Nucleocytoplasmic mRNA redistribution accompanies RNA binding protein mislocalisation in ALS motor neurons and is restored by VCP inhibition

Although the pathological hallmark of amyotrophic lateral sclerosis (ALS) is the nucleocytoplasmic mislocalisation of RNA binding proteins (RBPs), such as TDP-43 and FUS, the nucleocytoplasmic distribution of mRNA remains uncharacterised. Here, we used subcellular fractionation with RNA sequencing and proteomics to assess nucleocytoplasmic mRNA and protein localisation in human induced pluripotent stem cell-derived motor neurons (iPSNs) from ALS patients with TARDBP mutations, VCP mutations, and controls with VCP mutations knocked-in with CRISPR/Cas9. In each mutant group, we found substantial nucleocytoplasmic mRNA redistribution, particularly in transcripts involved in protein binding. Redistributed transcripts in ALS iPSNs were enriched in protein-coding biotypes, exhibited longer lengths, and had enhanced interactions with RBPs, including TDP-43 and FUS. Using mass spectrometry in TARDBP mutant, VCP mutant, and VCP mutant knock-in iPSNs, we reveal widespread protein mislocalisation, especially in RBPs. We find that mislocalised proteins exhibit substantially greater binding to redistributed transcripts than non-redistributed mRNAs, suggesting that RBP mislocalisation may be directly coupled to mRNA redistribution. Remarkably, treatment with the VCP D2 ATPase domain inhibitor ML240 restored both nucleocytoplasmic mRNA redistribution and protein mislocalisation not only in VCP mutants but also in TARDBP mutant iPSNs. Functional assays in VCP mutant iPSNs further confirmed that ML240 restores lysosomal localisation from the perinuclear region towards the neurites and reduces oxidative stress. Our findings demonstrate that ALS motor neurons exhibit concomitant nucleocytoplasmic mRNA redistribution and RBP

尽管肌萎缩侧索硬化症（amyotrophic lateral sclerosis, ALS）的病理标志性特征是RNA结合蛋白（RNA binding proteins, RBPs，如TDP-43与FUS）出现核质定位异常，但mRNA的核质分布状态仍未被阐明。本研究采用亚细胞分级分离结合RNA测序与蛋白质组学技术，对携带TARDBP突变、VCP突变的ALS患者来源的人类诱导多能干细胞衍生运动神经元（induced pluripotent stem cell-derived motor neurons, iPSNs），以及经CRISPR/Cas9敲入VCP突变的对照样本中的核质mRNA与蛋白质定位情况进行评估。在各突变组中，我们均发现了显著的核质mRNA重分布现象，尤其富集于参与蛋白质结合功能的转录本中。ALS患者iPSNs中发生重分布的转录本多为蛋白编码生物型，长度更长，且与包括TDP-43、FUS在内的RBPs存在更强的相互作用。通过对TARDBP突变、VCP突变及VCP突变敲入的iPSNs进行质谱分析，我们揭示了广泛的蛋白质定位异常现象，尤以RBPs为甚。我们发现，定位异常的蛋白质相较于未发生重分布的mRNA，与发生重分布的转录本的结合能力显著更强，这提示RBPs的定位异常可能直接与mRNA重分布相关联。值得注意的是，使用VCP D2 ATP酶结构域抑制剂ML240进行处理后，不仅在VCP突变样本中，同时在TARDBP突变的iPSNs中，核质mRNA重分布与蛋白质定位异常均得到了恢复。对VCP突变iPSNs的功能实验进一步证实，ML240可将溶酶体定位从核周区域向神经突方向恢复，并减轻氧化应激。本研究结果表明，ALS患者的运动神经元同时存在核质mRNA重分布与RBPs