5-(N-ethyl-N-isopropyl)-amiloride enhances SMN2 exon 7 inclusion and protein expression in spinal muscular atrophy cells

• Published in: Annals of neurology 2008-01, Vol.63 (1), p.26-34
• Main Authors: Yuo, Chung-Yee, Lin, Hui-Hua, Chang, Ya-Sian, Yang, Wen-Kuang, Chang, Jan-Gowth
• Format: Article
• Language: English
• Subjects: Alternative Splicing - drug effects; Alternative Splicing - genetics; Amiloride - analogs & derivatives; Amiloride - pharmacology; Amiloride - therapeutic use; Biological and medical sciences; Cell Line; Cell Survival - drug effects; Cell Survival - genetics; Cyclic AMP Response Element-Binding Protein - drug effects; Cyclic AMP Response Element-Binding Protein - genetics; Enzyme Inhibitors - pharmacology; Enzyme Inhibitors - therapeutic use; Exons - drug effects; Exons - genetics; Humans; Hydrogen-Ion Concentration - drug effects; Injuries of the nervous system and the skull. Diseases due to physical agents; Lymphocytes - drug effects; Lymphocytes - metabolism; Medical sciences; Molecular Biology - methods; Motor Neurons - drug effects; Motor Neurons - metabolism; Muscular Atrophy, Spinal - genetics; Muscular Atrophy, Spinal - therapy; Nerve Tissue Proteins - drug effects; Nerve Tissue Proteins - genetics; Neurology; Neuroprotective Agents - pharmacology; Neuroprotective Agents - therapeutic use; RNA Precursors - drug effects; RNA Precursors - genetics; RNA Splicing - drug effects; RNA Splicing - genetics; RNA-Binding Proteins - drug effects; RNA-Binding Proteins - genetics; SMN Complex Proteins; Sodium-Hydrogen Exchangers - antagonists & inhibitors; Sodium-Hydrogen Exchangers - metabolism; Survival of Motor Neuron 1 Protein; Survival of Motor Neuron 2 Protein; Transcription, Genetic - drug effects; Transcription, Genetic - genetics; Traumas. Diseases due to physical agents
• ISSN: 0364-5134; 1531-8249
• DOI: 10.1002/ana.21241

Objective Spinal muscular atrophy (SMA) is a common inherited neuromuscular disorder caused by homozygous loss of function of the survival motor neuron 1 (SMN1) gene. All SMA patients carry at least one copy of a nearly identical SMN2 gene. However, a critical nucleotide change in SMN2 results in alternative splicing and exclusion of exon 7 in the majority of SMN2 messenger RNA (mRNA), thus producing a low level of functional SMN protein. Increasing SMN protein production by promoting SMN2 exon 7 inclusion could be a therapeutic approach for SMA. It has been shown that cellular pH microenvironment can modulate pre‐mRNA alternative splicing in vivo. In this study, we tested whether inhibitors of the Na+/H+ exchanger can modulate the exon 7 splicing of SMN2 mRNA Methods We treated SMA lymphoid cell lines with Na+/H+ exchanger inhibitors and then measured SMN2 exon 7 splicing by reverse transcriptase polymerase chain reaction and SMN protein production by Western blotting and immunofluorescence Results We found that treatment with an Na+/H+ exchanger inhibitor, 5‐(N‐ethyl‐N‐isopropyl)‐amiloride (EIPA), significantly enhances SMN2 exon 7 inclusion and SMN protein production in SMA cells. In addition, EIPA increases the number of nuclear gems in SMA cells. We further explored the underlying mechanism, and our results suggest that EIPA may promote SMN2 exon 7 inclusion through upregulation of the splicing factor SRp20 in the nucleus Interpretation Our finding that EIPA, an inhibitor of the Na+/H+ exchanger, can increase SMN protein expression in SMA cells provides a new direction for the development of drugs for SMA treatment. However, further translational studies are needed to determine whether this finding is applicable for SMA treatment or just a proof of cellular pH effect on SMN splicing. Ann Neurol 2007