An integrated pharmacological/antioxidant approach for Duchenne Muscular Dystrophy: acid sphingomyelinase as new therapeutic target (1° annualità)

Duchenne muscular dystrophy (DMD) is characterized by chronic muscle damage that induces macrophage infiltration and generation of reactive oxigen species (ROS). Macrophages play crucial roles in DMD. Indeed, they are required for activation and differentiation of precursor cells and for matrix assembly but, at the same time, they actively amplify the damage through the generation of ROS. The innate immune response, consequent to muscle injury, is characterized, at the beginning, by the classical activation of macrophages to an M1 proinflammatory phenotype promoting muscle fiber damage. M1 macrophages reach their peak concentration at 4-5 weeks of age in mdx mice (a well established animal model of DMD). In a later phase M1 macrophages are replaced by a population of M2 macrophages that can attenuate the inflammatory response and promote tissue repair. Thus, strategy aimed at targeting M1 macrophages and driving the shift to M2 polarised macrophages may influence the progression of muscular dystrophy. In the past decade many studies have been focused on the role of bioactive sphingolipids in inflammatory associated disorders, highlighting the possibility of targeting them as a therapeutic option. Among the sphingolipid-metabolizing enzymes acid sphingomyelinase (A-SMase) activation is correlated with the induction of oxidative stress pathways in several diseases. Moreover, A-SMase activity in macrophages is triggered by inflammatory stimuli. Our preliminary results show a role for this protein in macrophages M1 polarization and an increased expression and activity of A-SMase in mdx muscles. Overall this evidence suggests the possible involvement of A-SMase in DMD pathogenesis and encourages further investigations. Our hypothesis is that A- SMase might be a major player in DMD and that modulation of its expression could be crucial to induce a decrease of oxidative stress in muscles by tuning macrophages, an important benefit when considering as complementary approach for therapy, such as gene therapy, currently in preclinical phase for DMD. Scientific objectives: The goal of this project is to understand the role of A-SMase as possible target for DMD treatment. In particular, our preliminary data indicate that the expression/activity of A-SMase is upregulated in muscles of mdx mice and that the treatment of mdx mice with an inhibitor of the enzyme activity ameliorate the muscle phenotype. Starting from these results, the main goal of the project is to define a new therapeutic strategy exploiting the modulation of A-SMase signaling and antioxidants that may synergize with A-SMase inhibition. We will pursue this objective through the following aims: AIM 1: Pharmacological inhibition of A-SMase in DMD; AIM 2: Antioxidant treatment in DMD: the role of A-SMase and its therapeutic value; AIM 3: Pharmacological/antioxidant inhibition of A-SMase pathway as therapeutic strategy for DMD. Methodology: In AIM 1 we will validate pharmacological treatments, known to inhibit A-SMase activity (FIASMA), by treating mdx mice, the mouse model f DMD. In AIM 2 we will test the effect of antioxidants on A-SMase activity and validate their therapeutic value in mdx mice. In AIM 3 we will inhibit A-SMase by drugs/antioxidants to investigate if their combination has further beneficial effects in mdx mice. In the animals of the three aims we will then analyze muscle morphology, muscle function, oxidative stress and inflammatory infiltrate.