PERSISTENT INHIBITION OF MITOCHONDRIAL BIOGENESIS IN DYSTROPHIC MICE: IDENTIFICATION OF NITRIC OXIDE-DEPENDENT SALVAGE PATHWAY

Muscular dystrophies are a group of genetic diseases showing muscle degeneration characterized by progressive skeletal muscle weakness, defects in muscle proteins fiber necrosis, and progressive substitution of fibers with connective and adipose tissue. The therapeutic protocols currently in use, based on corticosteroid administration, provide some delay in the progression of the disease, but they are associated with severe side effects. The genetic approaches (exon skipping and antisense oligonucleotides) currently being investigated show some degree of success, however they are directed to specific subsets of population and cannot restore fully the damage already caused by the disease to the muscle. Several studies had demonstrate that the pathophysiology of muscular dystrophies correlates with an altered synthesis of nitric oxide (NO), in fact neuronal nitric oxide synthase (nNOS) is absent from the sarcolemma and relocated to the cytosol, with total muscle NOS activity being thus reduced. During the years, nitric oxide donors were identified as good candidate molecules for Duchenne Muscular dystrophy therapy and recently, our group found that a NO donor, molsidomine, is able to slow disease progression and to restore the functional capacity of damaged muscle, significantly enhancing spontaneous and forced motor activities1. Nitric oxide regulates some mitochondrial functions, such as morphology and complexes activity; moreover mitochondrial dysfunction has long been suspected to be an important pathogenetic feature in muscular dystrophies even if their role is not fully understood. The aim of this project is to analyze the mitochondrial profile of alpha-Sarcoglican-null (a-SG) mice, a mouse model for Limb Girdle muscular dystrophy 2D (LGMD 2D) and to evaluate a possible effect of molsidomine on mitochondrial function. Our long term aim is to define approaches that limit muscle wasting, with a dual finality, on the one hand to ameliorate the dystrophic symptoms per se, on the other to increase the efficiency of cell/gene therapies, in a combined therapy for the disease. To this end elucidation of novel possible targets is necessary and this is the final goal of this project. We find out a severe reduction in mitochondrial content in both tibialis anterior and diaphragm accounting for a lower OxPhos capacity of these muscles. The respiratory rates relative to mitochondrial DNA suggest that mitochondrial content is the major determinant of the lower oxidative capacity of a-SG null muscles. The low mitochondrial content in dystophyc mice is due to a persistent inhibition of the mitochondrial biogenesis pathway. Unexpectedly, the treatment with the NO-donor molsidomine is not able to restore mitochondrial content in a-SG-/- mice, but it is able to improve significantly their oxidative capacity, triggering a therapeutic fiber switch and stimulating fatty acid oxidation rather than improving mitochondrial function per se. Molsidomine promotes in fact an important deacetylation and activation of peroxisome proliferator-activated receptor \u3b3 coactivator 1-gene \u3b1 (PGC-1\u3b1), the principal trascriptional co-activator involved in muscle fiber type determination. Deacetylation of PGC-1\u3b1 occurs through a nitric oxide-dependent AMP activated protein kinase (APMK) activation leading to an increase expression and activity of the deacetylase Sirt1. Altogether these results highlight for the first time a defective mitochondrial biogenesis in LGMD 2D impairing mitochondrial metabolism and define the increase in OxPhos capacity associated with fiber switch as rescue mechanism with a mechanism independent on mitochondrial biogenesis but focused on lipid metabolism

2-semiarcs in PG(2, q), q <= 13

A 2-semiarc is a pointset S-2 with the property that the number of tangent lines to S-2 at each of its points is two. Using some theoretical results and computer aided search, the complete classification of 2-semiarcs in PG(2, q) is given for q <= 7, the spectrum of their sizes is determined for q <= 9, and some results about the existence are proven for q = 11 and q = 13. For several sizes of 2-semiarcs in PG(2, q), q <= 7, classification results have been obtained by theoretical proofs

Pre-clinical validation of a selective anti-cancer stem cell therapy for Numb-deficient human breast cancers

The cell fate determinant Numb is frequently downregulated in human breast cancers (BCs), resulting in p53 inactivation and an aggressive disease course. In the mouse mammary gland, Numb/p53 downregulation leads to aberrant tissue morphogenesis, expansion of the stem cell compartment, and emergence of cancer stem cells (CSCs). Strikingly, CSC phenotypes in a Numb-knockout mouse model can be reverted by Numb/p53 restoration. Thus, targeting Numb/p53 dysfunction in Numb-deficient human BCs could represent a novel anti-CSC therapy. Here, using patient-derived xenografts, we show that expansion of the CSC pool, due to altered self-renewing divisions, is also a feature of Numb-deficient human BCs. In these cancers, using the inhibitor Nutlin-3 to restore p53, we corrected the defective self-renewal properties of Numb-deficient CSCs and inhibited CSC expansion, with a marked effect on tumorigenicity and metastasis. Remarkably, a regimen combining Nutlin-3 and chemotherapy induced persistent tumor growth inhibition, or even regression, and prevented CSC-driven tumor relapse after removal of chemotherapy. Our data provide a pre-clinical proof-of-concept that targeting Numb/p53 results in a specific anti-CSC therapy in human BCs

Insulin resistance in type 1 diabetes: what is ‘double diabetes’ and what are the risks?

In this review, we explore the concept of ‘double diabetes’, a combination of type 1 diabetes with features of insulin resistance and type 2 diabetes. After considering whether double diabetes is a useful concept, we discuss potential mechanisms of increased insulin resistance in type 1 diabetes before examining the extent to which double diabetes might increase the risk of cardiovascular disease (CVD). We then go on to consider the proposal that weight gain from intensive insulin regimens may be associated with increased CV risk factors in some patients with type 1 diabetes, and explore the complex relationships between weight gain, insulin resistance, glycaemic control and CV outcome. Important comparisons and contrasts between type 1 diabetes and type 2 diabetes are highlighted in terms of hepatic fat, fat partitioning and lipid profile, and how these may differ between type 1 diabetic patients with and without double diabetes. In so doing, we hope this work will stimulate much-needed research in this area and an improvement in clinical practice

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

Essential role for acid sphingomyelinase-inhibited autophagy in melanoma response to cisplatin

The sphingolipid metabolising enzyme Acid Sphingomyelinase (A-SMase) has been recently shown to inhibit melanoma progression and correlate inversely to tumour grade. In this study we have investigated the role of A-SMase in the chemo-resistance to anticancer treatmentusing mice with melanoma allografts and melanoma cells differing in terms of expression/activity of A-SMase. Since autophagy is emerging as a key mechanism in tumour growth and chemo-resistance, we have also investigated whether an action of A-SMase in autophagy can explain its role. Melanoma sensitivity to chemotherapeutic agent cisplatin in terms of cell viability/apoptosis, tumour growth, and animal survival depended directly on the A-SMase levels in tumoural cells. A-SMase action was due to inhibition of autophagy through activation of Akt/mammalian target of rapamycin (mTOR) pathway. Treatment of melanoma-bearing mice with the autophagy inhibitor chloroquine restored sensitivity to cisplatin of tumours expressing low levels of A-SMase while no additive effects were observed in tumours characterised by sustained A-SMase levels. The fact that A-SMase in melanomas affects mTOR-regulated autophagy and plays a central role in cisplatin efficacy encourages pre-clinical testing on the modulation of A-SMase levels/activity as possible novel anti-neoplastic strategy

Cumulative Risk, Age at Onset, and Sex-Specific Differences for Developing End-Stage Renal Disease in Young Patients With Type 1 Diabetes: A Nationwide Population-Based Cohort Study

OBJECTIVE This study aimed to estimate the current cumulative risk of end-stage renal disease (ESRD) due to diabetic nephropathy in a large, nationwide, population-based prospective type 1 diabetes cohort and specifically study the effects of sex and age at onset. RESEARCH DESIGN AND METHODS In Sweden, all incident cases of type 1 diabetes aged 0-14 years and 15-34 years are recorded in validated research registers since 1977 and 1983, respectively. These registers were linked to the Swedish Renal Registry, which, since 1991, collects data on patients who receive active uremia treatment. Patients with years duration of type 1 diabetes were included (n = 11,681). RESULTS During a median time of follow-up of 20 years, 127 patients had developed ESRD due to diabetic nephropathy. The cumulative incidence at 30 years of type 1 diabetes duration was low, with a male predominance (4.1% [95% CI 3.1-5.3] vs. 2.5% [1.7-3.5]). In both male and female subjects, onset of type I diabetes before 10 years of age was associated with the lowest risk of developing ESRD. The highest risk of ESRD was found in male subjects diagnosed at age 20-34 years (hazard ratio 3.0 [95% CI 1.5-5.7]). In female subjects with onset at age 20-34 years, the risk was similar to patients diagnosed before age 10 years. CONCLUSIONS The cumulative incidence of ESRD is exceptionally low in young type 1 diabetic patients in Sweden. There is a striking difference in risk for male compared with female patients. The different patterns of risk by age at onset and sex suggest a role for puberty and sex hormones