Federica Dabbeni-Sala

Cyclophilin D Modulates Mitochondrial F0F1-ATP Synthase by Interacting with the Lateral Stalk of the Complex

Blue native gel electrophoresis purification and immunoprecipitation of F0F1-ATP synthase from bovine heart mitochondria revealed that cyclophilin (CyP) D associates to the complex. Treatment of intact mitochondria with the membrane-permeable bifunctional reagent dimethyl 3,3-dithiobis-propionimidate (DTBP) cross-linked CyPD with the lateral stalk of ATP synthase, whereas no interactions with F1 sector subunits, the ATP synthase natural inhibitor protein IF1, and the ATP/ADP carrier were observed. The ATP synthase-CyPD interactions have functional consequences on enzyme catalysis and are modulated by phosphate (increased CyPD binding and decreased enzyme activity) and cyclosporin (Cs) A (decreased CyPD binding and increased enzyme activity). Treatment of MgATP submitochondrial particles or intact mitochondria with CsA displaced CyPD from membranes and activated both hydrolysis and synthesis of ATP sustained by the enzyme. No effect of CsA was detected in CyPD-null mitochondria, which displayed a higher specific activity of the ATP synthase than wild-type mitochondria. Modulation by CyPD binding appears to be independent of IF1, whose association to ATP synthase was not affected by CsA treatment. These findings demonstrate that CyPD association to the lateral stalk of ATP synthase modulates the activity of the complex.

Melatonin protects against 6-OHDA-induced neurotoxicity in rats: a role for mitochondrial complex I activity

Unilateral injection into the right substantia nigra of the catecholaminergic neurotoxin 6‐hydroxydopamine (6‐OHDA) produces extensive loss of dopaminergic cells (‘hemi‐parkinsonian rat’). The pineal hormone melatonin, which is a potent antioxidant against different reactive oxygen species and has been reported to be neuroprotective in vivo and in vitro, was evaluated for potential anti‐Parkinson effects in this model. Imbalance in dopaminergic innervation between the striata produced by intranigral administration of 6‐OHDAresults in a postural asymmetry causing rotation away from the nonlesioned side. Melatonin given systemically prevented apomorphine‐induced circling behavior in 6‐OHDA‐lesioned rats. Reduced activity of mitochondrial oxidative phosphorylation enzymes has been suggested in some neurodegenerative diseases; in particular, selective decrease in complex I activity is observed in the substantia nigra of Parkinsons disease patients. Analysis of mitochondrial oxidative phosphorylation enzyme activities in nigral tissue from 6‐OHDA‐lesioned rats by a novel BN‐PAGE histochemical procedure revealed a clear loss of complex I activity, which was protected against in melatonintreated animals. A good correlation between behavioral parameters and enzymatic (complex I) analysis was observed independent of melatonin administration. A deficit in mitochondrial complex I could conceivably contribute to cell death in parkinsonism via free radical mechanisms, both directly via reactive oxygen species production and by decreased ATP synthesis and energy failure. Melatonin may have potential utility in the treatment of neurodegenerative disorders where oxidative stress is a participant.—Dabbeni‐Sala, F., Di Santo, S., Franceschini, D., Skaper, S. D., Giusti, P. Melatonin protects against 6‐OHDA‐induced neurotoxicity in rats: a role for mitochondrial complex I activity. FASEB J. 15, 164–170 (2001)

Antioxidant defences in cybrids harboring mtDNA mutations associated with Leber's hereditary optic neuropathy

Oxidative stress and imbalance between free radical generation and detoxification may play a pivotal role in the pathogenesis of Lebers hereditary optic neuropathy (LHON). Mitochondria, carrying the homoplasmic 11778/ND4, 3460/ND1 and 14484/ND6 mtDNA point mutations associated with LHON, were used to generate osteosarcoma‐derived cybrids. Enhanced mitochondrial production of reactive oxygen species has recently been demonstrated in these cybrids [Beretta S, Mattavelli L, Sala G, Tremolizzo L, Schapira AHV, Martinuzzi A, Carelli V & Ferrarese C (2004) Brain127, 2183–2192]. The aim of this study was to characterize the antioxidant defences of these LHON‐affected cells. The activities of glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutases (SOD) and catalase, and the amounts of glutathione (GSH) and oxidized glutathione (GSSG) were measured in cybrids cultured both in glucose‐rich medium and galactose‐rich medium. The latter is known to cause oxidative stress and to trigger apoptotic death in these cells. In spite of reduced SOD activities in all LHON cybrids, and of low GPx and GR activities in cells with the most severe 3460/ND1 and 11778/ND4 mutations, GSH and GSSG content were not significantly modified in LHON cybrids cultured in glucose medium. In contrast, in galactose, GSSG concentrations increased significantly in all cells, indicating severe oxidative stress, whereas GR and MnSOD activities further decreased in all LHON cybrids. These data suggest that, in cells carrying LHON mutations, there is a decrease in antioxidant defences, which is especially evident in cells with mutations associated with the most severe clinical phenotype. This is magnified by stressful conditions such as exposure to galactose.

Regulation of the Inner Membrane Mitochondrial Permeability Transition by the Outer Membrane Translocator Protein (Peripheral Benzodiazepine Receptor)

We studied the properties of the permeability transition pore (PTP) in rat liver mitochondria and in mitoplasts retaining inner membrane ultrastructure and energy-linked functions. Like mitochondria, mitoplasts readily underwent a permeability transition following Ca2+ uptake in a process that maintained sensitivity to cyclosporin A. On the other hand, major differences between mitochondria and mitoplasts emerged in PTP regulation by ligands of the outer membrane translocator protein of 18 kDa, TSPO, formerly known as the peripheral benzodiazepine receptor. Indeed, (i) in mitoplasts, the PTP could not be activated by photo-oxidation after treatment with dicarboxylic porphyrins endowed with protoporphyrin IX configuration, which bind TSPO in intact mitochondria; and (ii) mitoplasts became resistant to the PTP-inducing effects of N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and of other selective ligands of TSPO. Thus, the permeability transition is an inner membrane event that is regulated by the outer membrane through specific interactions with TSPO.

Activation of (Na+ + K+)-dependent ATPase by lipid vesicles of negative phospholipids.

1. Kidney (Na+ + K+)-stimulated ATPase was depleted of phospholipids by extraction with lubrol and inserted in lipid structures of known composition. Both ouabain-sensitive ATPase and phosphatase reactions could be partially restored by lipid replacement. 2. Lipid vesicles of natural and synthetic negative phospholipids proved to be effective. The low activity of uncharged liposomes was increased when negative charges were included into the bilayer structure. 3. Reactivation by negative phospholipids was accompanied by spontaneous re-assembly of a stable lipid-protein complex. By contrast, the interaction of lipid deficient ATPase complex with uncharged lamellae was possible only after sonication of lipid-protein suspension. Reactivation did not ensue. 4. The ouabain-sensitive ATPase reactivated by synthetic dioleoylphosphatidylglycerol yielded curvilinear Arrhenius plots. The same pattern was seen with the original undepleted microsomal preparation. A discontinuity close to the temperature of fluid-order transition was found with dimyristoyl phosphatidylglycerol. 5. It is concluded that reassembly of lipid-deficient (Na+ + K+)-stimulated ATPase requires the addition of diacylphospholipids with fluid acyl-chains and negatively charged polar heads able to assemble in an expanded lamellar configuration.

Bilayer thickness and enzymatic activity in the mitochondrial cytochrome c oxidase and ATPase complex

Lipid-protein interactions are of great importance for the correct function and location of membrane-bound enzymes [ 1,2]. Nevertheless very little is known about the structure and folding of the polypeptide chains directly involved in the interaction with lipids. So far a low-resolution picture of polypeptide arrangement in the membrane has been obtained only for bacteriorhodopsin [3]. Few of the many integral membrane proteins, which have been isolated in pure form, have been sequenced. Most of these sequences show uninterrupted stretches of uncharged amino acids ,Z 18 residues long [4]. Hydrophobic photolabelling with photoreactive lipids has been used to identify which of these segments are in contact with lipids and hence to define the protein surface of the hydrophobic sector [5-71. A defined bilayer thickness is required for optimal activity of the sarcoplasmic reticulum Ca2+-ATPase and the (Na+, K+)-ATPase [8,9]. This investigation was based on the use of a homologous series of unsaturated phosphatidylcholines of different fatty acid chain length, which form bilayers of varying thickness [8]. Thus some information on the vertical dimension of the hydrophobic sector and on the forces involved in the

Switch from inhibition to activation of the mitochondrial permeability transition during hematoporphyrin-mediated photooxidative stress. Unmasking pore-regulating external thiols.

We have studied the mitochondrial permeability transition pore (PTP) under oxidizing conditions with mitochondria-bound hematoporphyrin, which generates reactive oxygen species (mainly singlet oxygen, (1)O(2)) upon UV/visible light-irradiation and promotes the photooxidative modification of vicinal targets. We have characterized the PTP-modulating properties of two major critical sites endowed with different degrees of photosensitivity: (i) the most photovulnerable site comprises critical histidines, whose photomodification by vicinal hematoporphyrin causes a drop in reactivity of matrix-exposed (internal), PTP-regulating cysteines thus stabilizing the pore in a closed conformation; (ii) the most photoresistant site coincides with the binding domains of (external) cysteines sensitive to membrane-impermeant reagents, which are easily unmasked when oxidation of internal cysteines is prevented. Photooxidation of external cysteines promoted by vicinal hematoporphyrin reactivates the PTP after the block caused by histidine photodegradation. Thus, hematoporphyrin-mediated photooxidative stress can either inhibit or activate the mitochondrial permeability transition depending on the site of hematoporphyrin localization and on the nature of the substrate; and selective photomodification of different hematoporphyrin-containing pore domains can be achieved by fine regulation of the sensitizer/light doses. These findings shed new light on PTP modulation by oxidative stress.

Kainic acid induces selective mitochondrial oxidative phosphorylation enzyme dysfunction in cerebellar granule neurons: protective effects of melatonin and GSH ethyl ester

Kainic acid (KA), a potent central excitotoxin, may elicit neuronal death via generation of reactive oxygen species (ROS). The present study was undertaken to further characterize KA neurotoxicity and its relationship to ROS production and mitochondrial dysfunction. Exposure of rat cerebellar granule neurons at 14 days in vitro to 0.5 mM KA for 30 min resulted in the death of 53% of cells 24 h later. ROS production, evaluated by 2′,7′‐dichlorofluorescein diacetate, increased in KA‐treated granule neurons. Resolution of mitochondrial oxidative phosphorylation enzymes by blue native polyacrylamide gel electrophoresis, followed by histochemical staining, showed that KA induced a strong decrease (‐40%, P < 0.01) in succinate dehydrogenase (SDH) activity of complex II. Western analysis revealed a marked reduction in quantity of the catalytic portion of complex II enzyme in KA‐treated cells. No significant changes were observed in the activities of other mitochondrial complex enzymes. The actions of KA at the mitochondrial level, as well as on ROS generation and cell viability, were prevented by the KA receptor‐selective antagonist 6,7‐dinitroquinoxaline‐2,3(1H, 4H)‐dione. Pretreatment of granule neurons with melatonin, a direct scavenger of ROS, or with the reduced glutathione (GSH) delivery agent GSH ethyl ester, before KA challenge prevented both the decrease in cell viability and complex II damage. The last result supports a link between KA‐induced mitochondrial oxidative enzyme dysfunction and ROS generation. Together the results suggest mitochondria to be a critical target in KA injury to neurons.

Reactivation of a phospholipid-depleted sodium, potassium-stimulated ATPase

Abstract 1. 1. The effect of phospholipids on (Na + -K + )-ATPase was studied in a particulate preparation from bovine heart. 2. 2. After treatment with ultrasonic oscillations the complex became partially soluble in a solution of nonionic detergent lubrol. Fractionation of “solubilized” ATPase with (NH 4 ) 2 SO 4 , followed by washing in NaCl solutions, resulted in a preparation depleted of phospholipids as well as cholesterol and largely devoid of ATPase activity. 3. 3. Total phospholipids from various sources as well as individual purified phospholipids restored the ATPase activity which was 90% inhibited by ouabain. Reconstruction was accompanied by reuptake of lost phospholipids. Differences among phospholipids were recorded: phosphatidylserine and disphosphatidylglycerol were the most effective but active preparations were also obtained with phosphatidylcholine and phosphatidylethanolamine. Cholesterol added as aqueous emulsion was ineffective. 4. 4. It is concluded that activation of (Na + -K + )-ATPase by phospholipids is a rather aspecific process. Although phosphatidylserine shows remarkable affinity for the enzyme complex and presumably plays an important role in maintaining the activity of the ATPase, it can not be considered as a specific activator. In agreement with recent observations on particulate mitochondrial ATPase from rat liver, acidic phospholipids showed greater effectiveness in the reactivation of transport ATPase and could be bound better.

The activation of mitochondrial particulate ATPase by liposomes of diacylphospholipids.

Abstract 1. 1. The stimulation of mitochondrial particulate ATPase by natural phosphatidylcholines was studied in a phospholipid-free preparation from bovine heart obtained by extraction with cholate. The low stimulatory activity of these phospholipids was increased by: (a) introduction of negatively charged amphiphatic substances into the zwitterionic liposomes; (b) addition of Cl − to the incubation medium. 2. 2. Liposomes of acidic phospholipids or phosphatidylcholine containing anionic amphipaths prevented the ATP plus Mg 2+ -induced decrease of ATPase activity in Mg-ATP particles which have a high content of ATPase inhibitor. 3. 3. Acidic phospholipids, in contrast to phosphatidylcholine, prevented the inhibition of soluble and particulate ATPase by purified ATPase inhibitor. 4. 4. It is concluded that the structure of the lipid-water interface is of primary importance in the stimulation produced by liposomes of diacylphospholipids on particulate mitochondrial ATPase. This effect is associated with the removal of inhibition induced by endogenous ATPase inhibitor on phospholipid-depleted preparations.