Abstract
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.
Highlights
Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease characterized by loss of motor neurons in the anterior horn of the spinal cord and skeletal muscle atrophy [1]
To facilitate the identification of novel targets and therapeutics that act on survival motor neuron (SMN) expression and on SMN function and downstream events induced by SMN deficiency, we developed a cell-based system that uses cell proliferation defects triggered by SMN deficiency in cultured mammalian cells as phenotypic readout of the functional levels of SMN produced from the SMN2 gene
The model system we developed is based on a mouse NIH3T3 fibroblast cell line (NIH3T3-SmnRNAi) in which regulated knockdown of endogenous mouse Smn triggers a severe cell proliferation defect [11], providing a direct phenotypic readout of SMN function
Summary
Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease characterized by loss of motor neurons in the anterior horn of the spinal cord and skeletal muscle atrophy [1]. In animal models of SMA, the disruption of snRNP biogenesis induced by SMN deficiency decreases snRNP levels [7,8,9] and causes splicing defects in genes that contribute to motor system dysfunction [10,11,12]. The two SMN genes are nearly identical, a C to T transition in exon 7 of SMN2 disrupts splicing regulatory elements resulting mainly in the production of transcripts lacking exon 7 (SMND7) with only a small proportion encoding full-length SMN [14,15,16,17]. As a consequence, reduced levels of full-length SMN protein produced from the SMN2 gene, while sufficient to prevent embryonic lethality, are not able to fully compensate for the loss of SMN1 resulting in motor neuron disease
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