Mitochondria, Oxidative Stress, cAMP Signalling and Apoptosis: A Crossroads in Lymphocytes of Multiple Sclerosis, a Possible Role of Nutraceutics

Multiple sclerosis (MS) is a complex inflammatory and neurodegenerative chronic disease that involves the immune and central nervous systems (CNS). The pathogenesis involves the loss of blood–brain barrier integrity, resulting in the invasion of lymphocytes into the CNS with consequent tissue damage. The MS etiology is probably a combination of immunological, genetic, and environmental factors. It has been proposed that T lymphocytes have a main role in the onset and propagation of MS, leading to the inflammation of white matter and myelin sheath destruction. Cyclic AMP (cAMP), mitochondrial dysfunction, and oxidative stress exert a role in the alteration of T lymphocytes homeostasis and are involved in the apoptosis resistance of immune cells with the consequent development of autoimmune diseases. The defective apoptosis of autoreactive lymphocytes in patients with MS, allows these cells to perpetuate, within the CNS, a continuous cycle of inflammation. In this review, we discuss the involvement in MS of cAMP pathway, mitochondria, reactive oxygen species (ROS), apoptosis, and their interaction in the alteration of T lymphocytes homeostasis. In addition, we discuss a series of nutraceutical compounds that could influence these aspect

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

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

PBMC of Multiple Sclerosis Patients Show Deregulation of OPA1 Processing Associated with Increased ROS and PHB2 Protein Levels

Multiple sclerosis (MS) is an autoimmune disease in which activated lymphocytes affect the central nervous system. Increase of reactive oxygen species (ROS), impairment of mitochondria-mediated apoptosis and mitochondrial alterations have been reported in peripheral lymphocytes of MS patients. Mitochondria-mediated apoptosis is regulated by several mechanisms and proteins. Among others, optic atrophy 1 (OPA1) protein plays a key role in the regulating mitochondrial dynamics, cristae architecture and release of pro-apoptotic factors. Very interesting, mutations in OPA1 gene, have been associated with multiple sclerosis-like disorder. We have analyzed OPA1 and some factors involved in its regulation. Fifteen patients with MS and fifteen healthy control subjects (HC) were enrolled into the study and peripheral blood mononuclear cells (PBMCs) were isolated. H2O2 level was measured spectrofluorimetrically, OPA1, PHB2, SIRT3, and OMA1 were analyzed by western blotting. Statistical analysis was performed using Student’s t-test. The results showed that PBMC of MS patients were characterized by a deregulation of OPA1 processing associated with increased H2O2 production, inactivation of OMA1 and increase of PHB2 protein level. The presented data suggest that the alteration of PHB2, OMA1, and OPA1 processing could be involved in resistance towards apoptosis. These molecular parameters could also be useful to assess disease activity

Resveratrol Treatment in Human Parkin-Mutant Fibroblasts Modulates cAMP and Calcium Homeostasis Regulating the Expression of Mitochondria-Associated Membranes Resident Proteins

Parkin plays an important role in ensuring efficient mitochondrial function and calcium homeostasis. Parkin-mutant human fibroblasts, with defective oxidative phosphorylation activity, showed high basal cAMP level likely ascribed to increased activity/expression of soluble adenylyl cyclase and/or low expression/activity of the phosphodiesterase isoform 4 and to a higher Ca2+ level. Overall, these findings support the existence, in parkin-mutant fibroblasts, of an abnormal Ca2+ and cAMP homeostasis in mitochondria. In our previous studies resveratrol treatment of parkin-mutant fibroblasts induced a partial rescue of mitochondrial functions associated with stimulation of the AMPK/SIRT1/PGC-1α pathway. In this study we provide additional evidence of the potential beneficial effects of resveratrol inducing an increase in the pre-existing high Ca2+ level and remodulation of the cAMP homeostasis in parkin-mutant fibroblasts. Consistently, we report in these fibroblasts higher expression of proteins implicated in the tethering of ER and mitochondrial contact sites along with their renormalization after resveratrol treatment. On this basis we hypothesize that resveratrol-mediated enhancement of the Ca2+ level, fine-tuned by the ER–mitochondria Ca2+ crosstalk, might modulate the pAMPK/AMPK pathway in parkin-mutant fibroblasts

Optimizing enzymatic photo‐redox cycles by a hybrid protein complex chain

The construction from scratch of artificial cells by means of a "bottom up" approach is one of the most ambitious challenges in synthetic biology. Artificial cells capable of imitating the light phase of photosynthesis can be considered photoautotrophs. In bacterial photosynthesis, the first step in the light energy transduction process is the enzymatic photo-redox cycle catalysed by two membrane protein complexes, that is, the photosynthetic reaction centre (RC) and the ubiquinol oxidase (bc1(B)). In this work we studied this process in a micellar suspension of both proteins but coupling a bacterial RC with an ortholog bc1 extracted from mammalian mitochondria (bc1(M)). With this hybrid protein complex chain, the light transduction efficiency turns out to be enhanced up to 90 % by tuning the enzymatic level ratio of the two protein complexes. These results pave the way towards the reconstitution of the entire photosynthetic machinery in artificial membranes for the realization of photoautotrophic artificial cells

Influence of melanocortin-1 receptor (MC1R) polymorphisms on clinical outcomes of patients with metastatic melanoma harboring the BRAF mutation and treated with BRAF inhibitors

Background: In the pigmentation of hair and skin a key role is played by the MC1R gene whose inherited variations are linked with a higher melanoma risk. In melanoma cells, MC1R has a central role because of its interaction with several intracellular signaling cascades such as the MAPK or mTOR pathways. Little heed has been paid to the role of its genetic polymorphisms in the management of metastatic melanoma. Our aim was to evaluate the effects of MC1R variants on the clinical outcomes of BRAF mutated patients treated with BRAF inhibitors. Methods: Fifty-three consecutive patients were evaluated according to their MC1R status (21 wild type and 32 with minor/major functional variants). First we explored eventual association between MC1R status and basal patient features. Than we evaluated the influence of MC1R status on therapeutic outcomes to anti BRAF inhibitors (ORR, PFS, OS). Finally, we studied the effect of vemurafenib in 2 BRAF V600 melanoma cell lines with and without MC1R variant. Results: Regarding the baseline patient features, we only found an association between MC1R polymorphisms and bone metastases (p 0.017). Moreover, MC1R variants were significantly associated with worse outcomes to BRAF inhibitors compared with MC1R wild type in terms of both ORR (59% vs 95% [p 0.011 in univariate, p 0.028 in multivariate analysis]) and PFS shorter than 6 months (72% vs 33% [p 0.012 in univariate, p 0.027 in multivariate analysis]). No significant difference was found in OS, probably due to the influence of further treatments. Data in vitro demonstrated a significantly different phosphorylation of Erk1/2 and p38 MAPK before and after treatment, associated with a greater increase of vemurafenib IC50 in cases of MC1R variant. Conclusions: Our data document, for the first time, detrimental effects of MC1R variants on BRAF inhibitor therapy. Moreover, these evidences concur to understand the resistance mechanisms to targeted therapy and may help to pinpoint new therapeutic strategies