SMN deficiency attenuates migration of U87MG astroglioma cells through the activation of RhoA Víctor Caraballo-Miralles a , Andrea Cardona-Rossinyol a , Ana Garcera b , Priam Villalonga c , Rosa M. Soler b , Gabriel Olmos a, 1 , Jerònia Lladó a, ⁎ , 1 a Grup de Neurobiologia Cel·lular, Institut Universitari d'Investigacions en Ciències de la Salut, IUNICS and Departament de Biologia, Universitat de les Illes Balears, Cra. de Valldemossa km 7.5, 07122-Palma de Mallorca, Spain b Unitat de Senyalització Neuronal, Departament de Ciències Mèdiques Bàsiques, Universitat de Lleida-IRBLLEIDA, Montserrat Roig, 2. 25008-Lleida, Spain c Cancer Cell Biology Group, IUNICS and Departament de Biologia Fonamental, Universitat de les Illes Balears, Spain abstract article info Article history: Received 22 July 2011 Revised 23 November 2011 Accepted 5 December 2011 Available online 16 December 2011 Keywords: Spinal muscular atrophy SMN Cell migration Profilin RhoA Spinal muscular atrophy (SMA) is a neurodegenerative disease that affects alpha motoneurons in the spinal cord caused by homozygous deletion or specific mutations in the survival motoneuron-1 (SMN1) gene. Cell migration is critical at many stages of nervous system development; to investigate the role of SMN in cell mi- gration, U87MG astroglioma cells were transduced with shSMN lentivectors and about 60% reduction in SMN expression was achieved. In a monolayer wound-healing assay, U87MG SMN-depleted cells exhibit reduced cell migration. In these cells, RhoA was activated and phosphorylated levels of myosin regulatory light chain (MLC), a substrate of the Rho kinase (ROCK), were found increased. The decrease in cell motility was related to activation of RhoA/Rho kinase (ROCK) signaling pathway as treatment with the ROCK inhibitor Y-27632 abrogated both the motility defects and MLC phosphorylation in SMN-depleted cells. As cell migration is reg- ulated by continuous remodeling of the actin cytoskeleton, the actin distribution was studied in SMN- depleted cells. A shift from filamentous to monomeric (globular) actin, involving the disappearance of stress fibers, was observed. In addition, profilin I, an actin-sequestering protein showed an increased expression in SMN-depleted cells. SMN is known to physically interact with profilin, reducing its actin-sequestering activ- ity. The present results suggest that in SMN-depleted cells, the increase in profilin I expression and the reduc- tion in SMN inhibitory action on profilin could lead to reduced filamentous actin polymerization, thus decreasing cell motility. We propose that the alterations reported here in migratory activity in SMN- depleted cells, related to abnormal activation of RhoA/ROCK pathway and increased profilin I expression could have a role in developing nervous system by impairing normal neuron and glial cell migration and thus contributing to disease pathogenesis in SMA. © 2011 Elsevier Inc. All rights reserved. Introduction Spinal muscular atrophy (SMA) is a neurodegenerative disease inherited in an autosomal recessive manner that affects alpha moto- neurons in the spinal cord and causes muscular atrophy of proximal limb and trunk muscles, paralysis, and in the most severe cases, death (Crawford and Pardo, 1996). The frequency of carriers of SMA is 1:35 and the incidence rate is approximately 1 per 6000 births (Pearn, 1978), being the most common genetic cause of childhood death (reviewed by Lorson et al., 2010). SMA is caused by the homo- zygous deletion or specific mutations in the survival motoneuron-1 (SMN1) gene, localized in the telomeric region of chromosome 5, cod- ifying for SMN protein (Lefebvre et al., 1995). In humans, an addition- al SMN2 gene also produces SMN; however, this gene produces about 90% of a highly unstable, truncated protein called SMNΔ7 due to de- fective mRNA maturation, and only 10% of normal (full-length) pro- tein (Cifuentes-Diaz et al., 2001). The severity of the disease depends on the number of copies of SMN2 gene; low number of SMN2 copies results in a more severe form of the disease. SMN func- tions in the assembly of small ribonucleoproteins, snRNPs, (Pellizzoni et al., 2002; Yong et al., 2004) and causes pre-mRNA splicing defects in the spinal cord and brain in a mouse model of SMA (Zhang et al., 2008). However, no causal link has been found between the aberrant- ly spliced targets and SMA pathogenesis. A role for SMN in neurite outgrowth, neuronal differentiation, axonal pathfinding and neuro- muscular maturation has been described (Fan and Simard, 2002; Kariya et al., 2008; Kong et al., 2009; McWhorter et al., 2003; Murray et al., 2008; Rossoll et al., 2003). In this sense, it has been Molecular and Cellular Neuroscience 49 (2012) 282–289 ⁎ Corresponding author at: Departament de Biologia, Ed. Guillem Colom, Universitat de les Illes Balears, Cra. de Valldemossa, km 7.5, E-07122 Palma de Mallorca, Illes Balears, Spain. Fax: + 34 971 173184. 1 Senior co-authors. 1044-7431/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2011.12.003 Contents lists available at SciVerse ScienceDirect Molecular and Cellular Neuroscience journal homepage: www.elsevier.com/locate/ymcne