Division of Neurology, Nemours Children's Hospital Delaware, Wilmington, Delaware (S.K., E.M., M.E.R.B.); Department of Biological Sciences, University of Delaware, Newark, Delaware (S.K., M.E.R.B.); Center for Pediatric Research, Nemours Biomedical Research, Nemours Children's Hospital Delaware, Wilmington, Delaware (M.E.R.B.); and Department of Pediatrics, Thomas Jefferson University, Philadelphia, Pennsylvania (M.E.R.B.).
Division of Neurology, Nemours Children's Hospital Delaware, Wilmington, Delaware (S.K., E.M., M.E.R.B.); Department of Biological Sciences, University of Delaware, Newark, Delaware (S.K., M.E.R.B.); Center for Pediatric Research, Nemours Biomedical Research, Nemours Children's Hospital Delaware, Wilmington, Delaware (M.E.R.B.); and Department of Pediatrics, Thomas Jefferson University, Philadelphia, Pennsylvania (M.E.R.B.)
Mol Pharmacol. 2022 Aug;102(2):92-105. doi: 10.1124/molpharm.122.000529. Epub 2022 Jun 6.
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.
脊髓性肌萎缩症（SMA）是一种常染色体隐性、儿科发病的疾病，由脊髓运动神经元丧失引起，从而导致肌肉萎缩。SMA 是由 基因的缺失或突变引起的。在人类中， 基因被复制，产生了同源的 基因。除了外显子 7 中的一个内含子剪接增强元件内的胞嘧啶到胸腺嘧啶的转换之外，这个同源基因几乎是相同的。因此，大多数 转录本缺乏外显子 7（SMNΔ7），从而产生截短和不稳定的 SMN 蛋白。由于 拷贝数与疾病严重程度呈反比，因此它是 SMA 治疗开发的既定靶点。5-（N-乙基-N-异丙基）amiloride（EIPA），一种钠/质子交换器（NHEs）抑制剂，先前已被证明可增加 SMA 细胞中外显子 7 的包含和 SMN 蛋白水平。在这项研究中，评估了 NHE 抑制剂调节 表达的能力。EIPA 以及 5-（N，N-己二基）amiloride（HMA）均在外显子 7 剪接报告系以及 SMA 成纤维细胞中增加外显子 7 的包含。EIPA 诱导的外显子 7 包含通过一种独特的机制发生，该机制不涉及先前鉴定的剪接因子。转录组分析确定了新的靶标，包括 和 ，以进一步进行表征。EIPA 和 HMA 对 NHE5 同工型的抑制更为选择性，该同工型在成纤维细胞以及神经元细胞中表达。这些结果表明，NHE5 抑制增加了 表达，可能是治疗开发的新靶点。 意义声明：本研究证明了钠离子-蛋白交换器抑制剂在脊髓性肌萎缩症细胞中增加 可变剪接的分子机制。NHE5 选择性抑制剂通过靶向 和 表达增加全长 mRNA 的包含，这与其他 可变剪接的小分子调节剂不同。这项研究提供了一种增加全长 表达的新方法和治疗开发的新靶点。
