ES Cell-Derived Motoneurons from SMA transgenic mice

描述（由申请人提供）：我们提出了一种依赖于胚胎干（ES）细胞分化为运动神经元的策略，以确定脊髓性肌萎缩症（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，并表明这些细胞系在骨骼肌存在下分化为运动神经元时经历轴突变性和最终细胞死亡。在具体目标1中，我们将严格定义SMA ES细胞衍生的运动神经元与共培养的骨骼肌形成功能性神经肌肉接头的能力。我们还将定义轴突运输和轴突或细胞死亡途径的活动异常。具体目标2将通过在与正常ES细胞衍生的运动神经元和SMA ES细胞衍生的运动神经元共培养的SMA骨骼肌中进行来自具体目标1的选定实验来定义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的空间要求，以通过排除差异定位于细胞核、细胞质和轴突的SMN衍生物来拯救异常运动神经元表型。具体目标4将定义SMN的时间要求，以通过利用在分化期间的各个阶段调节SMN2的外显子7纳入的药理学疗法来挽救运动神经元表型。具体目标5将定义SMA ES细胞衍生的运动神经元在卵内移植到鸡胚脊髓中后的表型，从而定义轴突延伸或寻路的异常。该项目将确定SMA运动神经元功能障碍的关键分子线索，并将确定潜在的策略来停止或逆转这种功能障碍。

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.