A study of the relationship between cAMP/PKA-dependent phosphorylation and oxidative damage of subunits of complex I of the mitochondrial respiratory chain is presented. It is shown that, in fibroblast cultures, PKA-mediated phosphorylation of the NDUFS4 subunit of complex I rescues the activity of the oxidatively damaged complex. Evidence is presented showing that this effect is mediated by phosphorylation-dependent exchange of carbonylated NDUFS4 subunit in the assembled complex with the de novo synthesized subunit. These results indicate a potential use for β-adrenoceptor agonists in preventing/reversing the detrimental effects of oxidative stress in the mitochondrial respiratory system.

Background: The poor response to chemotherapy and the brief response to vemurafenib in metastatic melanoma patients, make the identification of new therapeutic approaches an urgent need. Interestingly the increased expression and activity of the Aurora kinase B during melanoma progression suggests it as a promising therapeutic target. Methods: The efficacy of the Aurora B kinase inhibitor barasertib-HQPA was evaluated in BRAF mutated cells, sensitive and made resistant to vemurafenib after chronic exposure to the drug, and in BRAF wild type cells. The drug effectiveness has been evaluated as cell growth inhibition, cell cycle progression and cell migration. In addition, cellular effectors of drug resistance and response were investigated. Results: The characterization of the effectors responsible for the resistance to vemurafenib evidenced the increased expression of MITF or the activation of Erk1/2 and p-38 kinases in the newly established cell lines with a phenotype resistant to vemurafenib. The sensitivity of cells to barasertib-HQPA was irrespective of BRAF mutational status. Barasertib-HQPA induced the mitotic catastrophe, ultimately causing apoptosis and necrosis of cells, inhibited cell migration and strongly affected the glycolytic metabolism of cells inducing the release of lactate. In association i) with vemurafenib the gain in effectiveness was found only in BRAF(V600K) cells while ii) with nab-paclitaxel, the combination was more effective than each drug alone in all cells. Conclusions: These findings suggest barasertib as a new therapeutic agent and as enhancer of chemotherapy in metastatic melanoma treatment.

Background: Metastatic melanoma represents the most deadly form of skin cancer. The poor response to chemotherapy and the brief response to the anti-BRAF vemurafenib in selected population of patients, make the identification of new therapeutic approaches an urgent need. Our goal is the evaluation of the efficacy of barasertib, an aurora B kinase inhibitor impairing cytokinesis, in both mutated and non-mutated melanoma cell lines. Materials and methods: Panel of melanoma cells: BRAFV600E mutated cells (MBA72 and Hmel1), the same cell in which the resistance to vemurafenib was induced by chronic exposure to it (MBA72R and Hmel1R) and BRAF wt (HBL and LND1). Cells were characterized for vemurafenib and barasertib effectiveness on cell growth by MTT assay after 3 and 6 days of continuous exposure. Cell cycle was determined by flowcytometry and migration was evaluated by wound-healing assay. Results: Cells with BRAFV600E mutation are sensitive to vemurafenib conversely, those with BRAF wt and the resistant ones showed an IC50 of at least 10 folds higher. 3days-barasertib exposure strongly reduced cell growth (30-60% at 30 and 300nM, respectively) in all cell lines; when the drug was given together with vemurafenib, no gain in effectiveness was evident. Prolonged exposure to barasertib (6 days) showed a progressive increase of effectiveness particularly in cells BRAF wt. The analysis of cell death mechanisms involved in determining the effectiveness of barasertib and vemurafenib showed that the first drug induced both apoptosis and necrosis conversely, the latter mainly apoptosis. Cell cycle analysis demonstrated that barasertib induced an increase in cell size and in polyploidia, suggesting also the mitotic catastrophe as a further cell death mechanism. Moreover, the anti-metastatic behaviour of this agent has been evaluated in function of drug concentration and time exposure. Preliminary results showed a strong reduction of cell migration after drug exposure. Conclusions: The sensitivity of melanoma cells to barasertib is irrespective to BRAF mutational status; however, cells BRAF wt show an higher nuclear modification. In conclusion, our results suggest barasertib as a novel therapeutic approach in melanoma treatment irrespective of BRAFV600E mutation.

Dietary choline deprivation (CD) is associated with behavioral changes, but mechanisms underlying these detrimental effects are not well characterized. For instance, no literature data are available concerning the CD effects on brain mitochondrial function related to impairment in cognition. Therefore, we investigated brain mitochondrial function and redox status in male Wistar rats fed a CD diet for 28 d. Moreover, the CD behavioral phenotype was characterized. Compared with rats fed a control diet (CTRL), CD rats showed lower NAD-dependent mitochondrial state III and state IV respiration, 40% lower complex I activity, and significantly higher reactive oxygen species production. Total glutathione was oxidatively consumed more in CD than in CTRL rats and the rate of protein oxidation was 40% higher in CD than in CTRL rats, reflecting an oxidative stress condition. The mitochondrial concentrations of cardiolipin, a phospholipid required for optimal activity of complex I, was 20% lower in CD rats than in CTRL rats. Compared with CTRL rats, the behavioral phenotype of CD rats was characterized by impairment in motor coordination and motor learning assessed with the rotarod/accelerod test. Furthermore, compared with CTRL rats, CD rats were less capable of learning the active avoidance task and the number of attempts they made to avoid foot shock was fewer. The results suggest that CD-induced dysfunction in brain mitochondria may be responsible for impairment in cognition and underline that, similar to the liver, the brain also needs an adequate choline supply for its normal functioning.

Mitochondrial dysfunction and oxidative stress occur in Parkinson's 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-1α's 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.

A simple and fast method of lipid analysis of isolated intact mitochondria by means of MALDI-TOF mass spectrometry is described. Mitochondria isolated from bovine heart and yeast have been employed to set up and validate the new method of lipid analysis. The mitochondrial suspension is directly applied over the target and, after drying, covered by a thin layer of the 9-aminoacridine matrix solution. The lipid profiles acquired with this procedure contain all peaks previously obtained by analyzing the lipid extracts of isolated mitochondria by TLC and/or mass spectrometry. The novel procedure allows the quick, simple, precise, and accurate analysis of membrane lipids, utilizing only a tiny amount of isolated organelle; it has also been tested with intact membranes of the bacterium Paracoccus denitrificans for its evolutionary link to present-day mitochondria. The method is of general validity for the lipid analysis of other cell fractions and isolated organelles.

Mutations in the parkin gene are expected to play an essential role in autosomal recessive Parkinson's disease. Recent studies have established an impact of parkin mutations on mitochondrial function and autophagy. In primary skin fibroblasts from two patients affected by an early onset Parkinson's disease, we identified a hitherto unreported compound heterozygous mutation del exon2-3/del exon3 in the parkin gene, leading to the complete loss of the full-length protein. In both patients, but not in their heterozygous parental control, we observed severe ultrastructural abnormalities, mainly in mitochondria. This was associated with impaired energy metabolism, deregulated reactive oxygen species (ROS) production, resulting in lipid oxidation, and peroxisomal alteration. In view of the involvement of parkin in the mitochondrial quality control system, we have investigated upstream events in the organelles' biogenesis. The expression of the peroxisome proliferator-activated receptor gamma-coactivator 1-alpha (PGC-1α), a strong stimulator of mitochondrial biogenesis, was remarkably upregulated in both patients. However, the function of PGC-1α was blocked, as revealed by the lack of its downstream target gene induction. In conclusion, our data confirm the role of parkin in mitochondrial homeostasis and suggest a potential involvement of the PGC-1α pathway in the pathogenesis of Parkinson's disease. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.

PP-7 PGC1 alpha expression and correlation with BRAF mutational status I. Maida1 , A. Ferretta1 , T. Cocco1 , P. Zanna1 , R. Labarile1 , S. Guida3 , C. Grieco1 , L. Porcelli2 , A. Azzariti2 , S. Tommasi2 , A. Albano2 , S. Strippoli2 , M. Guida2 , R. Filotico3 , G. Guida1 1 Department of Basic Medical Sciences, Neurosciences and Sense Organs, School of Medicine, University of Bari, Bari, Italy; 2 Clinical Experimental Oncology Laboratory and Medical Oncology Department, National Cancer Institute, Bari, Bari, Italy; 3 Unit of Dermatology and Venereology, University of Bari, P.O. “A. Perrino”, Brindisi, Italy In this study we present the behaviour of the mitochondrial master regulator gene PGC1alpha in novel sporadic melanoma cell lines. The HBL and LND1 cell lines, wild type for BRAF, highly express PGC1alpha while hmel1, hmel9, hmel11, Mba72, M3, presenting BRAF mutations at the V600 residue, show a down regulation of this gene. Mutations in homo and/or heterozigosis of V600 in BRAF protein kinase always correspond to a down regulation of PGC1alpha as described for the cell lines A375, SKmel28, SKmel2, RPM7951, WM115 in Vasquez et al., 2013. MITF expression levels, measured by western blotting in our melanoma cell lines, were more abundant in HBL and LND1 cell lines with respect to the other cell lines harbouring BRAF mutations. There is a direct correspondence between PGC1alpha and MITF levels: higher levels of PGC1alpha are associated with an enhanced MITF quantity. The analysis of cAMP levels in our melanoma cell lines showed a similar trend, being higher in wt BRAF cell lines compared to the other cell lines. Our data confirm the key role of BRAF mutations, MITF and cAMP levels in melanoma biology, suggesting a very important association with the transcriptional co-activator PGC1alpha, involved in energy metabolism, in mitochondrial biogenesis but also in various physiological stimuli which are reprogrammed in melanoma cells. This factor, also involved in regulating angiogenetic mechanisms, could be an interesting gene for target therapy but also it could provide an important diagnostic role.

The NDUFS4 subunit of complex I of the mammalian respiratory chain has a fully conserved carboxy-terminus with a canonical RVSTK phosphorylation site. Immunochemical analysis with specific antibodies shows that the serine in this site of the protein is natively present in complex I in both the phosphorylated and non-phosphorylated state. Two-dimensional IEF/SDS-PAGE electrophoresis, (32)P labelling and immunodetection show that "in vitro" PKA phosphorylates the serine in the C-terminus of the NDUFS4 subunit in isolated bovine complex I. (32)P labelling and TLC phosphoaminoacid mapping show that PKA phosphorylates serine and threonine residues in the purified heterologous human NDUFS4 protein.

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 Parkinson's disease patient. Parkin, an E3 ubiquitin ligase, is the most frequently mutated gene in hereditary Parkinson's 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.

A study is presented on the effect of the beta-adrenoceptor agonist isoproterenol on mitochondrial oxygen metabolism in fibroblast and heart myoblast cultures. Isoproterenol treatment of serum-limited fibroblasts and proliferating myoblasts results in the promotion of mitochondrial complex I activity and decrease of the cellular level of reactive oxygen species. These effects of isoproterenol are associated with cAMP-dependent phosphorylation of complex I subunit(s). Addition of okadaic acid, inhibitor of protein phosphatase(s), reverses the decline of complex I activity in serum-limited fibroblast cultures and activates the complex in proliferating myoblast cultures. The effects of isoproterenol on complex I activity and reactive oxygen species balance can contribute to the therapeutic effect of the drug. (C) 2010 Elsevier B.V. All rights reserved.

Background: Metabolic reprogramming is commonly found in cancer but it is poorly understood in melanoma. Recent works [1,2] provided new insights concerning molecular mechanisms involved in mitochondrial biogenesis of melanoma. This work aims to find possible correlations between pathways involved in the onset and progression of the disease in order to provide supporting information in this field. In particular we studied the behaviour of the mitochondrial master regulator gene PGC1alpha in novel sporadic melanoma cell lines and its relations with BRAF mutational status. Materials and methods: We studied new cell lines extracted from sporadic metastatic melanomas (hmel1, M3, Mba72) and primary melanomas (hmel9, hmel11), genotyped for genes involved in melanoma development compared to control melanoma cell lines (HBL, LND1) wt for MC1R and BRAF genes. Hmel1, hmel9 and hmel11 have already been described in Zanna et al., 2011 [3] and Zanna et al., 2013 [4]. We evaluated PGC1a levels and some of its mitochondrial target genes and the mitochondrial respiratory capacity, the amount of ROS, and the lactate level. We related these data to BRAF mutational stauts and analyzed MITF and cAMP levels. Results: The HBL and LND1 cell lines, wt for BRAF, highly express PGC1alpha while hmel1, hmel9, hmel11, Mba72, M3, presenting BRAF mutations at the V600 residue, show a downregulation of this gene. MITF expression levels were more abundant in HBL and LND1 cell lines with respect to the other cell lines harbouring BRAF mutations. There is a direct correspondence between PGC1alpha and MITF levels: higher levels of PGC1alpha are associated with an enhanced MITF quantity. The analysis of cAMP levels in our melanoma cell lines showed a similar trend, being higher in wt BRAF cell lines compared to the other cell lines. Conclusions: Our data confirm the key role of BRAF mutations, MITF and cAMP levels in melanoma biology, suggesting a very important association with the transcriptional co-activator PGC1alpha, involved in energy metabolism and in mitochondrial biogenesis but also in various physiological stimuli that are reprogrammed in melanoma cells. These data support the divergent pathways hypothesis for melanoma, which may require a reappraisal of targeted cancer prevention and target therapeutic activities. References 1. Haq R, et al: Cancer Cell 2013, 23:302-315. 2. Vasquez F, et al: Cancer Cell 2013, 23:287-301. 3. Zanna P, et al: J Biol Regul Homeost Agents 2011, 25:239-247. 4. Zanna P, et al: J Biol Regul Homeost Agents 2013, 27:131-141.

The p66(Shc) protein mediates oxidative stress-related injury in multiple tissues. Steatohepatitis is characterized by enhanced oxidative stress-mediated cell damage. The role of p66(Shc) in redox signaling was investigated in human liver cells and alcoholic steatohepatitis. HepG2 cells with overexpression of wild-type or mutant p66(Shc), with Ser(36) replacement by Ala, were obtained through infection with recombinant adenoviruses. Reactive oxygen species and oxidation-dependent DNA damage were assessed by measuring dihydroethidium oxidation and 8-hydroxy-2'-deoxyguanosine accumulation into DNA, respectively. mRNA and protein levels of signaling intermediates were evaluated in HepG2 cells and liver biopsies from control and alcoholic steatohepatitis subjects. Exposure to H2O2 increased reactive oxygen species and phosphorylation of p66(Shc) on Ser(36) in HepG2 cells. Overexpression of p66(Shc) promoted reactive oxygen species synthesis and oxidation-dependent DNA damage, which were further enhanced by H2O2. p66(Shc) activation also resulted in increased Erk-1/2, Akt and FoxO3a phosphorylation. Blocking of Erk-1/2 activation inhibited p66(Shc) phosphorylation on Ser(36). Increased p66Shc expression was associated with reduced mRNA levels of anti-oxidant molecules, such as NF-E2-related factor 2 and its target genes. In contrast, overexpression of the phosphorylation defective p66(Shc) Ala(36) mutant inhibited p66(Shc) signaling, enhanced anti-oxidant genes, and suppressed reactive oxygen species and oxidation-dependent DNA damage. Increased p66(Shc) protein levels and Akt phosphorylation were observed in liver biopsies from alcoholic steatohepatitis compared to control subjects.

A study is presented on the effect of the β-adrenoceptor agonist isoproterenol on mitochondrial oxygen metabolism in fibroblast and heart myoblast cultures. Isoproterenol treatment of serum-limited fibroblasts and proliferating myoblasts results in the promotion of mitochondrial complex I activity and decrease of the cellular level of reactive oxygen species. These effects of isoproterenol are associated with cAMP-dependent phosphorylation of complex I subunit(s). Addition of okadaic acid, inhibitor of protein phosphatase(s), reverses the decline of complex I activity in serum-limited fibroblast cultures and activates the complex in proliferating myoblast cultures. The effects of isoproterenol on complex I activity and reactive oxygen species balance can contribute to the therapeutic effect of the drug.

Endothelial cells participate in inflammatory events leading to atherogenesis by regulating endothelial cell permeability via the expression of VE-Cadherin and β-catenin and leukocyte recruitment via the expression of E-Selectins and other adhesion molecules. The protein p66(Shc) acts as a sensor/inducer of oxidative stress and may promote vascular dysfunction. The objective of this study was to investigate the role of p66(Shc) in tumor necrosis factor TNFα-induced E-Selectin expression and function in human umbilical vein endothelial cells (HUVEC). Exposure of HUVEC to 50 ng/ml TNFα resulted in increased leukocyte transmigration through the endothelial monolayer and E-Selectin expression, in association with augmented phosphorylation of both p66(Shc) on Ser(36) and the stress kinase c-Jun NH2-terminal protein kinase (JNK)-1/2, and higher intracellular reactive oxygen species (ROS) levels. Overexpression of p66(Shc) in HUVEC resulted in enhanced p66(Shc) phosphorylation on Ser(36), increased ROS and E-Selectin levels, and amplified endothelial cell permeability and leukocyte transmigration through the HUVEC monolayer. Conversely, overexpression of a phosphorylation-defective p66(Shc) protein, in which Ser(36) was replaced by Ala, did not augment ROS and E-Selectin levels, nor modify cell permeability or leukocyte transmigration beyond those found in wild-type cells. Moreover, siRNA-mediated silencing of p66(Shc) resulted in marked reduction of E-Selectin expression and leukocyte transmigration. In conclusion, p66(Shc) acts as a novel intermediate in the TNFα pathway mediating endothelial dysfunction, and its action requires JNK-dependent phosphorylation of p66(Shc) on Ser(36).