Cellular and molecular pathogenic mechanisms involved in experimental spinal muscular atrophy

Resumen

Spinal muscular atrophy (SMA) is a neurodegenerative disease that affects alpha motoneurons in the spinal cord, being one of the leading genetic causes of death in childhood. The trigger of this disease is a reduction in the expression of the survival of motor neuron protein, SMN, owing to homozygous deletion or specific mutations in the survival motoneuron-1 (SMN1) gene. Although SMN is ubiquitously expressed, its lack specifically causes spinal motoneuron loss. Four functions have been assigned to SMN, three house-keeping functions, related to RNA splicing in all cells, and one neuron-specific, related to axonal transport of mRNA. SMN depletion has been described to cause alterations in cytoskeletal integrity. The actin cytoskeleton plays an important role in cell migration. Also, reduced expression of SMN induces impaired hippocampal neurogenesis, associated to alterations in the expression levels of proteins required for cell growth and migration. This suggests that alterations in cellular migration could be related to SMA. In the U87MG human astroglioma cell line depleted of SMN via shRNA, we characterized the change in migration dynamics induced by SMN depletion. We determined that SMN reduction causes reduced cell migration, associated to RhoA/ROCK signalling pathway activation and profilin overexpression. The activation of RhoA/ROCK leads to myosin light chain phosphorylation, which is known to promote interaction of myosin light chain with actin, increasing the speed of retrograde flow in filopodia and lamellipodia, thus impairing cell migration. In the lumbar spinal cord of the SMNΔ7 mouse model, we described pathological changes in motoneurons, such as soma reduction and neurofilament phosphorylation, previous to motoneuron loss at end-stage. In this model, we observed enhanced astrogliosis surrounding motoneurons at presymptomatic stage, and generalized astrogliosis in lumbar spinal cord at symptomatic stage. In contrast, microgliosis was only enhanced at symptomatic stage. In lumbar spinal cord motoneurons of this model, at symptomatic stage, we described a reduction in the number of pre-synaptic boutons, in which increased phosphorylation of myosin light chain was found. In addition, we observed increased expression of the neuronal isoform of the nitric oxide synthase in motoneurons and in interneurons, but the inducible isoform of nitric oxide synthase only was found in interneurons of the lumbar spinal cord at symptomatic stage. Notch signalling pathway and the overexpression of its ligands, Jagged and Delta, have been widely related to neuritogenesis. Moreover, neuritogenesis defects have been directly related to SMN reduction in models of SMA. However, the possible involvement of the Notch signalling pathway in the pathology of SMA has not been determined yet. After depletion of SMN in U87MG astroglioma cell line, we observed increased levels of Jagged and Delta ligands, Notch receptor and its active fragment, NICD. Thus, we used the SMNΔ7 mouse model to determine whether the glia or motoneurons in the lumbar spinal cord exhibited activation of the Notch signalling pathway. We found astroglial overexpression of Jagged and Delta ligands of Notch receptor at symptomatic stage. Increased levels of the active intracellular fragment of the Notch receptor, NICD, were also found in motoneurons, astroglia and microglia, associated to astroglial Jagged and Delta increased expression, at symptomatic stage. We observed that the motoneurons from SMNΔ7 mice at symptomatic stage, overexpressing the active fragment of Notch, expressed less Neurogenin, a protein known to be involved in neuritogenesis. The depletion of SMN protein in in vitro and in vivo models is associated to an increased activation of the RhoA/ROCK and Notch signalling pathways resulting in actin cytoskeleton disturbance and decreased Neurogenin expression, thus causing cell migration impairment, loss of synapses onto spinal motoneurons and soma reduction. In addition, the observed gliosis found in the SMNΔ7 mice could contribute to create a detrimental environment in SMA tissue. Therefore, our results suggest that SMA pathogenesis is not only a single process, but a convergence of pathogenic processes in motoneurons and glia which lead to motoneuron specific impairment and later death.