A cell system for phenotypic screening of modifiers of SMN2 gene expression and function.
A cell system for phenotypic screening of modifiers of SMN2 gene expression and function.
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DOI：
10.1371/journal.pone.0071965
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Pellizzoni L
中科院分区：
文献类型：
--
作者：
Li DK;Tisdale S;Espinoza-Derout J;Saieva L;Lotti F;Pellizzoni L
脊髓性肌萎缩症（SMA）是一种遗传性神经退行性疾病，由SMN1基因的纯合失活和运动神经元存活（SMN）蛋白水平降低引起。由于几乎相同的SMN2基因的较高拷贝数降低了疾病的严重程度，因此迄今为止，开发SMA治疗的大多数努力都集中在增强SMN表达上。替代治疗方法的鉴定在一定程度上受到潜在靶点知识有限和缺乏基于细胞的筛选试验（可作为SMN功能读数）的阻碍。在这里，我们建立了一个细胞系统，其中培养的小鼠成纤维细胞的增殖依赖于从SMN2基因产生的功能性SMN。为此，我们将整个人SMN2基因导入NIH3T3细胞系，其中内源性小鼠Smn的调控敲低严重降低了细胞增殖。我们发现，低SMN2拷贝数对Smn耗竭诱导的细胞增殖表型具有适度的影响，而高SMN2拷贝数具有强烈的保护作用。此外，细胞增殖与小核核糖核蛋白组装中SMN活性水平相关。在小型化为高通量形式之后，我们的基于细胞的表型测定准确地测量了导致SMN上调的药理学和遗传治疗的有益效果。该细胞模型为SMN 2基因表达和功能修饰剂的表型筛选提供了一个新的平台，该修饰剂通过多种机制发挥作用，并为SMN生物学和SMA治疗开发研究提供了一个强大的新工具。
Spinal muscular atrophy (SMA) is an inherited neurodegenerative disease caused by homozygous inactivation of the SMN1 gene and reduced levels of the survival motor neuron (SMN) protein. Since higher copy numbers of the nearly identical SMN2 gene reduce disease severity, to date most efforts to develop a therapy for SMA have focused on enhancing SMN expression. Identification of alternative therapeutic approaches has partly been hindered by limited knowledge of potential targets and the lack of cell-based screening assays that serve as readouts of SMN function. Here, we established a cell system in which proliferation of cultured mouse fibroblasts is dependent on functional SMN produced from the SMN2 gene. To do so, we introduced the entire human SMN2 gene into NIH3T3 cell lines in which regulated knockdown of endogenous mouse Smn severely decreases cell proliferation. We found that low SMN2 copy number has modest effects on the cell proliferation phenotype induced by Smn depletion, while high SMN2 copy number is strongly protective. Additionally, cell proliferation correlates with the level of SMN activity in small nuclear ribonucleoprotein assembly. Following miniaturization into a high-throughput format, our cell-based phenotypic assay accurately measures the beneficial effects of both pharmacological and genetic treatments leading to SMN upregulation. This cell model provides a novel platform for phenotypic screening of modifiers of SMN2 gene expression and function that act through multiple mechanisms, and a powerful new tool for studies of SMN biology and SMA therapeutic development.
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影响因子：
64.5
作者：
LEFEBVRE, S;BURGLEN, L;MELKI, J
通讯作者：
MELKI, J
影响因子：
46.9
作者：
通讯作者：
--
生存运动神经元影响塑蛋白3蛋白水平导致运动缺陷。
DOI：
10.1523/jneurosci.5808-11.2012
发表时间：
2012-04-11
期刊：
The Journal of neuroscience : the official journal of the Society for Neuroscience
影响因子：
--
作者：
Hao le T;Wolman M;Granato M;Beattie CE
通讯作者：
Beattie CE
影响因子：
3.5
作者：
Ackermann, Bastian;Kroeber, Sandra;Wirth, Brunhilde
通讯作者：
Wirth, Brunhilde
影响因子：
3.5
作者：
Hannus, S;Bühler, D;Fischer, U
通讯作者：
Fischer, U