The purpose of our project is to investigate the role of expression and splicing abnormalities in the pathogenesis of spinal muscular atrophy (SMA), one of the most common genetic neurological diseases causing infant mortality. SMA, which is characterized by the selective degeneration of spinal cord motor neurons (MNs), is caused by mutations in the survival of motor neuron 1 (SMN1) gene that are thought to alter RNA transcription/splicing. It remains unclear how these abnormalities cause MNs loss in SMA. We have previously analyzed both global gene and alternative splicing expression profiles of human SMA-induced pluripotent stem cell (iPSC)-derived MNs. The splicing array results were validated by real-time RT-PCR and proteomic analysis, highlighting the presence of multiple, differentially expressed/spliced genes of interest involved in RNA metabolism, MN development, and axonal guidance (Corti et al., submitted). Here we will extend this analysis in other SMA cell lines and investigate the pathogenetic role of these in vitro and in vivo expression/splicing alterations. WP1: Identify differentially expressed/spliced genes in SMA patient-specific MNs from multiple iPSC lines. 1.1-Generation of WT-iPSCs and SMA-iPSCs and their differentiation into MNs. We have successfully generated iPSCs free from vectors and transgenic sequences from two type 1 SMA patients and unaffected subjects and demonstrated that these cells differentiate into MNs. We would generate other wild-type and SMA patient-derived iPSCs and motor neurons in order to define molecular mechanisms that are uniquely dysregulated in SMA. These cells may be applied both in basic and translational research. 1.2 Identification of differentially spliced isoforms in SMA-MNs. We will perform whole RNA sequencing analysis in SMA/WT-MNs from other iPSC lines. Expression levels of mRNAs, snRNAs, and other genomic sequences will be profiled. As control, we will analyze the profile of SMA iPSC lines genetically co