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.

BackgroundNitric oxide (NO), generated in skeletal muscle mostly by the neuronal NO synthases (nNOSμ), has profound effects on both mitochondrial bioenergetics and muscle development and function. The importance of NO for muscle repair emerges from the observation that nNOS signalling is defective in many genetically diverse skeletal muscle diseases in which muscle repair is dysregulated. How the effects of NO/nNOSμ on mitochondria impact on muscle function, however, has not been investigated yet.MethodsIn this study we have examined the relationship between the NO system, mitochondrial structure/activity and skeletal muscle phenotype/growth/functions using a mouse model in which nNOSμ is absent. Also, NO-induced effects and the NO pathway were dissected in myogenic precursor cells.ResultsWe show that nNOSμ deficiency in mouse skeletal muscle leads to altered mitochondrial bioenergetics and network remodelling, and increased mitochondrial unfolded protein response (UPRmt) and autophagy. The absence of nNOSμ is also accompanied by an altered mitochondrial homeostasis in myogenic precursor cells with a decrease in the number of myonuclei per fibre and impaired muscle development at early stages of perinatal growth. No alterations were observed, however, in the overall resting muscle structure, apart from a reduced specific muscle mass and cross sectional areas of the myofibres. Investigating the molecular mechanisms we found that nNOSμ deficiency was associated with an inhibition of the Akt-mammalian target of rapamycin pathway. Concomitantly, the Akt-FoxO3-mitochondrial E3 ubiquitin protein ligase 1 (Mul-1) axis was also dysregulated. In particular, inhibition of nNOS/NO/cyclic guanosine monophosphate (cGMP)/cGMP-dependent-protein kinases induced the transcriptional activity of FoxO3 and increased Mul-1 expression. nNOSμ deficiency was also accompanied by functional changes in muscle with reduced muscle force, decreased resistance to fatigue and increased degeneration/damage post-exercise.ConclusionsOur results indicate that nNOSμ/NO is required to regulate key homeostatic mechanisms in skeletal muscle, namely mitochondrial bioenergetics and network remodelling, UPRmt and autophagy. These events are likely associated with nNOSμ-dependent impairments of muscle fibre growth resulting in a deficit of muscle performance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-014-0022-6) contains supplementary material, which is available to authorized users.

Purpose: In search of novel strategies to improve the outcome of advanced prostate cancer, we considered that prostate cancer cells rearrange iron homeostasis, favoring iron uptake and proliferation. We exploited this adaptation by exposing prostate cancer preclinical models to high-dose iron to induce toxicity and disrupt adaptation to androgen starvation. Experimental Design: We analyzed markers of cell viability and mechanisms underlying iron toxicity in androgen receptor–positive VCaP and LNCaP, castration-resistant DU-145 and PC-3, and murine TRAMP-C2 cells treated with iron and/or the antiandrogen bicalutamide. We validated the results in vivo in VCaP and PC-3 xenografts and in TRAMP-C2 injected mice treated with iron and/or bicalutamide. Results: Iron was toxic for all prostate cancer cells. In particular, VCaP, LNCaP, and TRAMP-C2 were highly iron sensitive. Toxicity was mediated by oxidative stress, which primarily affected lipids, promoting ferroptosis. In highly sensitive cells, iron additionally caused protein damage. High-basal iron content and oxidative status defined high iron sensitivity. Bicalutamide–iron combination exacerbated oxidative damage and cell death, triggering protein oxidation also in poorly iron-sensitive DU-145 and PC-3 cells. In vivo, iron reduced tumor growth in TRAMP-C2 and VCaP mice. In PC-3 xenografts, bicalutamide–iron combination caused protein oxidation and successfully impaired tumor expansion while single compounds were ineffective. Macrophages influenced body iron distribution but did not limit the iron effect on tumor expansion. Conclusions: Our models allow us to dissect the direct iron effect on cancer cells. We demonstrate the proof of principle that iron toxicity inhibits prostate cancer cell proliferation, proposing a novel tool to strengthen antiandrogen treatment efficacy.