Alejandro K. Samhan-Arias

Mitochondrial DNA Mutations Induce Mitochondrial Dysfunction, Apoptosis and Sarcopenia in Skeletal Muscle of Mitochondrial DNA Mutator Mice

Background Aging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established. Methodology/Principal Findings We investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase γ, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35–50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Δψm). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage. Conclusions/Significance These findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.

Kaempferol protects against rat striatal degeneration induced by 3‐nitropropionic acid

3‐Nitropropionic acid (NPA) produces degeneration of striatum and some neurological disturbances characteristic of Huntington’s disease in rodents and primates. We have shown that the flavonoid kaempferol largely reduced striatal damage induced by cerebral ischaemia‐reperfusion in rats ( Lopez‐Sanchez et al. 2007 ). In this work, we report that intraperitoneal (i.p.) administration of kaempferol affords an efficient protection against NPA‐induced neurodegeneration in Wistar rats. We studied the effects of daily i.p. injections of 7, 14 and 21 mg of kaempferol/kg body weight during the NPA‐treatment (25 mg/kg body weight/12 h i.p., for 5 days) on the neurological deficits, degeneration of rat striatum and oxidative stress markers. Intraperitoneal injections of 14–21 mg of kaempferol/kg body weight largely attenuated motor deficit and delayed mortality. The higher dose of kaempferol prevented the appearance of NPA‐induced striatal lesions up to the end of treatment, as revealed by haematoxylin‐eosin and TUNEL staining, and also NPA‐induced oxidative stress, because it blocked the fall of reduced glutathione and the increase of protein nitrotyrosines in NPA‐treated rats. It was found that striatal degeneration was associated with calpains activation and a large inactivation of creatine kinase, which were also prevented when the higher doses of kaempferol were administered.

Purified NADH-cytochrome b5 reductase is a novel superoxide anion source inhibited by apocynin: sensitivity to nitric oxide and peroxynitrite.

Cytochrome b5 reductase (Cb5R) is a pleiotropic flavoprotein that catalyzes multiple one-electron reduction reactions with various redox partners in cells. In earlier work from our laboratory, we have shown its implication in the generation of reactive oxygen species (ROS), primarily a superoxide anion overshoot peak, which plays a major role as a triggering event for the acceleration of apoptosis in cerebellar granule neurons in culture. However, the results obtained in that work did not allow us to exclude the possibility that this superoxide anion production could be derived from Cb5R acting in concert with other cellular components. In this work, we have purified Cb5R from pig liver and we have experimentally shown that this enzyme catalyzed NADH-dependent production of superoxide anion, assayed with cytochrome c and nitroblue tetrazolium as detection reagents for this particular ROS. The basic kinetic parameters for this novel NADH-dependent activity of Cb5R at 37°C are Vmax = 3.0 ± 0.5 μmol/min/mg of purified Cb5R and KM(NADH) = 2.8 ± 0.3 μM NADH. In addition, we report that apocynin, a widely used inhibitor of nonmitochondrial ROS production in mammalian cell cultures and tissues, is a potent inhibitor of purified Cb5R activity at the concentrations used in the experiments done with cell cultures. In the presence of apocynin the KM(NADH) value of Cb5R increases, and docking simulations indicate that apocynin can bind to a site near to or partially overlapping the NADH binding site of Cb5R. Other ROS, such as nitric oxide and peroxynitrite, have inhibitory effects on purified Cb5R, providing the basis for a feedback cellular protection mechanism through modulation of excessive extramitochondrial superoxide anion production by Cb5R. Both kinetic assays and docking simulations suggest that nitric oxide-induced nitrosylation (including covalent adduction of nitroso functional groups) of Cb5R cysteines and peroxynitrite-induced tyrosine nitration and cysteine oxidation modified the conformation of the NADH binding domain leading to a decreased affinity of Cb5R for NADH.

Hydrogen sulfide is a reversible inhibitor of the NADH oxidase activity of synaptic plasma membranes.

Hydrogen sulfide is now accepted as a neuromodulator, which can be involved in neuronal defence against oxidative stress insults in the brain. In this work we show that concentrations of H(2)S within the physiological range reported in the brain produce a reversible inhibition of the NADH oxidase activity and coupled superoxide anion production by synaptic plasma membranes from rat brain. At physiological pH 7 the concentration of H(2)S needed for 50% inhibition of the NADH oxidase activity is 5+/-1 microM, which is within the low range of the reported physiological H(2)S concentrations. Thus, the NADH oxidase activity of the neuronal plasma membrane can act as a sensor of local H(2)S depletion in neurones. H(2)S inhibition of the NADH oxidase activity of the neuronal plasma membrane can be accounted for direct reduction by H(2)S of cytochrome b(5). However, H(2)S fails to afford a significant protection against the inhibition of this activity by peroxynitrite. In conclusion, our results point out that H(2)S is more potent as inhibitor of reactive oxygen species formation than as a sacrificial antioxidant.

Early disruption of the actin cytoskeleton in cultured cerebellar granule neurons exposed to 3-morpholinosydnonimine-oxidative stress is linked to alterations of the cytosolic calcium concentration.

Cytoskeleton damage is a frequent feature in neuronal cell death and one of the early events in oxidant-induced cell injury. This work addresses whether actin cytoskeleton reorganization is an early event of SIN-1-induced extracellular nitrosative/oxidative stress in cultured cerebellar granule neurons (CGN). The actin polymerization state, i.e. the relative levels of G-/F-actin, was quantitatively assessed by the ratio of the fluorescence intensities of microscopy images obtained from CGN double-labelled with Alexa594-DNase-I (for actin monomers) and Bodipy-FL-phallacidin (for actin filaments). Exposure of CGN to a flux of peroxynitrite as low as 0.5-1μM/min during 30min (achieved with 0.1mM SIN-1) was found to promote alterations of the actin cytoskeleton dynamics as it increases the G-actin/F-actin ratio. Because L-type voltage-operated Ca(2+) channels (L-VOCC) are primary targets in CGN exposed to SIN-1, the possible role of Ca(2+) dynamics on the perturbation of the actin cytoskeleton was also assessed from the cytosolic Ca(2+) concentration response to the L-VOCCs agonist FPL-64176 and to the L-VOCCs blocker nifedipine. The results showed that SIN-1 induced changes in the actin polymerization state correlated with its ability to decrease Ca(2+) influx through L-VOCC. Combined analysis of cytosolic Ca(2+) concentration and G-actin/F-actin ratio alterations by SIN-1, cytochalasin D, latrunculin B and jasplakinolide support that disruption of the actin cytoskeleton is linked to cytosolic calcium concentration changes.

L-type calcium channels and cytochrome b5 reductase are components of protein complexes tightly associated with lipid rafts microdomains of the neuronal plasma membrane

The presence of cytosolic calcium microcompartments in neurons is well established. L-type voltage calcium channels play a leading role in the rise of cytosolic calcium in the neuronal soma and are sensitive to redox modulation. In a recent work [Samhan-Arias, A.K., García-Bereguiaín, M.A., Martín-Romero, F.J. and Gutiérrez-Merino, C. (2009) Mol. and Cell. Neurosci. 40, 14-26], we have shown that cytochrome b(5) reductase, whose deregulation leads to an overshot of superoxide anion production at the neuronal plasma membrane that triggers apoptosis in primary cultures of cerebellar granule neurons in culture, forms a large mesh of redox centres associated with lipid rafts in these neurons. In this work, we have implemented the use of fluorescent antibodies as reagents for quantitative Förster resonance energy transfer measurements and analysis using fluorescence microscopy images of cerebellar granule neurons in culture. The results of this study show that L-type voltage-operated calcium channels are also enriched in lipid rafts associated protein microdomains at a distance between 10 and 100 nm from cytochrome b(5) reductase. The methodological improvements done in this work can be also valuable for the study of proteins compartmentalization within other subcellular microdomains in any cell type in culture.

Stimulation and clustering of cytochrome b5 reductase in caveolin-rich lipid microdomains is an early event in oxidative stress-mediated apoptosis of cerebellar granule neurons.

The apoptosis of cerebellar granule neurons (CGN) induced by low potassium in the extracellular medium is a model of neuronal apoptosis where an overshot of reactive oxygen species (ROS) triggers the neuronal death. In this work, using dihydroethidium and L-012 as specific dyes for superoxide anion detection we show that this ROS overshot can be accounted by an increased release of superoxide anion to the extracellular medium. The amplitude and time course of the increase of superoxide anion observed early during apoptosis correlated with the increase of the content of soluble cytochrome b(5), a substrate of the NADH-dependent oxidase activity of the cytochrome b(5) reductase associated with lipid rafts in CGN. Western blotting and immunofluorescence microscopy approaches, including fluorescence energy transfer, pointed out an enhanced clustering of cytochrome b(5) reductase within caveolins-rich lipid rafts microdomains. Protein/protein docking analysis suggests that cytochrome b(5) reductase can form complexes with caveolins 1α, 1β and 2, playing electrostatic interactions a major role in this association. In conclusion, our results indicate that overstimulation of cytochrome b(5) reductase associated with lipid rafts can account for the overshot of plasma membrane-focalized superoxide anion production that triggers the entry of CGN in the irreversible phase of apoptosis. This article is part of a Special Issue entitled: Proteomics: The clinical link.

Topography of human cytochrome b 5 /cytochrome b 5 reductase interacting domain and redox alterations upon complex formation

Cytochrome b5 is the main electron acceptor of cytochrome b5 reductase. The interacting domain between both human proteins has been unidentified up to date and very little is known about its redox properties modulation upon complex formation. In this article, we characterized the protein/protein interacting interface by solution NMR and molecular docking. In addition, upon complex formation, we measured an increase of cytochrome b5 reductase flavin autofluorescence that was dependent upon the presence of cytochrome b5. Data analysis of these results allowed us to calculate a dissociation constant value between proteins of 0.5±0.1μM and a 1:1 stoichiometry for the complex formation. In addition, a 30mV negative shift of cytochrome b5 reductase redox potential in presence of cytochrome b5 was also measured. These experiments suggest that the FAD group of cytochrome b5 reductase increase its solvent exposition upon complex formation promoting an efficient electron transfer between the proteins.