Development of new therapeutic strategies for spinal muscular atrophy

Resumen

Spinal Muscular Atrophy (SMA) is a severe neurodegenerative disease and the first genetic cause of infant death. It is originated by the deletion or mutation of Survival Motor Neuron 1 (SMN1) gene causing a Survival Motor Neuron (SMN) protein deficiency. The reduction of this protein predominantly leads to the degeneration of spinal cord motoneurons (MNs) and consequently produces skeletal muscle atrophy and weakness. The exact cellular and molecular mechanisms responsible for MN loss of function are only partially known. SMN reduction causes neurite degeneration and cell death without classical apoptotic features. Autophagy is an important and highly regulated process, essential for the removal of damaged organelles and toxic substances or proteins through lysosome degradation. This mechanism is specifically important in post-mitotic cells like MNs where autophagosome accumulation causes axonal transport disruption, interference of intracellular space trafficking, and neurite degeneration. What is well known in SMA is that intracellular SMN protein levels are critical to define the disease onset and severity, and this is partially determined by the number of copies of SMN2, the centromeric duplication of the SMN gene and the main modifier of SMA. For that reason, understanding the processes of SMN stability and degradation to identify compounds that increase protein levels is a major goal in SMA therapeutics development. Calpains are a family of calcium-dependent proteases that have been related to muscle disorders and neurodegenerative diseases. Specifically, it has been described in muscle that SMN can be a proteolytic target of calpain. Calpain activity is also involved in autophagy regulation by modulation of multiple proteins involved in the process. The objectives in the present work have been to analyze the autophagy deregulation and determine the involvement of calpain in SMN protein regulation on MNs, in order to deepen in the origin of neurodegeneration and to develop a new therapeutic approach for SMA disease. To this end, we have analyzed autophagic markers in different mouse and human SMA in vitro models. The results showed that autophagosomes and LC3 levels were increased in SMA samples compared to controls, suggesting a deregulation of the autophagy process throughout the disease progression. Moreover, calpain knockdown using an shRNA approach showed an increase of both, Smn and LC3 levels and prevented neurite degeneration occurred in SMA affected mouse MNs. Similar results were obtained in in vitro experiments using a pharmacological calpain inhibitor, calpeptin. Likewise, calpain activation produced by depolarized conditions induced α-fodrin and SMN proteolysis, confirming that calpain directly regulates the SMN protein level in MNs. Additionally, calpeptin in vivo treatment significantly improved the lifespan and motor function of two severe SMA mouse models, demonstrating the potential utility of calpain inhibitors in SMA therapeutics. Finally, the analysis of calpain pathway members in mice and human cellular SMA models indicated an increase of calpain activity in SMN-reduced MNs. Thus, our results show that calpain activity is increased in SMA MNs and its inhibition may have a beneficial effect on the SMA phenotype through the increase of SMN and the regulation of the autophagy process in spinal cord MNs.