ES Cell-Derived Motoneurons from SMA transgenic mice

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DESCRIPTION (provided by applicant): We propose a strategy that relies upon the differentiation of embryonic stem (ES) cells to motoneurons in order to define molecular and cellular abnormalities in Spinal Muscular Atrophy (SMA). We have generated ES cell lines from SMA transgenic mice and have shown that these lines undergo axonal degeneration and ultimately cellular death when differentiated into motoneurons in the presence of skeletal muscle. In Specific Aim 1, we will rigorously define the ability of SMA ES cell-derived motoneurons to form functional neuromuscular junctions with co-cultured skeletal muscle. We will also define abnormalities in axonal transport and the activity of axonal- or cell-death pathways. Specific Aim 2 will define the importance of SMA skeletal muscle in the observed motoneuron phenotype by carrying out selected experiments from Specific Aim 1 with SMA skeletal muscle in co-culture with normal ES cell-derived motoneurons and with SMA ES cell-derived motoneurons. Specific Aim 3 will determine the spatial requirements of SMN to rescue the abnormal motoneuron phenotype by transfecting SMN derivatives that differentially localize to the nucleus, cytoplasm and axons. Specific Aim 4 will define the temporal requirements of SMN to rescue the motoneuron phenotype by utilizing pharmacologic therapies that modulate exon-7 inclusion from SMN2 at various stages during differentiation. Specific Aim 5 will define the phenotype of SMA ES cell-derived motoneurons in ovo following transplantation into embryonic chick spinal cord, thereby defining an abnormality of axon extension or pathfinding. This project will identify critical molecular clues to motoneuron dysfunction in SMA and will identify potential strategies to halt or reverse this dysfunction.