Department of Biological Science, University of Delaware, Newark, DE, USA.
BMC Neurosci. 2011 Mar 8;12:25. doi: 10.1186/1471-2202-12-25.
Deletion or mutation(s) of the survival motor neuron 1 (SMN1) gene causes spinal muscular atrophy (SMA), a neuromuscular disease characterized by spinal motor neuron death and muscle paralysis. Complete loss of the SMN protein is embryonically lethal, yet reduced levels of this protein result in selective death of motor neurons. Why motor neurons are specifically targeted by SMN deficiency remains to be determined. In this study, embryonic stem (ES) cells derived from a severe SMA mouse model were differentiated into motor neurons in vitro by addition of retinoic acid and sonic hedgehog agonist. Proteomic and western blot analyses were used to probe protein expression alterations in this cell-culture model of SMA that could be relevant to the disease.
When ES cells were primed with Noggin/fibroblast growth factors (bFGF and FGF-8) in a more robust neural differentiation medium for 2 days before differentiation induction, the efficiency of in vitro motor neuron differentiation was improved from 25% to ~50%. The differentiated ES cells expressed a pan-neuronal marker (neurofilament) and motor neuron markers (Hb9, Islet-1, and ChAT). Even though SMN-deficient ES cells had marked reduced levels of SMN (20% of that in control ES cells), the morphology and differentiation efficiency for these cells are comparable to those for control samples. However, proteomics in conjunction with western blot analyses revealed 6 down-regulated and 14 up-regulated proteins with most of them involved in energy metabolism, cell stress-response, protein degradation, and cytoskeleton stability. Some of these activated cellular pathways showed specificity for either undifferentiated or differentiated cells. Increased p21 protein expression indicated that SMA ES cells were responding to cellular stress. Up-regulation of p21 was confirmed in spinal cord tissues from the same SMA mouse model from which the ES cells were derived.
SMN-deficient ES cells provide a cell-culture model for SMA. SMN deficiency activates cellular stress pathways, causing a dysregulation of energy metabolism, protein degradation, and cytoskeleton stability.
生存运动神经元 1 (SMN1) 基因的缺失或突变导致脊髓性肌萎缩症（SMA），这是一种以脊髓运动神经元死亡和肌肉瘫痪为特征的神经肌肉疾病。SMN 蛋白的完全缺失在胚胎期是致命的，但这种蛋白水平的降低会导致运动神经元的选择性死亡。为什么运动神经元会被 SMN 缺乏特异性靶向，仍有待确定。在这项研究中，通过添加视黄酸和 sonic hedgehog 激动剂，从严重 SMA 小鼠模型中分离的胚胎干细胞（ES 细胞）在体外分化为运动神经元。蛋白质组学和 Western blot 分析用于探测这种 SMA 细胞培养模型中与疾病相关的蛋白质表达变化。
当 ES 细胞在用 Noggin/成纤维细胞生长因子（bFGF 和 FGF-8）在更强大的神经分化培养基中预培养 2 天后再进行诱导分化时，体外运动神经元分化的效率从约 25%提高到约 50%。分化的 ES 细胞表达了一种泛神经元标志物（神经丝）和运动神经元标志物（Hb9、Islet-1 和 ChAT）。尽管 SMN 缺陷型 ES 细胞的 SMN 水平明显降低（约为对照 ES 细胞的 20%），但这些细胞的形态和分化效率与对照样本相当。然而，蛋白质组学结合 Western blot 分析显示，有 6 种下调蛋白和 14 种上调蛋白，其中大多数与能量代谢、细胞应激反应、蛋白质降解和细胞骨架稳定性有关。其中一些激活的细胞通路对未分化或分化细胞具有特异性。p21 蛋白表达增加表明 SMA ES 细胞对细胞应激有反应。同样，从同一 SMA 小鼠模型中分离的 ES 细胞中证实了 p21 的上调。
SMN 缺陷型 ES 细胞为 SMA 提供了一个细胞培养模型。SMN 缺乏激活细胞应激途径，导致能量代谢、蛋白质降解和细胞骨架稳定性的失调。
