Amyotrophic lateral sclerosis (ALS) presents with focal muscle weakness due to motor neuron degeneration that becomes generalized, leading to death from respiratory failure within 3-5 years from symptom onset. Despite the heterogeneity of aetiology, TDP-43 proteinopathy is a common pathological feature that is observed in >95% of ALS and tau-negative frontotemporal dementia (FTD) cases. TDP-43 is a DNA/RNA-binding protein that in ALS and FTD translocates from being predominantly nuclear to form detergent-resistant, hyperphosphorylated aggregates in the cytoplasm of affected neurons and glia. Mutations in TARDBP account for 1-4% of all ALS cases and almost all arise in the low complexity C-terminal domain that does not affect RNA binding and processing. Here we report an ALS/FTD kindred with a novel K181E TDP-43 mutation that is located in close proximity to the RRM1 domain. To offer predictive gene testing to at-risk family members, we undertook a series of functional studies to characterize the properties of the mutation. Spectroscopy studies of the K181E protein revealed no evidence of significant misfolding. Although it is unable to bind to or splice RNA, it forms abundant aggregates in transfected cells. We extended our study to include other ALS-linked mutations adjacent to the RRM domains that also disrupt RNA binding and greatly enhance TDP-43 aggregation, forming detergent-resistant and hyperphosphorylated inclusions. Lastly, we demonstrate that K181E binds to, and sequesters, wild-type TDP-43 within nuclear and cytoplasmic inclusions. Thus, we demonstrate that TDP-43 mutations that disrupt RNA binding greatly enhance aggregation and are likely to be pathogenic as they promote wild-type TDP-43 to mislocalize and aggregate acting in a dominant-negative manner. This study highlights the importance of RNA binding to maintain TDP-43 solubility and the role of TDP-43 aggregation in disease pathogenesis.

中文翻译：

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RRM 相邻 TARDBP 突变破坏 RNA 结合并增强 TDP-43 蛋白病。

肌萎缩侧索硬化症 (ALS) 表现为全身运动神经元变性导致的局灶性肌肉无力，导致症状出现后 3-5 年内因呼吸衰竭而死亡。尽管病因具有异质性，但 TDP-43 蛋白病是一种常见的病理特征，在 >95% 的 ALS 和 tau 阴性额颞叶痴呆 (FTD) 病例中观察到。TDP-43 是一种 DNA/RNA 结合蛋白，在 ALS 和 FTD 中，它从主要是核易位，在受影响的神经元和神经胶质的细胞质中形成抗去污剂、过度磷酸化的聚集体。TARDBP 中的突变占所有 ALS 病例的 1-4%，并且几乎所有突变都出现在不影响 RNA 结合和加工的低复杂性 C 末端结构域中。在这里，我们报告了一个具有新型 K181E TDP-43 突变的 ALS/FTD 亲属，该突变位于靠近 RRM1 域的位置。为了向有风险的家庭成员提供预测性基因测试，我们进行了一系列功能研究来表征突变的特性。K181E 蛋白的光谱学研究显示没有明显错误折叠的证据。尽管它不能与 RNA 结合或剪接，但它在转染的细胞中会形成丰富的聚集体。我们将研究扩展到包括与 RRM 结构域相邻的其他 ALS 相关突变，这些突变也会破坏 RNA 结合并大大增强 TDP-43 聚集，形成抗去污剂和过度磷酸化的内含物。最后，我们证明 K181E 在核和细胞质包涵体中结合并隔离野生型 TDP-43。因此，我们证明了破坏 RNA 结合的 TDP-43 突变极大地增强了聚集，并且可能是致病的，因为它们促进野生型 TDP-43 错误定位并以显性负性方式聚集。本研究强调了 RNA 结合对维持 TDP-43 溶解度的重要性以及 TDP-43 聚集在疾病发病机制中的作用。