The Fate of Oxidative Strand Breaks in Mitochondrial DNA.

Mitochondrial DNA (mtDNA) is particularly vulnerable to somatic mutagenesis. Potential mechanisms include DNA polymerase γ (POLG) errors and the effects of mutagens, such as reactive oxygen species. Here, we studied the effects of transient hydrogen peroxide (HO pulse) on mtDNA integrity in cultured HEK 293 cells, applying Southern blotting, ultra-deep short-read and long-read sequencing.

Molecular and Functional Effects of Loss of Cytochrome c Oxidase Subunit 8A.

In this work we studied molecular and functional effects of the loss of the smallest nuclear encoded subunit of cytochrome c oxidase COX8A in fibroblasts from a patient with a homozygous splice site mutation and in CRISPR/Cas9 genome-edited HEK293T cells. In both cellular model systems, between 20 to 30% of the residual enzymatic activity of cytochrome c oxidase (COX) was detectable. In immunoblots of BN-PAGE separated mitochondria from both cellular models almost no monomers and dimers of the fully assembled COX could be visualized.

Heart failure after pressure overload in autosomal-dominant desminopathies: Lessons from heterozygous DES-p.R349P knock-in mice.

Background: Mutations in the human desmin gene (DES) cause autosomal-dominant and -recessive cardiomyopathies, leading to heart failure, arrhythmias, and AV blocks. We analyzed the effects of vascular pressure overload in a patient-mimicking p.R349P desmin knock-in mouse model that harbors the orthologue of the frequent human DES missense mutation p.

Linear mitochondrial DNA is rapidly degraded by components of the replication machinery.

Emerging gene therapy approaches that aim to eliminate pathogenic mutations of mitochondrial DNA (mtDNA) rely on efficient degradation of linearized mtDNA, but the enzymatic machinery performing this task is presently unknown. Here, we show that, in cellular models of restriction endonuclease-induced mtDNA double-strand breaks, linear mtDNA is eliminated within hours by exonucleolytic activities. Inactivation of the mitochondrial 5'-3'exonuclease MGME1, elimination of the 3'-5'exonuclease activity of the mitochondrial DNA polymerase POLG by introducing the p.

Mitochondrial Liver Toxicity of Valproic Acid and Its Acid Derivatives Is Related to Inhibition of α-Lipoamide Dehydrogenase.

The liver toxicity of valproic acid (VPA) is an established side effect of this widely used antiepileptic drug, which is extremely problematic for patients with metabolic epilepsy and particularly epilepsy due to mitochondrial dysfunction. In the present report, we investigated the reason for liver mitochondrial toxicity of VPA and several acid and amide VPA analogues. While the pyruvate and 2-oxoglutarate oxidation rates of rat brain mitochondria were nearly unaffected by VPA, rat liver mitochondrial pyruvate and 2-oxoglutarate oxidation was severely impaired by VPA concentrations above 100 µM.

Homozygous mutation in TXNRD1 is associated with genetic generalized epilepsy.

Increased oxidative stress has been widely implicated in the pathogenesis in various forms of human epilepsy. Here, we report a homozygous mutation in TXNRD1 (thioredoxin reductase 1) in a family with genetic generalized epilepsy. TXNRD1 is an essential selenium-containing enzyme involved in detoxification of reactive oxygen species (ROS) and redox signaling.

Loss of UCP2 attenuates mitochondrial dysfunction without altering ROS production and uncoupling activity.

Although mitochondrial dysfunction is often accompanied by excessive reactive oxygen species (ROS) production, we previously showed that an increase in random somatic mtDNA mutations does not result in increased oxidative stress. Normal levels of ROS and oxidative stress could also be a result of an active compensatory mechanism such as a mild increase in proton leak. Uncoupling protein 2 (UCP2) was proposed to play such a role in many physiological situations.

Hemin inhibits the large conductance potassium channel in brain mitochondria: a putative novel mechanism of neurodegeneration.

Intracerebral hemorrhage (ICH) is a pathological condition that accompanies certain neurological diseases like hemorrhagic stroke or brain trauma. Its effects are severely destructive to the brain and can be fatal. There is an entire spectrum of harmful factors which are associated with the pathogenesis of ICH.

Microglial CD33-related Siglec-E inhibits neurotoxicity by preventing the phagocytosis-associated oxidative burst.

Sialic acid-binding Ig-like lectins (Siglecs) are members of the Ig superfamily that recognize sialic acid residues of glycoproteins. Siglec-E is a mouse CD33-related Siglec that preferentially binds to sialic acid residues of the cellular glycocalyx. Here, we demonstrate gene transcription and protein expression of Siglec-E by cultured mouse microglia.

Mitofusin 2 mutations affect mitochondrial function by mitochondrial DNA depletion.

Charcot-Marie-Tooth neuropathy type 2A (CMT2A) is associated with heterozygous mutations in the mitochondrial protein mitofusin 2 (Mfn2) that is intimately involved with the outer mitochondrial membrane fusion machinery. The precise consequences of these mutations on oxidative phosphorylation are still a matter of dispute. Here, we investigate the functional effects of MFN2 mutations in skeletal muscle and cultured fibroblasts of four CMT2A patients applying high-resolution respirometry.

The contribution of thioredoxin-2 reductase and glutathione peroxidase to H(2)O(2) detoxification of rat brain mitochondria.

Brain mitochondria are not only major producers of reactive oxygen species but they also considerably contribute to the removal of toxic hydrogen peroxide by the glutathione (GSH) and thioredoxin-2 (Trx2) antioxidant systems. In this work we estimated the relative contribution of both systems and catalase to the removal of intrinsically produced hydrogen peroxide (H(2)O(2)) by rat brain mitochondria. By using the specific inhibitors auranofin and 1-chloro-2,4-dinitrobenzene (DNCB), the contribution of Trx2- and GSH-systems to reactive oxygen species (ROS) detoxification in rat brain mitochondria was determined to be 60±20% and 20±15%, respectively.

Changes in mitochondrial reactive oxygen species synthesis during differentiation of skeletal muscle cells.

Myogenesis is accompanied by an intensive metabolic remodeling. We investigated the mitochondrial reactive oxygen species (ROS) generation at different levels of skeletal muscle differentiation: in C2C12 myoblasts, in C2C12 myotubes and in adult mouse skeletal muscle. Differentiation was accompanied by an increase in mitochondrial content and respiratory chain activity.

N-acetyl cysteine treatment rescues cognitive deficits induced by mitochondrial dysfunction in G72/G30 transgenic mice.

Genetic studies have implicated the evolutionary novel, anthropoid primate-specific gene locus G72/G30 in psychiatric diseases. This gene encodes the protein LG72 that has been discussed to function as a putative activator of the peroxisomal enzyme D-amino-acid-oxidase (DAO) and as a mitochondrial protein. We recently generated 'humanized' bacterial artificial chromosome transgenic mice (G72Tg) expressing G72 transcripts in cells throughout the brain.

Complex III-dependent superoxide production of brain mitochondria contributes to seizure-related ROS formation.

Brain seizure activity is characterised by intense activation of mitochondrial oxidative phosphorylation. This stimulation of oxidative phosphorylation is in the low magnesium model of seizure-like events accompanied by substantial increase in formation of reactive oxygen species (ROS). However, it has remained unclear which ROS-generating sites can be attributed to this phenomenon.

Single channel studies of the ATP-regulated potassium channel in brain mitochondria.

Mitochondrial potassium channels in the brain have been suggested to have an important role in neuroprotection. The single channel activity of mitochondrial potassium channels was measured after reconstitution of the purified inner membrane from rat brain mitochondria into a planar lipid bilayer. In addition to a large conductance potassium channel that was described previously, we identified a potassium channel that has a mean conductance of 219 +/- 15 pS.

Parkinson phenotype in aged PINK1-deficient mice is accompanied by progressive mitochondrial dysfunction in absence of neurodegeneration.

Background: Parkinson's disease (PD) is an adult-onset movement disorder of largely unknown etiology. We have previously shown that loss-of-function mutations of the mitochondrial protein kinase PINK1 (PTEN induced putative kinase 1) cause the recessive PARK6 variant of PD.

Methodology/principal Findings: Now we generated a PINK1 deficient mouse and observed several novel phenotypes: A progressive reduction of weight and of locomotor activity selectively for spontaneous movements occurred at old age.

Calcium ions regulate K⁺ uptake into brain mitochondria: the evidence for a novel potassium channel.

The mitochondrial response to changes of cytosolic calcium concentration has a strong impact on neuronal cell metabolism and viability. We observed that Ca(2+) additions to isolated rat brain mitochondria induced in potassium ion containing media a mitochondrial membrane potential depolarization and an accompanying increase of mitochondrial respiration. These Ca(2+) effects can be blocked by iberiotoxin and charybdotoxin, well known inhibitors of large conductance potassium channel (BK(Ca) channel).

Dose-dependent memory effects and cerebral volume changes after in utero exposure to valproate in the rat.

Purpose: Recent clinical studies raised concern of a cognitive teratogenicity of the major antiepileptic drug valproate. To investigate possible cerebral correlates, we established a forced self-application schedule by diluting valproate in the drinking water of pregnant Wistar rats.

Methods: After application of medium (MD) and high doses (HDs) with mean daily intakes of about 470 and 720 mg/kg during the entire pregnancy, we analyzed effects on offspring performance in a series of behavioral paradigms as well as brain volumetric changes by magnetic resonance imaging (MRI).

Chapter 23 Quantification of superoxide production by mouse brain and skeletal muscle mitochondria.

The production of reactive oxygen species (ROS) has been implicated for numerous pathologic alterations, including neurodegeneration and aging. They are formed to a considerable extent by mitochondria by single electron reduction of molecular oxygen by competent electron donors like flavoproteins and semiubiqunone species. In this chapter, we evaluate quantitative methods for the detection of hydrogen peroxide and superoxide production.

Mitochondrial involvement in temporal lobe epilepsy.

Mitochondrial dysfunction has been identified as a potential cause of epileptic seizures and therapy-resistant forms of severe epilepsy. Thus, a broad variety of mutation in mitochondrial DNA or nuclear genes leading to the impairment of mitochondrial respiratory chain or of mitochondrial ATP synthesis has been associated with epileptic phenotypes. Additionally, with a variety of different methods impaired mitochondrial function has been reported for the seizure focus of patients with temporal lobe epilepsy and Ammon's horn sclerosis and of animal models of temporal lobe epilepsy.

BK channel openers inhibit ROS production of isolated rat brain mitochondria.

To delineate the potential mechanism of neuroprotective effects of potassium channel openers we have investigated, how Ca(2+)-activated large conductance potassium channel (BK(Ca) channel) openers influence the production of reactive oxygen species (ROS) by rat brain mitochondria, since mitochondrial generation of ROS is known to have a crucial influence on neuronal survival. We studied the effects of BK(Ca) channel openers CGS 7184 and NS 1619 on hydrogen peroxide production rate of isolated rat brain mitochondria. In K(+)-containing media 3 microM of both channel openers reduced the hydrogen peroxide production rates by approximately 20%.

Sites of generation of reactive oxygen species in homogenates of brain tissue determined with the use of respiratory substrates and inhibitors.

Reactive oxygen species (ROS) have been widely implicated in the pathogenesis of various neurological diseases and aging. But the exact sites of ROS generation in brain tissue remained so far elusive. Here, we provide direct experimental evidence that at least 50% of total ROS generation in succinate-oxidizing homogenates of brain tissue can be attributed to complex I of mitochondrial respiratory chain.

Subfield-specific loss of hippocampal N-acetyl aspartate in temporal lobe epilepsy.

Purpose: In patients with mesial temporal lobe epilepsy (MTLE) it remains an unresolved issue whether the interictal decrease in N-acetyl aspartate (NAA) detected by proton magnetic resonance spectroscopy ((1)H-MRS) reflects the epilepsy-associated loss of hippocampal pyramidal neurons or metabolic dysfunction.

Methods: To address this problem, we applied high-resolution (1)H-MRS at 14.1 Tesla to measure metabolite concentrations in ex vivo tissue slices from three hippocampal subfields (CA1, CA3, dentate gyrus) as well as from the parahippocampal region of 12 patients with MTLE.

Human epidermal keratinocytes accumulate superoxide due to low activity of Mn-SOD, leading to mitochondrial functional impairment.

The energy metabolism of the epidermis has been the subject of controversy; thus we characterized the mitochondrial phenotype of human primary keratinocytes and fibroblasts, in cell culture and in human skin sections. We found that keratinocytes respire as much as fibroblasts, however, maximal activities of the respiratory chain (RC) complexes were 2- to 5-fold lower, whereas expression levels of RC proteins were similar. Maximal activities of aconitase and isocitrate dehydrogenase, two mitochondrial enzymes especially vulnerable to superoxide, were lower than in fibroblasts.