title: 5-(N-ethyl-N-isopropyl)-amiloride enhances SMN2 exon 7 inclusion and protein expression in spinal muscular atrophy cells.
abstract: 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
  
  
  CONCLUSIONS
  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.
authors.affiliation: Faculty of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan, Republic of China.
authors.forename: Chung-Yee
authors.initials: CY
authors.lastname: Yuo
authors.forename: Hui-Hua
authors.initials: HH
authors.lastname: Lin
authors.forename: Ya-Sian
authors.initials: YS
authors.lastname: Chang
authors.forename: Wen-Kuang
authors.initials: WK
authors.lastname: Yang
authors.forename: Jan-Gowth
authors.initials: JG
authors.lastname: Chang
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects / Q000235:genetics; D000584:Amiloride / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D017362:Cyclic AMP Response Element-Binding Protein / Q000187:drug effects* / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D005091:Exons / Q000187:drug effects / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods; D009046:Motor Neurons / Q000187:drug effects / Q000378:metabolism; D009134:Muscular Atrophy, Spinal / Q000235:genetics* / Q000628:therapy*; D009419:Nerve Tissue Proteins / Q000187:drug effects* / Q000235:genetics*; D018696:Neuroprotective Agents / Q000494:pharmacology / Q000627:therapeutic use; D012322:RNA Precursors / Q000187:drug effects / Q000235:genetics; D012326:RNA Splicing / Q000187:drug effects* / Q000235:genetics; D016601:RNA-Binding Proteins / Q000187:drug effects* / Q000235:genetics*; D055532:SMN Complex Proteins; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; D018696:Neuroprotective Agents; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D055532:SMN Complex Proteins; C526471:SMN1 protein, human; C526477:SMN2 protein, human; D017923:Sodium-Hydrogen Exchangers; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein; D000584:Amiloride; C039614:ethylisopropylamiloride