Regulation of SMN expression by DcpS inhibition in spinal muscular atrophy
Date
2016
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Spinal muscular atrophy (SMA) is a neurodegenerative disorder that is characterized by the loss of motor neurons in the anterior horn of the spinal cord and brainstem, resulting in progressive muscle weakness and atrophy. SMA is caused by the loss or mutations of the survival motor neuron 1 (SMN1) gene, which encodes the SMN protein. SMA patients retain at least one copy of the survival motor neuron 2 (SMN2) gene. SMN2 differs from SMN1 by 11 nucleotides; only one of which is functionally relevant and it is a C-to-T inversion in exon 7. This single nucleotide transition causes SMN2 to predominantly produce an incorrectly spliced mRNA that excludes exon 7. SMN2 transcripts that lack exon 7 are termed SMNΔ7 and encode for a truncated form of the SMN protein that is not fully functional. There is currently no effective treatment available for SMA, however one of the potential therapeutic approaches is to develop small molecules that promote an increase in the transcription of SMN2 gene. Ultrahigh-throughput screening identified a series of C5-substituted 2,4-diaminoquinazoline compounds (2,4-DAQs) that increase SMN2 gene expression. In this study, we investigated the effects of four 2,4-DAQs—D156844, D158872, D157161, and D157495—as potential therapeutics for SMA. We show that the compounds increase SMN2 promoter activity in the motor neuron-like, NSC-34 reporter assay. D157495 had the lowest EC50, proving that it is the most effective of the four compounds at inducing SMN2 transcription. However, when fibroblasts derived from SMA type II patients were treated with varying concentrations of 2,4-DAQs, the compounds had no significant effects on SMN2 mRNA or SMN protein levels in these cell lines. The 2,4-DAQs also showed no significant differences in the levels of Smn mRNA and protein in the motor neuron-like NSC-34 cell line. Using protein microarrays, these compounds were found to bind to and inhibit the activity of the mRNA scavenger decapping enzyme, DcpS. This protein is known to be a modulator of RNA metabolism and functions to hydrolyze the resulting cap structure following mRNA decay. DcpS was previously found to modulate the expression levels of several RNAs in SH-SY5Y retinoblastoma cells and HEK293T cells. These include, a member of the basic helix-loop-helix family of transcription factors (ATOH7) and two long non-coding RNAs (DRNT1 and DRNT2). We were able to validate these findings using fibroblasts derived from SMA type II patients as well as non-SMA fibroblasts. Treatment of these fibroblast cell lines with all four compounds resulted in an increase in the expression levels of ATOH7, DRNT1, and DRNT2. Treatment of the motor neuron-like NSC-34 cells also resulted in an increase in Atoh7. Additionally, we found that the basal level of ATOH7 is significantly decreased in SMA type II patients when compared to non-SMA fibroblasts. Collectively, our data demonstrate that the 2,4-DAQs do not increase SMN expression in patient-derived fibroblasts however, these compounds proved to be effective at increasing SMN2 promoter activation in the NSC-34 cell-based promoter assay. The property of these compounds of inhibiting DcpS may indirectly influence SMN expression by modulating the levels of ATOH7, DRNT1, and DRNT2. Our findings suggest that the 2,4-DAQs have beneficial effects that are independent of SMN expression and the DcpS-inducible transcripts could be the actual molecular targets of these compounds.