Daniela P. Converso

HO-1 is located in liver mitochondria and modulates mitochondrial heme content and metabolism

This study investigated whether inducible HO‐1 is targeted to mitochondria and its putative effects on oxidative metabolism in rat liver. Western blot and immune‐electron microscopy in whole purified and fractionated organelles showed basal expression of HO‐1 protein in both microsomes and mitochondria (inner membrane), accompanied by a parallel HO activity. Inducers of HO‐1 increased HO‐1 targeting to the inner mitochondrial membrane, which also contained biliverdin reductase, supporting that both enzymes are in the same compartmentalization. Induction of mitochondrial HO‐1 was associated with a decrease of mitochondrial heme content and selective reduction of protein expression of cytochrome oxidase (COX) subunit I, which is coded by the mitochondrial genome and synthesized in the mitochondria depending on heme availability; these changes resulted in decreased COX spectrum and activity. Mitochondrial HO‐1 induction was also associated with down‐regulation of mitochondrial‐targeted NO synthase expression and activity, resulting in a reduction of NO‐dependent mitochondrial oxidant yield; inhibition of HO‐1 activity reverted these effects. In conclusion, we demonstrated for the first time localization of HO‐1 protein in mitochondria. It is surmised that mitochondrial HO‐1 has important biological roles in regulating mitochondrial heme protein turnover and in protecting against conditions such as hypoxia, neurodegenerative diseases, or sepsis, in which substantially increased mitochondrial NO and oxidant production have been implicated.—Converso, D. P., Taille, C., Carreras, M. C., Jaitovich, A., Poderoso, J. J., Boczkowski, J. HO‐1 is located in liver mitochondria and modulates mitochondrial heme content and metabolism. FASEB J. 20, E482–E492 (2006)

A mitochondrial kinase complex is essential to mediate an ERK1/2-dependent phosphorylation of a key regulatory protein in steroid biosynthesis.

ERK1/2 is known to be involved in hormone-stimulated steroid synthesis, but its exact roles and the underlying mechanisms remain elusive. Both ERK1/2 phosphorylation and steroidogenesis may be triggered by cAMP/cAMP-dependent protein kinase (PKA)-dependent and-independent mechanisms; however, ERK1/2 activation by cAMP results in a maximal steroidogenic rate, whereas canonical activation by epidermal growth factor (EGF) does not. We demonstrate herein by Western blot analysis and confocal studies that temporal mitochondrial ERK1/2 activation is obligatory for PKA-mediated steroidogenesis in the Leydig-transformed MA-10 cell line. PKA activity leads to the phosphorylation of a constitutive mitochondrial MEK1/2 pool with a lower effect in cytosolic MEKs, while EGF allows predominant cytosolic MEK activation and nuclear pERK1/2 localization. These results would explain why PKA favors a more durable ERK1/2 activation in mitochondria than does EGF. By means of ex vivo experiments, we showed that mitochondrial maximal steroidogenesis occurred as a result of the mutual action of steroidogenic acute regulatory (StAR) protein –a key regulatory component in steroid biosynthesis-, active ERK1/2 and PKA. Our results indicate that there is an interaction between mitochondrial StAR and ERK1/2, involving a D domain with sequential basic-hydrophobic motifs similar to ERK substrates. As a result of this binding and only in the presence of cholesterol, ERK1/2 phosphorylates StAR at Ser232. Directed mutagenesis of Ser232 to a non-phosphorylable amino acid such as Ala (StAR S232A) inhibited in vitro StAR phosphorylation by active ERK1/2. Transient transfection of MA-10 cells with StAR S232A markedly reduced the yield of progesterone production. In summary, here we show that StAR is a novel substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric protein kinase complex that regulates cholesterol transport. The role of MAPKs in mitochondrial function is underlined.

Mitochondrial extracellular signal‐regulated kinases 1/2 (ERK1/2) are modulated during brain development

Intracellular activation and trafficking of extracellular signal‐regulated protein kinases (ERK) play a significant role in cell cycle progression, contributing to developmental brain activities. Additionally, mitochondria participate in cell signalling through energy‐linked functions, redox metabolism and activation of pro‐ or anti‐apoptotic proteins. The purpose of the present study was to analyze the presence of ERK1/2 in mitochondria during rat brain development. Immunoblotting, immune electron microscopy and activity assays demonstrated that ERK1/2 are present in fully active brain mitochondria at the outer membrane/intermembrane space fraction. Besides, it was observed that ERK1/2 translocation to brain mitochondria follows a developmental pattern which is maximal between E19‐P2 stages and afterwards declines at P3, just before maximal translocation to nucleus, and up to adulthood. Most of mitochondrial ERK1/2 were active; upstream phospho‐MAPK/ERK kinases (MEK1/2) were also detected in the brain organelles. Mitochondrial phospho‐ERK1/2 increased at 1 μm hydrogen peroxide (H2O2) concentration, but it decreased at higher 50–100 μm H2O2, almost disappearing after the organelles were maximally stimulated to produce H2O2 with antimycin. Our results suggest that developmental mitochondrial activation of ERK1/2 cascade contributes to its nuclear translocation effects, providing information about mitochondrial energetic and redox status to the proliferating/differentiating nuclear pathways.

Mitochondrial Nitric Oxide Synthase: A Masterpiece of Metabolic Adaptation, Cell Growth, Transformation, and Death

Mitochondria are specialized organelles that control energy metabolism and also activate a multiplicity of pathways that modulate cell proliferation and mitochondrial biogenesis or, conversely, promote cell arrest and programmed cell death by a limited number of oxidative or nitrative reactions. Nitric oxide (NO) regulates oxygen uptake by reversible inhibition of cytochrome oxidase and the production of superoxide anion from the mitochondrial electron transfer chain. In this sense, NO produced by mtNOS will set the oxygen uptake level and contribute to oxidation-reduction reaction (redox)–dependent cell signaling. Modulation of translocation and activation of neuronal nitric oxide synthase (mtNOS activity) under different physiologic or pathologic conditions represents an adaptive response properly modulated to adjust mitochondria to different cell challenges.

Tumor Cell Phenotype Is Sustained by Selective MAPK Oxidation in Mitochondria

Mitochondria are major cellular sources of hydrogen peroxide (H2O2), the production of which is modulated by oxygen availability and the mitochondrial energy state. An increase of steady-state cell H2O2 concentration is able to control the transition from proliferating to quiescent phenotypes and to signal the end of proliferation; in tumor cells thereby, low H2O2 due to defective mitochondrial metabolism can contribute to sustain proliferation. Mitogen-activated protein kinases (MAPKs) orchestrate signal transduction and recent data indicate that are present in mitochondria and regulated by the redox state. On these bases, we investigated the mechanistic connection of tumor mitochondrial dysfunction, H2O2 yield, and activation of MAPKs in LP07 murine tumor cells with confocal microscopy, in vivo imaging and directed mutagenesis. Two redox conditions were examined: low 1 µM H2O2 increased cell proliferation in ERK1/2-dependent manner whereas high 50 µM H2O2 arrested cell cycle by p38 and JNK1/2 activation. Regarding the experimental conditions as a three-compartment model (mitochondria, cytosol, and nuclei), the different responses depended on MAPKs preferential traffic to mitochondria, where a selective activation of either ERK1/2 or p38-JNK1/2 by co-localized upstream kinases (MAPKKs) facilitated their further passage to nuclei. As assessed by mass spectra, MAPKs activation and efficient binding to cognate MAPKKs resulted from oxidation of conserved ERK1/2 or p38-JNK1/2 cysteine domains to sulfinic and sulfonic acids at a definite H2O2 level. Like this, high H2O2 or directed mutation of redox-sensitive ERK2 Cys214 impeded binding to MEK1/2, caused ERK2 retention in mitochondria and restricted shuttle to nuclei. It is surmised that selective cysteine oxidations adjust the electrostatic forces that participate in a particular MAPK-MAPKK interaction. Considering that tumor mitochondria are dysfunctional, their inability to increase H2O2 yield should disrupt synchronized MAPK oxidations and the regulation of cell cycle leading cells to remain in a proliferating phenotype.

Mitochondrial nitric oxide synthase drives redox signals for proliferation and quiescence in rat liver development

Mitochondrial nitric oxide synthase (mtNOS) is a fine regulator of oxygen uptake and reactive oxygen species that eventually modulates the activity of regulatory proteins and cell cycle progression. From this perspective, we examined liver mtNOS modulation and mitochondrial redox changes in developing rats from embryonic days 17–19 and postnatal day 2 (proliferating hepatocyte phenotype) through postnatal days 15–90 (quiescent phenotype). mtNOS expression and activity were almost undetectable in fetal liver, and progressively increased after birth by tenfold up to adult stage. NO‐dependent mitochondrial hydrogen peroxide (H2O2) production and Mn‐superoxide dismutase followed the developmental modulation of mtNOS and contributed to parallel variations of cytosolic H2O2 concentration ([H2O2]ss) and cell fluorescence. mtNOS‐dependent [H2O2]ss was a good predictor of extracellular signal–regulated kinase (ERK)/p38 activity ratio, cyclin D1, and tissue proliferation. At low 10−11–10−12 M [H2O2]ss, proliferating phenotypes had high cyclin D1 and phospho‐ERK1/2 and low phospho‐p38 mitogen‐activated protein kinase, while at 10−9 M [H2O2]ss, quiescent phenotypes had the opposite pattern. Accordingly, leading postnatal day 2–isolated hepatocytes to embryo or adult redox conditions with H2O2 or NO‐H2O2 scavengers, or with ERK inhibitor U0126, p38 inhibitor SB202190 or p38 activator anisomycin resulted in correlative changes of ERK/p38 activity ratio, cyclin D1 expression, and [3H] thymidine incorporation in the cells. Accordingly, p38 inhibitor SB202190 or N‐acetyl‐cysteine prevented H2O2 inhibitory effects on proliferation. In conclusion, the results suggest that a synchronized increase of mtNOS and derived H2O2 operate on hepatocyte signaling pathways to support the liver developmental transition from proliferation to quiescence. (HEPATOLOGY 2004;40:157–166.)

The LRRK2 G2019S mutation in a series of Argentinean patients with Parkinson's disease: clinical and demographic characteristics.

OBJECTIVES To determine clinical characteristics and frequency of leucine-rich repeat kinase 2 gene (LRRK2) mutations in a cohort of patients with Parkinsons disease (PD) from Argentina. BACKGROUND Variation in the LRRK2 gene represents the most common genetic determinant of PD, only few data are available from Latin-America. DESIGN/METHODS Informed consent was obtained and all studies were approved by the Institutional Review Boards. Fifty five consecutive PD patients were recruited. A structured interview and neurological examination were used to collect demographic and clinical information. Blood samples were obtained and DNA extracted from patient venous blood. All LRRK2 exons from 25 exon to 51 exon were screened in all patients. RESULTS Clinical and molecular data of 55 patients with PD were analyzed. Mean age was 68.8±10.6 years. Jewish and Basque ancestries were found positive in 9 and 7 patients, respectively; family history of PD was identified in 16 patients. The G2019S mutation was present in 3 Ashkenazi Jewish subjects (5.45%); all of them reported family history of PD in first-degree relatives. Although Argentina possesses one of the most important Basque communities outside Spain, non R1414G mutation was identified in this cohort. Eleven single polymorphisms (SNP) were identified in this cohort. The mean age at onset was higher in G2019S mutation carriers than non-carriers (66.67 vs 58.78 years). Asymmetrical tremor as initial symptom and non-motor symptoms occurred at similar frequencies in both groups. The G2019S mutation carriers showed a non significant increase in dyskinesias, and 2/3 developed Dopamine Dysregulation Syndrome and visual hallucinations. Systemic disorder identified in G2019S mutation carriers included: celiac disease, hypothyroidism, Hashimotos Thyroiditis and arterial hypertension. CONCLUSIONS The prevalence of LRRK2 G2019S mutation in this Argentinean cohort was similar to other international series, with a higher prevalence in Ashkenazi Jewish. The phenotype was indistinguishable from patients with idiopathic PD. Interestingly, we identified immune mediated disorders in two PD patients carrying the G2019S mutation. Within this context, recent studies have identified full-length LRRK2 as a relatively common constituent of many cell types in the immune system including human peripheral blood mononuclear cells. Nevertheless, a casual association could not be excluded and the analysis of more extensive series is required.

Hypoxia induces complex I inhibition and ultrastructural damage by increasing mitochondrial nitric oxide in developing CNS

NO‐mediated toxicity contributes to neuronal damage after hypoxia; however, the molecular mechanisms involved are still a matter of controversy. Since mitochondria play a key role in signalling neuronal death, we aimed to determine the role of nitrative stress in hypoxia‐induced mitochondrial damage. Therefore, we analysed the biochemical and ultrastructural impairment of these organelles in the optic lobe of chick embryos after in vivo hypoxia–reoxygenation. Also, we studied the NO‐dependence of damage and examined modulation of mitochondrial nitric oxide synthase (mtNOS) after the hypoxic event. A transient but substantial increase in mtNOS content and activity was observed at 0–2 h posthypoxia, resulting in accumulation of nitrated mitochondrial proteins measured by immunoblotting. However, no variations in nNOS content were observed in the homogenates, suggesting an increased translocation to mitochondria and not a general de novo synthesis. In parallel with mtNOS kinetics, mitochondria exhibited prolonged inhibition of maximal complex I activity and ultrastructural phenotypes associated with swelling, namely, fading of cristae, intracristal dilations and membrane disruption. Administration of the selective nNOS inhibitor 7‐nitroindazole 20 min before hypoxia prevented complex I inhibition and most ultrastructural damage. In conclusion, we show here for the first time that hypoxia induces NO‐dependent complex I inhibition and ultrastructural damage by increasing mitochondrial NO in the developing brain.