PEL is a B-cell non-Hodgkin lymphoma, occurring predominantly as a lymphomatous effusion in body cavities, characterized by aggressive clinical course, with no standard therapy. Based on previous reports that PEL cells display a Warburg phenotype, we hypothesized that the highly hypoxic environment in which they grow in vivo makes them more reliant on glycolysis, and more vulnerable to drugs targeting this pathway. We established here that indeed PEL cells in hypoxia are more sensitive to glycolysis inhibition. Furthermore, since PI3K/Akt/mTOR has been proposed as a drug target in PEL, we ascertained that pathway-specific inhibitors, namely the dual PI3K and mTOR inhibitor, PF-04691502, and the Akt inhibitor, Akti 1/2, display improved cytotoxicity to PEL cells in hypoxic conditions. Unexpectedly, we found that these drugs reduce lactate production/extracellular acidification rate, and, in combination with the glycolysis inhibitor 2-deoxyglucose (2-DG), they shift PEL cells metabolism from aerobic glycolysis towards oxidative respiration. Moreover, the associations possess strong synergistic cytotoxicity towards PEL cells, and thus may reduce adverse reaction in vivo, while displaying very low toxicity to normal lymphocytes. Finally, we showed that the association of 2-DG and PF-04691502 maintains its cytotoxic and proapoptotic effect also in PEL cells co-cultured with human primary mesothelial cells, a condition known to mimic the in vivo environment and to exert a protective and pro-survival action. All together, these results provide a compelling rationale for the clinical development of new therapies for the treatment of PEL, based on combined targeting of glycolytic metabolism and constitutively activated signaling pathways.

STAT3 is an oncogenic transcription factor exerting its functions both as a canonical transcriptional activator and as a non-canonical regulator of energy metabolism and mitochondrial functions. While both activities are required for cell transformation downstream of different oncogenic stimuli, they rely on different post-translational activating events, namely phosphorylation on either Y705 (nuclear activities) or S727 (mitochondrial functions). Here, we report the discovery of the unexpected STAT3 localization to the endoplasmic reticulum (ER), from where it modulates ER-mitochondria Ca2+ release by interacting with the Ca2+ channel IP3R3 and facilitating its degradation. The release of Ca2+ is of paramount importance for life/death cell decisions, as excessive Ca2+ causes mitochondrial Ca2+ overload, the opening of the mitochondrial permeability transition pore, and the initiation of the intrinsic apoptotic program. Indeed, STAT3 silencing enhances ER Ca2+ release and sensitivity to apoptosis following oxidative stress in STAT3-dependent mammary tumor cells, correlating with increased IP3R3 levels. Accordingly, basal-like mammary tumors, which frequently display constitutively active STAT3, show an inverse correlation between IP3R3 and STAT3 protein levels. These results suggest that STAT3-mediated IP3R3 downregulation in the ER crucially contributes to its anti-apoptotic functions via modulation of Ca2+ fluxes.

Mutations in the inositol 5-phosphatase OCRL cause Lowe syndrome and Dent's disease. Although OCRL, a direct clathrin interactor, is recruited to late-stage clathrin-coated pits, clinical manifestations have been primarily attributed to intracellular sorting defects. Here we show that OCRL loss in Lowe syndrome patient fibroblasts impacts clathrin-mediated endocytosis and results in an endocytic defect. These cells exhibit an accumulation of clathrin-coated vesicles and an increase in U-shaped clathrin-coated pits, which may result from sequestration of coat components on uncoated vesicles. Endocytic vesicles that fail to lose their coat nucleate the majority of the numerous actin comets present in patient cells. SNX9, an adaptor that couples late-stage endocytic coated pits to actin polymerization and which we found to bind OCRL directly, remains associated with such vesicles. These results indicate that OCRL acts as an uncoating factor and that defects in clathrin-mediated endocytosis likely contribute to pathology in patients with OCRL mutations.

The mitochondrial permeability transition pore (mPTP) has long been known to have a role in mitochondrial calcium (Ca(2+)) homeostasis under pathological conditions as a mediator of the mitochondrial permeability transition and the activation of the consequent cell death mechanism. However, its role in the context of mitochondrial Ca(2+) homeostasis is not yet clear. Several studies that were based on PPIF inhibition or knock out suggested that mPTP is involved in the Ca(2+) efflux mechanism, while other observations have revealed the opposite result. The c subunit of the mitochondrial F1/FO ATP synthase has been recently found to be a fundamental component of the mPTP. In this work, we focused on the contribution of the mPTP in the Ca(2+) efflux mechanism by modulating the expression of the c subunit. We observed that forcing mPTP opening or closing did not impair mitochondrial Ca(2+) efflux. Therefore, our results strongly suggest that the mPTP does not participate in mitochondrial Ca(2+) homeostasis in a physiological context in HeLa cells.

Activation by extracellular ligands of G protein-coupled (GPCRs) and tyrosine kinase receptors (RTKs), results in the generation of second messengers that in turn control specific cell functions. Further, modulation/amplification or inhibition of the initial signalling events, depend on the recruitment onto the plasma membrane of soluble protein effectors.High throughput methodologies to monitor quantitatively second messenger production, have been developed over the last years and are largely used to screen chemical libraries for drug development. On the contrary, no such high throughput methods are yet available for the other aspect of GPCRs regulation, i.e. protein translocation to the plasma membrane, despite the enormous interest of this phenomenon for the modulation of receptor downstream functions. Indeed, to date, the experimental procedures available are either inadequate or complex and expensive.

Malignant mesothelioma (MM) is a very aggressive type of cancer, with a dismal prognosis and inherent resistance to chemotherapeutics. Development and evaluation of new therapeutic approaches is highly needed. Immunosuppressant FTY720, approved for multiple sclerosis treatment, has recently raised attention for its anti-tumor activity in a variety of cancers. However, its therapeutic potential in MM has not been evaluated yet.

Gait and mobility impairments affect the quality of life (QoL) of patients with progressive multiple sclerosis (MS). Robot-assisted gait training (RAGT) is an effective rehabilitative treatment but evidence of its superiority compared to other options is lacking. Furthermore, the response to rehabilitation is multidimensional, person-specific and possibly involves functional reorganization processes. The aims of this study are: (1) to test the effectiveness on gait speed, mobility, balance, fatigue and QoL of RAGT compared to conventional therapy (CT) in progressive MS and (2) to explore changes of clinical and circulating biomarkers of neural plasticity.

Tissue engineering-based bone graft is an emerging viable treatment modality to repair and regenerate tissues damaged as a result of diseases or injuries. The structure and composition of scaffolds should modulate the classical osteogenic pathways in human stem cells. The osteoinductivity properties of the hydroxylapatite-collagen hybrid scaffold named Coll/Pro Osteon 200 were investigated in an in vitro model of human adipose mesenchymal stem cells (hASCs), whereas the clinical evaluation was carried out in maxillofacial patients. Differentially expressed genes (DEGs) induced by the scaffold were analyzed using the Osteogenesis RT2 PCR Array. The osteoinductivity potential of the scaffold was also investigated by studying the alkaline phosphatase (ALP) activity, matrix mineralization, osteocalcin (OCN), and CLEC3B expression protein. Fifty patients who underwent zygomatic augmentation and bimaxillary osteotomy were evaluated clinically, radiologically, and histologically during a 3-year follow-up. Among DEGs, osteogenesis-related genes, including BMP1/2, ALP, BGLAP, SP7, RUNX2, SPP1, and EGFR, which play important roles in osteogenesis, were found to be upregulated. The genes to cartilage condensation SOX9, BMPR1B, and osteoclast cells TNFSF11 were detected upregulated at every time point of the investigation. This scaffold has a high osteoinductivity revealed by the matrix mineralization, ALP activity, OCN, and CLEC3B expression proteins. Clinical evaluation evidences that the biomaterial promotes bone regrowth. Histological results of biopsy specimens from patients showed prominent ossification. Experimental data using the Coll/Pro Osteon 200 indicate that clinical evaluation of bone regrowth in patients, after scaffold implantation, was supported by DEGs implicated in skeletal development as shown in "in vitro" experiments with hASCs.

Dementia is a neurocognitive disorder characterized by a progressive memory loss and impairment in cognitive and functional abilities. Autophagy and mitophagy are two important cellular processes by which the damaged intracellular components are degraded by lysosomes. To investigate the contribution of autophagy and mitophagy in degenerative diseases, we investigated the serum levels of specific autophagic markers (ATG5 protein) and mitophagic markers (Parkin protein) in a population of older patients by enzyme-linked immunosorbent assay. Two hundred elderly (≥65 years) outpatients were included in the study: 40 (20 F and 20 M) with mild-moderate late onset Alzheimer's disease (AD); 40 (20 F and 20 M) affected by vascular dementia (VAD); 40 with mild cognitive impairment (MCI); 40 (20 F and 20 M) with "mixed" dementia (MD); 40 subjects without signs of cognitive impairment were included as sex-matched controls. Our data indicated that, in serum samples, ATG5 and Parkin were both elevated in controls, and that VAD compared with AD, MCI and MD (all p < 0.01). Patients affected by AD, MD, and MCI showed significantly reduced circulating levels of both ATG5 and Parkin compared to healthy controls and VAD individuals, reflecting a significant down-regulation of autophagy and mitophagy pathways in these groups of patients. The measurement of serum levels of ATG5 and Parkin may represent an easily accessible diagnostic tool for the early monitoring of patients with cognitive decline.

FBXL2 targets IP3R3 for ubiquitin-mediated degradation to limit Ca2+ flux to mitochondria and, consequently, apoptosis. Efficient replication of hepatitis C virus (HCV) requires geranylgeranylation of FBXL2. Here, we show that the viral protein NS5A forms a trimeric complex with IP3R3 and FBXL2, unmasking IP3R3's degron in the absence of inositol 1,4,5-trisphosphate (IP3) stimulation. FBXL2 knockdown or expression of a stable IP3R3 mutant causes persistent Ca2+ flux and sensitizes cells to apoptosis, resulting in the inhibition of viral replication. Importantly, the effect of FBXL2 silencing is rescued by depleting IP3R3, but not p85β, another established FBXL2 substrate, indicating that the anti-HCV effect of FBXL2 knockdown is largely due to IP3R3 stabilization. Finally, disruption of the FBXL2-NS5A-IP3R3 complex using somatic cell genetics or pharmacologic inhibition results in IP3R3 stabilization and suppression of HCV replication. This study reveals an IP3-independent molecular mechanism through which HCV promotes IP3R3 degradation, thereby inhibiting virus-induced apoptosis and establishing chronic infection.

Mitochondria are dynamic organelles that have essential metabolic activity and are regarded as signalling hubs with biosynthetic, bioenergetics and signalling functions that orchestrate key biological pathways. However, mitochondria can influence all processes linked to oncogenesis, starting from malignant transformation to metastatic dissemination. In this review, we describe how alterations in the mitochondrial metabolic status contribute to the acquisition of typical malignant traits, discussing the most recent discoveries and the many unanswered questions. We also highlight that expanding our understanding of mitochondrial regulation and function mechanisms in the context of cancer cell metabolism could be an important task in biomedical research, thus offering the possibility of targeting mitochondria for the treatment of cancer.

SEPN1-related myopathy (SEPN1-RM) is a muscle disorder due to mutations of the SEPN1 gene, which is characterized by muscle weakness and fatigue leading to scoliosis and life-threatening respiratory failure. Core lesions, focal areas of mitochondria depletion in skeletal muscle fibers, are the most common histopathological lesion. SEPN1-RM underlying mechanisms and the precise role of SEPN1 in muscle remained incompletely understood, hindering the development of biomarkers and therapies for this untreatable disease. To investigate the pathophysiological pathways in SEPN1-RM, we performed metabolic studies, calcium and ATP measurements, super-resolution and electron microscopy on in vivo and in vitro models of SEPN1 deficiency as well as muscle biopsies from SEPN1-RM patients. Mouse models of SEPN1 deficiency showed marked alterations in mitochondrial physiology and energy metabolism, suggesting that SEPN1 controls mitochondrial bioenergetics. Moreover, we found that SEPN1 was enriched at the mitochondria-associated membranes (MAM), and was needed for calcium transients between ER and mitochondria, as well as for the integrity of ER-mitochondria contacts. Consistently, loss of SEPN1 in patients was associated with alterations in body composition which correlated with the severity of muscle weakness, and with impaired ER-mitochondria contacts and low ATP levels. Our results indicate a role of SEPN1 as a novel MAM protein involved in mitochondrial bioenergetics. They also identify a systemic bioenergetic component in SEPN1-RM and establish mitochondria as a novel therapeutic target. This role of SEPN1 contributes to explain the fatigue and core lesions in skeletal muscle as well as the body composition abnormalities identified as part of the SEPN1-RM phenotype. Finally, these results point out to an unrecognized interplay between mitochondrial bioenergetics and ER homeostasis in skeletal muscle. They could therefore pave the way to the identification of biomarkers and therapeutic drugs for SEPN1-RM and for other disorders in which muscle ER-mitochondria cross-talk are impaired.

The precise molecular mechanisms that coordinate apoptosis and autophagy in cancer remain to be determined. Here, we provide evidence that the tumor suppressor promyelocytic leukemia protein (PML) controls autophagosome formation at mitochondria-associated membranes (MAMs) and, thus, autophagy induction. Our in vitro and in vivo results demonstrate how PML functions as a repressor of autophagy. PML loss promotes tumor development, providing a growth advantage to tumor cells that use autophagy as a cell survival strategy during stress conditions. These findings demonstrate that autophagy inhibition could be paired with a chemotherapeutic agent to develop anticancer strategies for tumors that present PML downregulation.

Abnormal levels of pyruvate and lactate were reported in multiple sclerosis (MS). We studied the response of markers of mitochondrial function to rehabilitation in relation to type, intensity and endurance performance in severely disabled MS patients. Forty-six progressive MS patients were randomized to receive 12 walking sessions of robot-assisted gait training (RAGT, n = 23) or conventional overground therapy (CT, n = 23). Ten healthy subjects were also studied. Blood samples were collected to determine lactate, pyruvate, and glutathione levels and lactate/pyruvate ratio pre-post rehabilitation. In vivo muscle metabolism and endurance walking capacity were assessed by resting muscle oxygen consumption (rmVO2) using near-infrared spectroscopy and by six-minute walking distance (6MWD), respectively. The levels of mitochondrial biomarkers and rmVO2, altered at baseline with respect to healthy subjects, improved after rehabilitation in the whole population. In the two groups, an enhanced response was observed after RAGT compared to CT for lactate (p = 0.012), glutathione (<0.001), lactate/pyruvate ratio (p = 0.08) and rmVO2 (p = 0.07). Metabolic biomarkers and 6MWD improvements were exclusively correlated with a training speed markedly below individual gait speed. In severely disabled MS patients, rehabilitation rebalanced altered serum metabolic and muscle parameters, with RAGT being more effective than CT. A determinable slow training speed was associated with better metabolic and functional recovery. Trial Registration: ClinicalTrials.gov NCT02421731.

Preclinical models of ischemia/reperfusion injury (RI) demonstrate the deleterious effects of permeability transition pore complex (PTPC) opening in the first minutes upon revascularization of the occluded vessel. The ATP synthase c subunit (Csub) influences PTPC activity in cells, thus impacting tissue injury. A conserved glycine-rich domain in Csub is classified as critical because, when mutated, it modifies ATP synthase properties, protein interaction with the mitochondrial calcium (Ca2+) uniporter complex, and the conductance of the PTPC. Here, we document the role of a naturally occurring mutation in the Csub-encoding ATP5G1 gene at the G87 position found in two ST-segment elevation myocardial infarction (STEMI) patients and how PTPC opening is related to RI in patients affected by the same disease. We report a link between the expression of ATP5G1G87E and the response to hypoxia/reoxygenation of human cardiomyocytes, which worsen when compared to those expressing the wild-type protein, and a positive correlation between PTPC and RI.

Membrane contact sites between virtually any known organelle have been documented and, in the last decades, their study received momentum due to their importance for fundamental activities of the cell and for the subtle comprehension of many human diseases. The lack of tools to finely image inter-organelle proximity hindered our understanding on how these subcellular communication hubs mediate and regulate cell homeostasis. We develop an improved and expanded palette of split-GFP-based contact site sensors (SPLICS) for the detection of single and multiple organelle contact sites within a scalable distance range. We demonstrate their flexibility under physiological conditions and in living organisms.

Leber's hereditary optic neuropathy (LHON), a disease associated with a mitochondrial DNA mutation, is characterized by blindness due to degeneration of retinal ganglion cells (RGCs) and their axons, which form the optic nerve. We show that a sustained pathological autophagy and compartment-specific mitophagy activity affects LHON patient-derived cells and cybrids, as well as induced pluripotent-stem-cell-derived neurons. This is variably counterbalanced by compensatory mitobiogenesis. The aberrant quality control disrupts mitochondrial homeostasis as reflected by defective bioenergetics and excessive reactive oxygen species production, a stress phenotype that ultimately challenges cell viability by increasing the rate of apoptosis. We counteract this pathological mechanism by using autophagy regulators (clozapine and chloroquine) and redox modulators (idebenone), as well as genetically activating mitochondrial biogenesis (PGC1-α overexpression). This study substantially advances our understanding of LHON pathophysiology, providing an integrated paradigm for pathogenesis of mitochondrial diseases and druggable targets for therapy.

Selenoprotein N (SELENON) is an endoplasmic reticulum (ER) protein whose loss of function leads to human SELENON-related myopathies. SelenoN knockout (KO) mouse limb muscles, however, are protected from the disease, and display no major alterations in muscle histology or contractile properties. Interestingly, we find that the highly active diaphragm muscle shows impaired force production, in line with the human phenotype. In addition, after repeated stimulation with a protocol which induces muscle fatigue, also hind limb muscles show altered relaxation times. Mechanistically, muscle SELENON loss alters activity-dependent calcium handling selectively impinging on the Ca2+ uptake of the sarcoplasmic reticulum and elicits an ER stress response, including the expression of the maladaptive CHOP-induced ERO1. In SELENON-devoid models, ERO1 shifts ER redox to a more oxidised poise, and further affects Ca2+ uptake. Importantly, CHOP ablation in SelenoN KO mice completely prevents diaphragm dysfunction, the prolonged limb muscle relaxation after fatigue, and restores Ca2+ uptake by attenuating the induction of ERO1. These findings suggest that SELENON is part of an ER stress-dependent antioxidant response and that the CHOP/ERO1 branch of the ER stress response is a novel pathogenic mechanism underlying SELENON-related myopathies.

Mutations in C19orf12 have been identified in patients affected by Neurodegeneration with Brain Iron Accumulation (NBIA), a clinical entity characterized by iron accumulation in the basal ganglia. By using western blot analysis with specific antibody and confocal studies, we showed that wild-type C19orf12 protein was not exclusively present in mitochondria, but also in the Endoplasmic Reticulum (ER) and MAM (Mitochondria Associated Membrane), while mutant C19orf12 variants presented a different localization. Moreover, after induction of oxidative stress, a GFP-tagged C19orf12 wild-type protein was able to relocate to the cytosol. On the contrary, mutant isoforms were not able to respond to oxidative stress. High mitochondrial calcium concentration and increased H2O2 induced apoptosis were found in fibroblasts derived from one patient as compared to controls. C19orf12 protein is a 17 kDa mitochondrial membrane-associated protein whose function is still unknown. Our in silico investigation suggests that, the glycine zipper motifs of C19orf12 form helical regions spanning the membrane. The N- and C-terminal regions with respect to the transmembrane portion, on the contrary, are predicted to rearrange in a structural domain, which is homologs to the N-terminal regulatory domain of the magnesium transporter MgtE, suggesting that C19orf12 may act as a regulatory protein for human MgtE transporters. The mutations here described affect respectively one glycine residue of the glycine zipper motifs, which are involved in dimerization of transmembrane helices and predicted to impair the correct localization of the protein into the membranes, and one residue present in the regulatory domain, which is important for protein-protein interaction.

Mounting evidence links neurodegenerative disorders such as Parkinson disease and Alzheimer disease with mitochondrial dysfunction, and recent emphasis has focused on mitochondrial dynamics and quality control. Mitochondrial dynamics and mtDNA maintenance is another link recently emerged, implicating mutations in the mitochondrial fusion genes OPA1 and MFN2 in the pathogenesis of multisystem syndromes characterized by neurodegeneration and accumulation of mtDNA multiple deletions in postmitotic tissues. Here, we report 2 Italian families affected by dominant chronic progressive external ophthalmoplegia (CPEO) complicated by parkinsonism and dementia.