ALS1 family with incomplete penetrance. PATIENTS AND METHODS Clinical data including age at onset, initial topography, progression and survival were available in three affected members. Erythrocyte SOD1 activity was measured in four individuals. Analysis of the SOD1 gene was performed by PCR and direct sequencing. RESULTS A novel missense mutation in the exon 5 of the SOD1 gene, an A-to-C transversion at nucleotide position 1485 leading to N139H residue change, was identified in three family members. The phenotype was similar in all cases, with initial symptoms in the distal limb muscles and a mean survival time of around 4 years. Incomplete penetrance was observed in our family, as two obligate carriers did not develop any symptoms of amyotrophic lateral sclerosis (ALS). CONCLUSIONS N139H is the fifth SOD1 gene mutation reported in Spain, and the first one presenting with incomplete penetrance. Genetic counseling for at-risk relatives in these low-penetrance families could be difficult as some individuals harbouring the mutation remain asymptomatic throughout their lives. Further genetic characterisation of ALS1 families should provide information regarding the distribution of SOD1 mutants in different ethnic groups.