背景:脊髓性肌萎缩症(SMA)是一种罕见的神经肌肉疾病,也是婴儿死亡的主要遗传原因。SMA主要由存活运动神经元1(SMN1)基因的缺失引起,留下重复基因SMN2作为SMN蛋白的唯一来源。SMN2的剪接缺陷(外显子7跳跃)导致SMN蛋白的量不足。因此,纠正这种SMN2拼接缺陷被认为是治疗SMA的一种有前途的方法。
目的:本研究旨在鉴定植物资源中的活性化合物和提取物,以通过校正SMN2剪接来挽救SMA表型。
方法:可用的植物资源,使用基于稳健荧光素酶的SMN2剪接报告分子筛选具有SMA相关传统医学信息的候选人.进一步评估初级命中纠正SMA患者来源的成纤维细胞中剪接缺陷和由此产生的SMN活性增加的能力。最后测试确认的命中以确定对严重Δ7SMA小鼠的有益效果。
方法:使用基于荧光素酶的SMN2剪接报告分子和随后的SMN2mRNA的RT-PCR分析SMN2剪接。SMA表型通过生存率进行评估,体重,Δ7SMA小鼠的正正反射。
结果:在对492种植物提取物的筛选中,我们发现鸦胆子提取物及其主要成分BruceaD具有SMN2剪接校正活性。在SMA成纤维细胞中进一步证实了它们纠正剪接缺陷的能力和由此产生的SMN活性增加。重要的是,两种B.javanica和BruceineD显着改善了表型缺陷,尤其是肌肉功能,在SMA小鼠中。异质核核糖核蛋白A1(hnRNPA1)的表达降低有助于修正爪哇芽孢杆菌的剪接。
结论:我们的工作表明,javanica和BruceineD纠正了SMN2剪接缺陷并改善了小鼠SMA的症状。这些资源将为开发植物来源的SMA药物候选物提供另一种可能性。
BACKGROUND: Spinal muscular atrophy (SMA) is a rare neuromuscular disease and a leading genetic cause of infant mortality. SMA is caused primarily by the deletion of the survival motor neuron 1 (SMN1) gene, which leaves the duplicate gene SMN2 as the sole source of SMN protein. The splicing defect (exon 7 skipping) of SMN2 leads to an insufficient amount of SMN protein. Therefore, correcting this SMN2 splicing defect is considered to be a promising approach for the treatment of SMA.
OBJECTIVE: This study aimed to identify active compounds and extracts from plant resources to rescue SMA phenotypes through the correction of SMN2 splicing.
METHODS: Of available plant resources, candidates with SMA-related traditional medicine information were selected for screening using a robust luciferase-based SMN2 splicing reporter. Primary hits were further evaluated for their ability to correct the splicing defect and resultant increase of SMN activity in SMA patient-derived fibroblasts. Confirmed hits were finally tested to determine the beneficial effects on the severe Δ7 SMA mouse.
METHODS: SMN2 splicing was analyzed using a luciferase-based SMN2 splicing reporter and subsequent RT-PCR of SMN2 mRNAs. SMA phenotypes were evaluated by the survival, body weights, and righting reflex of Δ7 SMA mice.
RESULTS: In a screen of 492 selected plant extracts, we found that Brucea javanica extract and its major constituent Bruceine D have SMN2 splicing-correcting activity. Their ability to correct the splicing defect and the resulting increased SMN activity were further confirmed in SMA fibroblasts. Importantly, both B. javanica and Bruceine D noticeably improved the phenotypic defects, especially muscle function, in SMA mice. Reduced expression of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) contributed to the correction of splicing by B. javanica.
CONCLUSIONS: Our work revealed that B. javanica and Bruceine D correct the SMN2 splicing defect and improve the symptoms of SMA in mice. These resources will provide another possibility for development of a plant-derived SMA drug candidate.