Marco Di Paola

Antioxidants, reactive oxygen and nitrogen species, gene induction and mitochondrial function.

Redox-sensitive cell signalling Thiol groups and the regulation of gene expression Redox-sensitive signal transduction pathways Protein kinases Protein phosphatases Lipids and phospholipases Antioxidant (electrophile) response element Intracellular calcium signalling Transcription factors NF-?B AP-1 p53 Cellular responses to oxidative stress Cellular responses to change in redox state Proliferation Cell death Immune cell function Reactive oxygen and nitrogen species – good or bad? Reactive oxygen species and cell death Reactive oxygen species and inflammation Are specific reactive oxygen species and antioxidants involved in modulating cellular responses? Specific effects of dietary antioxidants in cell regulation Carotenoids Vitamin E Flavonoids Inducers of phase II enzymes Disease states affected Oxidants, antioxidants and mitochondria Introduction Mitochondrial generation of reactive oxygen and nitrogen species Mitochondria and apoptosis Mitochondria and antioxidant defences Key role of mitochondrial GSH in the defence against oxidative damage Mitochondrial oxidative damage Direct oxidative damage to the mitochondrial electron transport chain Nitric oxide and damage to mitochondria Effects of nutrients on mitochondria Caloric restriction and antioxidants Lipids Antioxidants Techniques and approaches Mitochondrial techniques cDNA microarray approaches Proteomics approaches Transgenic mice as tools in antioxidant research Gene knockout and over expression Transgenic reporter mice Conclusions Future research needs

Effect of resveratrol on mitochondrial function: implications in parkin-associated familiar Parkinson's disease

Mitochondrial dysfunction and oxidative stress occur in Parkinsons disease (PD), but the molecular mechanisms controlling these events are not completely understood. Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) is a transcriptional coactivator known as master regulator of mitochondrial functions and oxidative metabolism. Recent studies, including one from our group, have highlighted altered PGC-1α activity and transcriptional deregulation of its target genes in PD pathogenesis suggesting it as a new potential therapeutic target. Resveratrol, a natural polyphenolic compound proved to improve mitochondrial activity through the activation of several metabolic sensors resulting in PGC-1α activation. Here we have tested in vitro the effect of resveratrol treatment on primary fibroblast cultures from two patients with early-onset PD linked to different Park2 mutations. We show that resveratrol regulates energy homeostasis through activation of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) and raise of mRNA expression of a number of PGC-1αs target genes resulting in enhanced mitochondrial oxidative function, likely related to a decrease of oxidative stress and to an increase of mitochondrial biogenesis. The functional impact of resveratrol treatment encompassed an increase of complex I and citrate synthase activities, basal oxygen consumption, and mitochondrial ATP production and a decrease in lactate content, thus supporting a switch from glycolytic to oxidative metabolism. Moreover, resveratrol treatment caused an enhanced macro-autophagic flux through activation of an LC3-independent pathway. Our results, obtained in early-onset PD fibroblasts, suggest that resveratrol may have potential clinical application in selected cases of PD-affected patients.

Role of mitochondria and reactive oxygen species in dendritic cell differentiation and functions

Dendritic cells (DC) are potent antigen-presenting cells capable of inducing T and B responses and immune tolerance. We have characterized some aspects of energy metabolism accompanying the differentiation process of human monocytes into DC. Compared to precursor monocytes, DC exhibited a much larger number of mitochondria and consistently (i) a higher endogenous respiratory activity and (ii) a more than sixfold increase in ATP content and an even larger increase in the activity of the mitochondrial marker enzyme citrate synthase. The presence in the culture medium of rotenone, an inhibitor of the respiratory chain Complex I, prevented the increase in mitochondrial number and ATP level, without affecting cell viability. Rotenone inhibited DC differentiation, as revealed by the observation that the expression of CD1a, which is a specific surface marker of DC differentiation, was strongly reduced. Cells cultured in the presence of rotenone displayed a lower content of growth factor-induced, mitochondrially generated, hydrogen peroxide. A similar drop in ROS was observed upon addition of catalase, which caused functional effects similar to those produced by rotenone treatment. These results suggest that ROS play a crucial role in DC differentiation and that mitochondria are an important source of ROS in this process.

Effect of dietary restriction and N-acetylcysteine supplementation on intestinal mucosa and liver mitochondrial redox status and function in aged rats.

The age-related changes of glutathione (GSH) levels and the effect of hypocaloric regimen and N-acetylcysteine (NAC) supplementation were investigated in intestinal mucosa and liver mitochondria of 28 months rats. Old rats exhibited lower proteins, GSH and protein sulphydrils (PSH) concentrations, higher GSH-peroxidase (GSH-Px) activity and protein carbonyl deposit, partial inhibition of succinate stimulated mitochondrial state III respiration and decreased mitochondrial nitrosothiols (RSNO) concentration. Lower electric potential and current intensity were found in the colonic mucosa. Old rats undergone hypocaloric regimen showed higher intestinal concentrations of GSH, lower oxidized protein accumulation and GSH-Px activity and higher mitochondrial RSNO levels. Mitochondrial state III respiration and intestinal transport were improved. NAC supplementation enhanced GSH and PSH levels in the ileal but not in the colonic mucosa, GSH and RSNO in liver mitochondria, while GSH-Px and protein carbonyls were decreased everywhere. Mitochondrial respiration ameliorated. In conclusion, ageing is characterized by a spread decrease of GSH concentrations, increased protein oxidation and decreased mitochondrial NO content. Hypocaloric diet ameliorated intestinal transport and, as well as NAC, was effective in enhancing GSH levels but at different extent according to the investigated districts. Both interventions reduced the age-associated increase of GSH-Px and protein carbonyls and improved mitochondrial respiration.

Arachidonic acid induces specific membrane permeability increase in heart mitochondria

Micromolar concentrations of arachidonic acid cause in Ca2+ loaded heart mitochondria matrix swelling and Ca2+ release. These effects appear to be unrelated to the classical membrane permeability transition (MPT), as they are CsA insensitive, membrane potential independent and can also be activated by Sr2+. Atractyloside potentiated and ATP inhibited the arachidonic acid induced swelling. These observations suggest that the ATP/ADP translocator (ANT) may be involved in the AA induced, CsA insensitive membrane permeability increase. Under the same experimental conditions used for heart mitochondria, arachidonic acid induced the classical CsA sensitive, ADP inhibitable MPT in liver mitochondria.

The monocarboxylate carrier from rat liver mitochondria: Purification and kinetic characterization in a reconstituted system

The monocarboxylate (pyruvate) carrier was extracted from rat liver mitochondria with Triton X-100 in the presence of asolectin and partially purified by chromatography on HTP. The HTP eluate reconstituted in liposomes was shown to catalyze active pyruvatein/acetoacetateout, and acetoacetatein/pyruvateout counter-exchange. Kinetic characterization of the reconstituted pyruvate carrier was achieved by an original spectrophotometric method consisting of determination of substrate release from proteoliposomes with a coupled enzymatic assay.The monocarboxylate (pyruvate) carrier was extracted from rat liver mitochondria with Triton X‐100 in the presence of asolectin and partially purified by chromatography on HTP. The HTP eluate reconstituted in liposomes was shown to catalyze active pyruvatein/acetoacetateout, and acetoacetatein/pyruvateout counter‐exchange. Kinetic characterization of the reconstituted pyruvate carrier was achieved by an original spectrophotometric method consisting of determination of substrate release from proteoliposomes with a coupled enzymatic assay.

Steady-State Proton Translocation in Bovine Heart Mitochondrial bc1 Complex Reconstituted into Liposomes

The effect of different anions on the steady-state proton translocation in bovine bc1 complex reconstituted in liposomes was studied. The H+/e− ratio for vectorial proton translocation is at the steady state definitely lower than that measured at level flow, (0.3 vs. 1.0). The presence of azide or arachidonate at micro- and submicromolar concentrations, respectively, gave a substantial reactivation of the proton pumping activity at the steady state, without any appreciable effect on respiration-dependent transmembrane pH difference. Addition of azide to turning-over bc1 vesicles also caused a transition of b cytochromes toward oxidation. The results are discussed in terms of possible involvement of an acidic residue in the protonation of the semiquinone/quinol couple at the N side of the membrane.

Localization of acidic residues involved in the proton pumping activity of the bovine heart mitochondrial bc1 complex

Chemical modification of carboxyl residues in polypeptide subunits of the mitochondrial bc1 complex causes a decoupling effect, that is inhibition of the proton pumping activity, without affecting the rate of electron transfer to ferricytochrome c. The study presented here is aimed at localizing and identifying the residues whose modification results in decoupling of the complex. Glutamate‐53 in subunit IX (the DCCD‐binding protein) and aspartate‐166 in the Rieske iron‐sulfur protein are the residues modified by N,N′‐dicyclohexylcarbodiimide (DCCD) and N‐(ethoxycarbonyl)‐2‐ethoxy‐1,2‐dihydroquinoline (EEDQ), respectively. The results obtained also suggest that the carboxy‐terminal sequence of the Core protein II, which is fairly rich in acidic residues, may also play a role in the vectorial proton translocation activity of the complex.

Resveratrol Modulation of Protein Expression in parkin-Mutant Human Skin Fibroblasts: A Proteomic Approach

In this study, we investigated by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) analysis the effects of resveratrol treatment on skin primary fibroblasts from a healthy subject and from a parkin-mutant early onset Parkinsons disease patient. Parkin, an E3 ubiquitin ligase, is the most frequently mutated gene in hereditary Parkinsons disease. Functional alteration of parkin leads to impairment of the ubiquitin-proteasome system, resulting in the accumulation of misfolded or aggregated proteins accountable for the neurodegenerative process. The identification of proteins differentially expressed revealed that resveratrol treatment can act on deregulated specific biological process and molecular function such as cellular redox balance and protein homeostasis. In particular, resveratrol was highly effective at restoring the heat-shock protein network and the protein degradation systems. Moreover, resveratrol treatment led to a significant increase in GSH level, reduction of GSSG/GSH ratio, and decrease of reduced free thiol content in patient cells compared to normal fibroblasts. Thus, our findings provide an experimental evidence of the beneficial effects by which resveratrol could contribute to preserve the cellular homeostasis in parkin-mutant fibroblasts.