Abstract
Spinal muscular atrophy (SMA) is a neuromuscular genetic disease caused by reduced survival motor neuron (SMN) protein. SMN is ubiquitous and deficient levels cause spinal cord motoneurons (MNs) degeneration and muscle atrophy. Nevertheless, the mechanism by which SMN reduction in muscle contributes to SMA disease is not fully understood. Therefore, studies evaluating atrophy mechanisms in SMA muscles will contribute to strengthening current knowledge of the pathology. Here we propose to evaluate autophagy in SMA muscle, a pathway altered in myotube atrophy. We analized autophagy proteins and mTOR in muscle biopsies, fibroblasts, and lymphoblast cell lines from SMA patients and in gastrocnemius muscles from a severe SMA mouse model. Human MNs differentiated from SMA and unaffected control iPSCs were also included in the analysis of the autophagy. Muscle biopsies, fibroblasts, and lymphoblast cell lines from SMA patients showed reduction of the autophagy marker LC3-II. In SMA mouse gastrocnemius, we observed lower levels of LC3-II, Beclin 1, and p62/SQSTM1 proteins at pre-symptomatic stage. mTOR phosphorylation at Ser2448 was decreased in SMA muscle cells. However, in mouse and human cultured SMA MNs mTOR phosphorylation and LC3-II levels were increased. These results suggest a differential regulation in SMA of the autophagy process in muscle cells and MNs. Opposite changes in autophagy proteins and mTOR phosphorylation between muscle cells and neurons were observed. These differences may reflect a specific response to SMN reduction, which could imply diverse tissue-dependent reactions to therapies that should be taken into account when treating SMA patients.
Highlights
Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder characterized by progressive muscle weakness and atrophy [1]
LC3‐II autophagosome marker is decreased in SMA muscle and fibroblasts, and increased in human SMA MNs LC3-II isoform is incorporated to the autophagosome membrane and the levels reflect the number of autophagosomes [30]
Western blot analysis of protein extracts obtained from SMA patients muscle biopsies (SMAI, SMA type I; and SMAII, SMA type II) showed reduced levels of LC3-II compared to non-affected control (Fig. 1A)
Summary
Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder characterized by progressive muscle weakness and atrophy [1]. Autophagy is a highly regulated pathway responsible for the degradation of cytoplasmic proteins and organelles captured by the autophagosomes. Oxidative stress induced by overexpression of a mutant superoxide dismutase protein (SODG93A) causes muscle atrophy by activating autophagy [9]. Muscle-specific inactivation of genes encoding autophagy-related proteins demonstrate the essential role of autophagy in muscle homeostasis in mice [10]. Protein synthesis and degradation in skeletal muscle are largely regulated by the mammalian target of rapamycin (mTOR) as part of the complex mTORC1 [13]. MTOR regulates many fundamental cell processes, including negative modulation of the autophagy process [14]. The positive regulation of mTOR pathway may be involved in muscle protection in SMA pathology [15, 16]
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