ES Cell-Derived Motoneurons from SMA transgenic mice

描述(申请人提供)：我们提出了一种策略，依赖于胚胎干细胞向运动神经元的分化，以确定脊髓肌萎缩症(SMA)的分子和细胞异常。我们已经从SMA转基因小鼠中产生了ES细胞系，并表明当这些细胞系在骨骼肌存在的情况下分化为运动神经元时，会经历轴突退化和最终细胞死亡。在具体目标1中，我们将严格定义SMA ES细胞来源的运动神经元与共培养的骨骼肌形成功能性神经肌肉连接的能力。我们还将定义轴突运输的异常和轴突或细胞死亡途径的活动。特定目标2将通过从特定目标1与SMA骨骼肌与正常ES细胞来源的运动神经元和SMA ES细胞来源的运动神经元共同培养的选定实验来确定SMA骨骼肌在观察到的运动神经元表型中的重要性。具体目标3将确定SMN的空间需求，通过转染差异定位于细胞核、细胞质和轴突的SMN衍生物来挽救异常的运动神经元表型。具体目标4将确定SMN的时间要求，通过利用药物疗法在分化的不同阶段调节SMN的外显子7包涵体来挽救运动神经元表型。具体目标5将确定移植到鸡胚胎脊髓中的SMA ES细胞衍生运动神经元在OVO中的表型，从而定义轴突延伸或路径发现的异常。该项目将确定SMA运动神经元功能障碍的关键分子线索，并将确定阻止或逆转这一功能障碍的潜在策略。

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.