Effects of Inhibitors of SLC9A-Type Sodium-Proton Exchangers on () mRNA Splicing and Expression.

Spinal muscular atrophy (SMA) is an autosomal recessive, pediatric-onset disorder caused by the loss of spinal motor neurons, thereby leading to muscle atrophy. SMA is caused by the loss of or mutations in the () gene. is duplicated in humans to give rise to the paralogous () gene. This paralog is nearly identical except for a cytosine to thymine transition within an exonic splicing enhancer element within exon 7. As a result, the majority of transcripts lack exon 7 (SMNΔ7), which produces a truncated and unstable SMN protein. Since copy number is inversely related to disease severity, it is a well established target for SMA therapeutics development. 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of sodium/proton exchangers (NHEs), has previously been shown to increase exon 7 inclusion and SMN protein levels in SMA cells. In this study, NHE inhibitors were evaluated for their ability to modulate expression. EIPA as well as 5-(N,N-hexamethylene)amiloride (HMA) increase exon 7 inclusion in splicing reporter lines as well as in SMA fibroblasts. The EIPA-induced exon 7 inclusion occurs via a unique mechanism that does not involve previously identified splicing factors. Transcriptome analysis identified novel targets, including and , for further characterization. EIPA and HMA are more selective at inhibiting the NHE5 isoform, which is expressed in fibroblasts as well as in neuronal cells. These results show that NHE5 inhibition increases expression and may be a novel target for therapeutics development. SIGNIFICANCE STATEMENT: This study demonstrates a molecular mechanism by which inhibitors of the sodium-protein exchanger increase the alternative splicing of in spinal muscular atrophy cells. NHE5 selective inhibitors increase the inclusion of full-length mRNAs by targeting and expression, which is distinct from other small molecule regulators of alternative splicing. This study provides a novel means to increase full-length expression and a novel target for therapeutics development.