Pharmacotherapy alleviates pathological changes in human direct reprogrammed neuronal cell model of myotonic dystrophy type 1.
Pharmacotherapy alleviates pathological changes in human direct reprogrammed neuronal cell model of myotonic dystrophy type 1.
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DOI：
10.1371/journal.pone.0269683
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发表时间：
2022
期刊：
影响因子：
3.7
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中科院分区：
文献类型：
--
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强直性肌营养不良1型（DM1）是一种影响多个器官的三核苷酸重复疾病。然而，大多数研究都集中在研究和治疗其肌肉症状。另一方面，尽管神经系统症状对患者的生活质量有显著影响，但由于DM 1脑特异性动物模型的重现性不足，没有研究药物治疗。为建立DM1神经元模型，采用慢病毒介导的针对多聚嘧啶结合蛋白（PTBP）的小发夹RNA（shRNA）将人皮肤成纤维细胞直接转化为神经元。我们发现，与对照人类诱导神经元（cohohiNeurons）相比，DM1人类诱导神经元（DM1 hiNeurons）的变性更快，表现为从病毒感染（DPI）后10天开始的较低活力和在15 DPI时的异常轴突生长。在10 DPI时，大多数DM1 hiNeuron中存在核RNA灶。此外，DM1 hiNeurons模拟了10 DPI时MBNL1和2、MAPT、CSNK1D和MPRIP的异常剪接。我们测试了两种在非神经元模型中对DM1有效的药物，发现用100 nM或200 nM放线菌素D（ACT）处理DM1 hiNeurons 24 h导致每个细胞核的RNA灶数量以剂量依赖性方式减少50%以上，在200 nM浓度下，含RNA灶的细胞核数量减少16.5%，用35 μM或65 μM红霉素处理48 h，MBNL 1外显子5和MBNL 2外显子5和8的剪接分别高达17.5%、10%和8.5%。此外，红霉素挽救了MAPT外显子2，CSNK1D外显子9和MPRIP外显子9的异常剪接，最高分别为46.4%，30.7%和19.9%。这些结果证明我们的模型是神经系统详细病因检查和新药筛选的一个有前途的工具。
Myotonic dystrophy type 1 (DM1) is a trinucleotide repeat disorder affecting multiple organs. However, most of the research is focused on studying and treating its muscular symptoms. On the other hand, despite the significant impact of the neurological symptoms on patients’ quality of life, no drug therapy was studied due to insufficient reproducibility in DM1 brain-specific animal models. To establish DM1 neuronal model, human skin fibroblasts were directly converted into neurons by using lentivirus expressing small hairpin RNA (shRNA) against poly-pyrimidine tract binding protein (PTBP). We found faster degeneration in DM1 human induced neurons (DM1 hiNeurons) compared to control human induced neurons (ctrl hiNeurons), represented by lower viability from 10 days post viral-infection (DPI) and abnormal axonal growth at 15 DPI. Nuclear RNA foci were present in most of DM1 hiNeurons at 10 DPI. Furthermore, DM1 hiNeurons modelled aberrant splicing of MBNL1 and 2, MAPT, CSNK1D and MPRIP at 10 DPI. We tested two drugs that were shown to be effective for DM1 in non-neuronal model and found that treatment of DM1 hiNeurons with 100 nM or 200 nM actinomycin D (ACT) for 24 h resulted in more than 50% reduction in the number of RNA foci per nucleus in a dose dependent manner, with 16.5% reduction in the number of nuclei containing RNA foci at 200 nM and treatment with erythromycin at 35 μM or 65 μM for 48 h rescued mis-splicing of MBNL1 exon 5 and MBNL 2 exons 5 and 8 up to 17.5%, 10% and 8.5%, respectively. Moreover, erythromycin rescued the aberrant splicing of MAPT exon 2, CSNK1D exon 9 and MPRIP exon 9 to a maximum of 46.4%, 30.7% and 19.9%, respectively. These results prove that our model is a promising tool for detailed pathogenetic examination and novel drug screening for the nervous system.
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