Derangement of ATP- and NAD+- related pathways in α-sarcoglycanopathy: identification of new therapeutic strategies

Sarcoglycanopathies, limb-girdle muscular dystrophies (LGMD), are rare genetic disorders caused by loss-of-function mutations of the membrane proteins sarcoglycans (SGs). These diseases are characterized by progressive degeneration of skeletal muscle aggravated by immune-mediated damage, whose triggering and perpetuating molecular mechanisms are not fully elucidated yet. In the first part of the study, we investigated extracellular adenosine triphosphate (eATP) effects in primary cultures of myoblasts isolated from patients with LGMDR3 (α-sarcoglycanopathy) and in immortalized cells isolated from a patient with LGMDR5 (γ-sarcoglycanopathy). eATP is known to activate purinergic receptors. In vivo blockade of the eATP/P2X7 purinergic pathway was previously demonstrated to ameliorate muscle disease progression. P2X7R inhibition improved the dystrophic process by restraining the activity of P2X7 receptors on immune cells. Whether P2X7R blockade could display a direct action on muscle cells was not known yet. Our results demonstrated that, owing to a reduced ecto-ATPase activity and/or an enhanced release of ATP, patient cells are exposed to increased juxtamembrane concentrations of eATP and display a higher susceptivity to eATP signals. The purinoceptor P2Y2, which proved to be overexpressed in patient cells, was identified as a pivotal receptor responsible for the enhanced ATP-induced or UTP-induced Ca2+ increase in affected myoblasts. Moreover, P2Y2R stimulation in LDMDR3 muscle cells induced the release of interleukin-8 and chemotaxis of immune cells. In the second part, we aimed to define the effects of P2X7R genetic ablation in α-sarcoglycanopathy. For this purpose, we generated a double knockout mouse model Sgca-/- p2x7-/-. Sgca-/- mice expressing the wild-type form of P2X7R were used as controls (Sgca-/- p2x7+/+). Diaphragmatic muscle is early and severely damaged in Sgca-/- mice, the animal model of LGMDR3. Thus, we analyzed diaphragmatic muscle from Sgca-/- p2x7+/+ and Sgca-/- p2x7-/- mice: when compared to age-matched Sgca-/- p2x7+/+, 24 weeks old male Sgca-/- p2x7-/- animals, displayed increased extracellular matrix deposition and augmented cellularity in fibrotic areas. Moreover, immunofluorescence stainings showed high number of CD3+ lymphocytes and Iba1+ macrophages in diaphragms from Sgca-/- p2x7-/- mice. Notably, we observed an intense P2X4R signal, which co-localized with CD3+ and Iba1+ cells, confirming its expression by the infiltrating immune cells. No differences in the dystrophic phenotype were observed histologically in quadriceps of 24 weeks old Sgca-/- p2x7-/- and Sgca-/- p2x7+/+ mice, although the fibrotic reaction was less enhanced than in diaphragms, suggesting a differential microenvironment influence on the receptor functions. Accordingly, flow cytometric data of limb muscle infiltrating immune cells did not reveal any difference between the two genotypes. Lastly in the third part, we focused on nicotinamide adenine dinucleotide (NAD+), an essential cofactor whose repletion ameliorates muscular dystrophy by improving the mitochondrial and muscle stem cell function. Indeed, NAD+ deficit represents a deleterious feature of the dystrophic muscle resulting in muscle degeneration. The NAD+-ase CD38, the major consumer of extracellular NAD+, was demonstrated to exert a key role in the pathophysiology of Duchenne Muscular Dystrophy. In SGCs the role of NAD+/CD38 axis in the pathophysiology was unknown. Our data demonstrate that in skeletal muscles of Sgca-/- mice, NAD(P)(H) and ATP content are significantly lower than in WT animals. NAD+ decrease is associated to a lower expression of Nampt, the enzyme mediating NAD+ synthesis through the salvage pathway for Nicotinamide, and increased NAD+-ase activity and CD38 expression in muscle infiltrating cells of Sgca-/- mice. RNA sequencing was performed in 17 patients affected by α-SGC, classified as severe or mild on the basis of clinical parameters, and 10 unaffected individuals. Unsupervised hierarchical clustering analysis of 5000 genes with the highest variance demonstrates that the gene expression profile for severe α-SGC is strongly different compared to controls, whereas mild α-SGC shows no consistent differences to controls. The analysis of genes related to NAD+ metabolism reveals significant downregulation of NAD+ salvage pathway enzymes, whereas NAD+ consuming enzymes are overexpressed in severe a-SGC patients compared to controls. In conclusion, a higher eATP concentration and sensitivity in primary human muscle cells carrying different α-SG or γ-SG loss-of-function mutations indicate that eATP/P2Y2 is an enhanced signaling axis in cells from patients with α-/γ-sarcoglycanopathy. In addition, this study highlighted controversial effects of p2x7 genetic ablation in α-sgc-/- dystrophic tissue. Pharmacological interventions adopting P2X7R inhibitors for dystrophic immune-mediate damage need the definition of a precise time window and dosage administration to avoid detrimental effects. Finally, this study provides a first picture of alterations in NAD+-related metabolic pathways, suggesting novel promising therapeutic targets for α-SGC patients.