Spinal muscular atrophy (SMA) is the most common genetic disease resulting in infant mortality due to severe loss of alpha-motor neurons. SMA is caused by mutations or deletions of the ubiquitously expressed survival motor neuron (SMN) gene. However, why alpha-motor neurons of SMA patients are specifically affected is not clear. We demonstrate here that Smn knockdown in PC12 cells alters the expression pattern of profilin II, resulting in an increase in the neuronal-specific profilin IIa isoform. Moreover, the depletion of Smn, a known interacting partner of profilin IIa, further contributes to the increased profilin IIa availability. Altogether, this leads to an increased formation of ROCK/profilin IIa complex and an inappropriate activation of the RhoA/ROCK pathway, resulting in altered cytoskeletal integrity and a subsequent defect in neuritogenesis. This study represents the first description of a mechanism underlying SMA pathogenesis and highlights new targets for therapeutic intervention for this devastating disorder.

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Deletions or mutations in survival of motor neuron 1 (SMN1) cause motor neuron loss and spinal muscular atrophy (SMA), a neuromuscular disorder, with the most severe type manifesting in utero. Whether SMA is a disease of defects in neurodevelopment and/or neuromaintenance remains unclear. We performed an analysis of Smn gene and protein expression during murine embryogenesis. Furthermore, we examined Smn(-/-);SMN2 mice, a model of very severe SMA, for developmental, morphological, and molecular abnormalities. We demonstrate that Smn transcript levels are regulated in a tissue- and developmental stage-specific manner and that the Smn protein expression pattern generally followed that of the Smn mRNA. Cell death and pathological foci were observed in E10.5 Smn-depleted embryos, and this increased in the telencephalon at E14.5. Furthermore, we show an altered morphology of cranial nerves as well as truncated lumbar spinal nerves in a subset of E10.5 Smn(-/-);SMN2 embryos. Finally, we compared the splicing of a subset of genes shown recently to be aberrantly spliced in phenotypic-stage SMA mice. Changes in alternative splicing of the Slc38a5 and Uspl1 genes were detectable in prephenotypic-stage embryos and neonates but became more pronounced with the severity of the phenotype. By comparison, Hif3a alternative splicing was affected only at the end stage of disease. This result suggests that alterations in mRNA splicing in SMA occur, in part, as a result of disease progression. Overall, we conclude that Smn depletion affects developmental processes, which ultimately may also contribute to SMA pathogenesis.

Mutations in the survival motor neuron (SMN) gene cause spinal muscular atrophy (SMA), a neuromuscular disease associated with muscle weakness that progresses to paralysis, respiratory distress, and ultimately death. Both neurons and muscle are severely affected in this disease. Tandem affinity purification (TAP) has emerged as a useful tool for studying protein complexes in vitro. We have used this purification system to discover novel SMN interacting partners in C2C12 muscle and PC12 neuronal cells. To do so, we fused a TAP-tag, consisting of a HIS hexamer and FLAG epitope separated by the tobacco etch virus (TEV) protease cleavage site, to either the N- or C-terminal region of SMN. Interestingly, the profile of SMN interacting proteins varies depending on the cell type and stage. We have identified a number of novel SMN interacting proteins in both C2C12 and PC12 cells, and from among these we have validated Annexin II and myosin regulatory light chain (MRLC). The discovery of these proteins will lead to a better understanding of the mechanisms underlying the pathophysiology of SMA.