Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by aberrant alternative splicing (AS). Nuclear loss and cytoplasmic accumulation of the splicing factor TDP-43 in motor neurons (MN) are hallmarks of ALS at late stages of the disease. However, it is unknown if altered AS is present before TDP-43 pathology occurs. Here, we investigate altered AS and its origins in early stages of ALS using human induced pluripotent stem cell-derived motor neurons (MNs) from sporadic and familial ALS patients. We find high levels of the RNA-binding proteins NOVA1, NOVA2, and RBFOX2 in the insoluble protein fractions and observe that AS events in ALS-associated MNs are enriched for binding sites of these proteins. Our study points to an early disrupted function of NOVA1 that drives AS changes in a complex fashion, including events caused by a consistent loss of NOVA1 function. NOVA1 exhibits increased cytoplasmic protein levels in early stage MNs without TDP-43 pathology in ALS postmortem tissue. As nuclear TDP-43 protein level depletes, NOVA1 is reduced. Potential indications for a reduction of NOVA1 also came from mice over-expressing TDP-43 lacking its nuclear localization signal and iPSC-MN stressed with puromycin. This study highlights that additional RBP-RNA perturbations in ALS occur in parallel to TDP-43.

肌萎缩侧索硬化症 (ALS) 是一种以异常可变剪接 (AS) 为特征的致命疾病。运动神经元 (MN) 中剪接因​​子 TDP-43 的核损失和细胞质积累是 ALS 在疾病晚期的标志。然而，尚不清楚在 TDP-43 病理发生之前是否存在改变的 AS。在这里，我们使用来自散发性和家族性 ALS 患者的人类诱导的多能干细胞衍生的运动神经元 (MN) 研究改变的 AS 及其在 ALS 早期阶段的起源。我们在不溶性蛋白质组分中发现高水平的 RNA 结合蛋白 NOVA1、NOVA2 和 RBFOX2，并观察到 ​​ALS 相关 MN 中的 AS 事件富含这些蛋白质的结合位点。我们的研究指出了 NOVA1 的早期破坏功能，它以复杂的方式驱动 AS 变化，包括由 NOVA1 功能持续丧失引起的事件。NOVA1 在 ALS 中没有 TDP-43 病理学的早期 MN 中表现出细胞质蛋白水平升高死后组织。随着核 TDP-43 蛋白水平的消耗，NOVA1 减少。NOVA1 减少的潜在迹象也来自过度表达缺乏其核定位信号的 TDP-43 的小鼠和用嘌呤霉素应激的 iPSC-MN。这项研究强调了 ALS 中的额外 RBP-RNA 扰动与 TDP-43 并行发生。