Muscle-specific Drp1 overexpression impairs skeletal muscle growth via translational attenuation.

Mitochondrial fission and fusion are essential processes in the maintenance of the skeletal muscle function. The contribution of these processes to muscle development has not been properly investigated in vivo because of the early lethality of the models generated so far. To define the role of mitochondrial fission in muscle development and repair, we have generated a transgenic mouse line that overexpresses the fission-inducing protein Drp1 specifically in skeletal muscle. These mice displayed a drastic impairment in postnatal muscle growth, with reorganisation of the mitochondrial network and reduction of mtDNA quantity, without the deficiency of mitochondrial bioenergetics. Importantly we found that Drp1 overexpression activates the stress-induced PKR/eIF2α/Fgf21 pathway thus leading to an attenuated protein synthesis and downregulation of the growth hormone pathway. These results reveal for the first time how mitochondrial network dynamics influence muscle growth and shed light on aspects of muscle physiology relevant in human muscle pathologies.

Acid sphingomyelinase determines melanoma progression and metastatic behaviour via the microphtalmia-associated transcription factor signalling pathway.

Melanoma is a rapidly growing and highly metastatic cancer with high mortality rates, for which a resolutive treatment is lacking. Identification of novel therapeutic strategies and biomarkers of tumour stage is thus of particular relevance. We report here on a novel biomarker and possible candidate therapeutic target, the sphingolipid metabolising enzyme acid sphingomyelinase (A-SMase). A-SMase expression correlates inversely with tumour stage in human melanoma biopsies. Studies in a mouse model of melanoma and on cell lines derived from mouse and human melanomas demonstrated that A-SMase levels of expression actually determine the malignant phenotype of melanoma cells in terms of pigmentation, tumour progression, invasiveness and metastatic ability. The action of A-SMase is mediated by the activation of the extracellular signal-regulated kinase, the subsequent proteasomal degradation of the Microphtalmia-associated transcription factor (Mitf) and inhibition of cyclin-dependent kinase 2, Bcl-2 and c-Met, downstream targets of Mitf involved in tumour cell proliferation, survival and metastatisation.

Placental mitochondrial content and function in intrauterine growth restriction and preeclampsia.

Intrauterine growth restriction (IUGR) and pregnancy hypertensive disorders such as preeclampsia (PE) associated with IUGR share a common placental phenotype called "placental insufficiency", originating in early gestation when high availability of energy is required. Here, we assess mitochondrial content and the expression and activity of respiratory chain complexes (RCC) in placental cells of these pathologies. We measured mitochondrial (mt)DNA and nuclear respiratory factor 1 (NRF1) expression in placental tissue and cytotrophoblast cells, gene and protein expressions of RCC (real-time PCR and Western blotting) and their oxygen consumption, using the innovative technique of high-resolution respirometry. We analyzed eight IUGR, six PE, and eight uncomplicated human pregnancies delivered by elective cesarean section. We found lower mRNA levels of complex II, III, and IV in IUGR cytotrophoblast cells but no differences at the protein level, suggesting a posttranscriptional compensatory regulation. mtDNA was increased in IUGR placentas. Both mtDNA and NRF1 expression were instead significantly lower in their isolated cytotrophoblast cells. Finally, cytotrophoblast RCC activity was significantly increased in placentas of IUGR fetuses. No significant differences were found in PE placentas. This study provides genuine new data into the complex physiology of placental oxygenation in IUGR fetuses. The higher mitochondrial content in IUGR placental tissue is reversed in cytotrophoblast cells, which instead present higher mitochondrial functionality. This suggests different mitochondrial content and activity depending on the placental cell lineage. Increased placental oxygen consumption might represent a limiting step in fetal growth restriction, preventing adequate oxygen delivery to the fetus.

Nitric oxide and sphingolipids control apoptosis and autophagy with a significant impact on Alzheimer's disease.

Aberrant regulation of signalling pathways promoting and regulating apoptosis and autophagy contributes to the development of most human neurodegenerative diseases characterised by progressive dysfunction and death of neuronal and glial cells. Both in central and peripheral nervous systems cell death is either apoptotic or autophagic, depending on the cellular setting and the initial pathogenic cue. While some mixed phenotypes have been reported, apoptosis and autophagy tend to develop into mutually exclusive ways to such an extent that they inhibit each other. The sphingolipid ceramide is a key intracellular signalling molecule involved in many cellular processes leading to either survival or death; in most of these processes also the short-lived gaseous messenger nitric oxide (NO) plays a crucial role. The crosstalk between these two messengers and their downstream mediators has been thus extensively investigated and we now have a deep understanding of it and of its multiple feedback controls. What we provide here are details on how NO- and sphingolipid-dependent signalling and their crosstalk impact on degenerative brain diseases, in particular Alzheimer’s disease; we also describe how the ability of these molecules to regulate autophagy and apoptosis plays a significant role in determining the pathogenic evolution of these diseases. The evidence reported in this review suggests that targeting the NO and sphingolipid-dependent signalling pathways is worth exploiting in therapeutic perspective. In order to pursue these strategies, however, we still need to understand conclusively how the crosstalk between the NO and ceramide/sphingolipid pathways balances towards beneficial vs. toxic effects. In view of the nature of the signalling pathways involved and their multiple roles, the type of crosstalk involved is complex and intermingled with other signalling pathways.

Autophagy as a new therapeutic target in Duchenne muscular dystrophy.

A resolutive therapy for Duchene muscular dystrophy, a severe degenerative disease of the skeletal muscle, is still lacking. Because autophagy has been shown to be crucial in clearing dysfunctional organelles and in preventing tissue damage, we investigated its pathogenic role and its suitability as a target for new therapeutic interventions in Duchenne muscular dystrophy (DMD). Here we demonstrate that autophagy is severely impaired in muscles from patients affected by DMD and mdx mice, a model of the disease, with accumulation of damaged organelles. The defect in autophagy was accompanied by persistent activation via phosphorylation of Akt, mammalian target of rapamycin (mTOR) and of the autophagy-inhibiting pathways dependent on them, including the translation-initiation factor 4E-binding protein 1 and the ribosomal protein S6, and downregulation of the autophagy-inducing genes LC3, Atg12, Gabarapl1 and Bnip3. The defective autophagy was rescued in mdx mice by long-term exposure to a low-protein diet. The treatment led to normalisation of Akt and mTOR signalling; it also reduced significantly muscle inflammation, fibrosis and myofibre damage, leading to recovery of muscle function. This study highlights novel pathogenic aspects of DMD and suggests autophagy as a new effective therapeutic target. The treatment we propose can be safely applied and immediately tested for efficacy in humans.

Nitric oxide inhibition of Drp1-mediated mitochondrial fission is critical for myogenic differentiation.

During myogenic differentiation the short mitochondria of myoblasts change into the extensively elongated network observed in myotubes. The functional relevance and the molecular mechanisms driving the formation of this mitochondrial network are unknown. We now show that mitochondrial elongation is required for myogenesis to occur and that this event depends on the cellular generation of nitric oxide (NO). Inhibition of NO synthesis in myogenic precursor cells leads to inhibition of mitochondrial elongation and of myogenic differentiation. This is due to the enhanced activity, translocation and docking of the pro-fission GTPase dynamin-related protein-1 (Drp1) to mitochondria, leading also to a latent mitochondrial dysfunction that increased sensitivity to apoptotic stimuli. These effects of NO inhibition were not observed in myogenic precursor cells containing a dominant-negative form of Drp1. Both NO-dependent repression of Drp1 action and maintenance of mitochondrial integrity and function were mediated through the soluble guanylate cyclase. These data uncover a novel level of regulation of differentiation linking mitochondrial morphology and function to myogenic differentiation.

Effective salvage chemotherapy in relapsed or refractory non-Hodgkin's lymphoma.

BACKGROUND: Over the last few years, high-dose chemotherapy has been extensively investigated in relapsing/refractory non-Hodgkin's lymphoma (NHL). However, this approach is reserved to a limited subset of cases and new conventional-dose second-line chemotherapies need to be investigated. PATIENTS AND METHODS: Thirty consecutive out-patients with refractory or recurrent NHL were given polychemotherapy in a regimen consisting of ifosfamide, mitoxantrone and etoposide on day 1 and vindesine, cisplatinum and cytosine arabinoside on day 15: courses were repeated every 29 days. Five patients had refractory disease following first-line chemotherapy and 25 were relapsing. RESULTS: The median number of administered cycles was 4 (range 2-8). We observed 16 complete (53%; 95% confidence interval, 34%-72%) and 3 partial remissions, for an overall remission rate of 63% (95% confidence interval, 44%-80%). Responses were seen only among patients who achieved at least a partial response during first-line therapy. The median duration of complete remission was 15 months (range 5-47+), whereas median survival of the treated patients was 26 months (range 2-50+). Five patients were long-term responders after 34+, 35+, 46+, 46+ and 47+ months. No-life threatening toxicity was observed. The main side effects were myelosuppression, nausea/vomiting and alopecia. CONCLUSIONS: The proposed regimen is feasible and effective in terms of complete remission rate and disease-free survival, suggesting that this treatment may be potentially curative in a subgroup of relapsed patients with limited tumor burden and normal LDH values. A more aggressive approach is needed in refractory patients.

Assessment of myocardial oxidative stress in patients after myocardial revascularization.

A homogeneous group of six patients, who underwent coronary artery bypass surgery, was studied to determine the presence of oxidative stress caused by oxygen-derived free radicals and its relationship with reperfusion cell damage. Biopsies were performed before ischemia and 10 minutes after reperfusion. The samples were assayed for hydroperoxide-initiated chemiluminescence and histochemical succinic dehydrogenase activity; the specimens were also studied by electron microscopy. The preischemic biopsy specimens showed chemiluminescence of 40 +/- 2 (cpm/mg protein) x 10(3), normal succinic dehydrogenase activity (grade 4), and generally preserved ultrastructure (necrotic/normal cells 5/100). However, the reperfusion biopsy specimens showed an increase in chemiluminescence to 91 +/- 19 (cpm/mg protein) x 10(3) (p less than 0.025), a partial loss of enzymatic activity (grade 2.6), and ultrastructural changes characterized by mitochondrial swelling and focal myofibrillar disorganization (necrotic/normal cells: 15/100; p less than 0.001). These observations seem to indicate the presence of oxidative stress during reoxygenation, a situation that may play a major role in the genesis of reperfusion injury. It appears to be the first observation relating free radical-induced oxidative stress to reperfusion injury in humans.

[Electrolytic changes in febrile convulsions]. / Alterazioni elettrolitiche in corso di convulsioni febbrili.

The authors valued some metabolic parameters (sodium, potassium, calcium, blood levels of glucose, proteins, nitrogen, creatinine) in 159 children with febrile convulsions and compared the results with those of a group of children with fever (50 subjects) and of a group of health, fever free children (50 subjects). In the patients with febrile convulsions serum levels of sodium, calcium and osmolarity resulted significantly lower than those obtained in both control groups. The electrolytic modification (overall hyponatremia, probably due to a SIADH) may have a role in short-term relapses of febrile convulsions. Hyponatremia is present also in some children with high fever, without seizures; it may be that hyponatremia, in predisposed subjects, lower the threshold of neuromuscular excitability.