The key pathological signature of ALS/ FTLD is the mis-localization of endogenous TDP-43 from the nucleus to the cytoplasm. However, TDP-43 gain of function in the cytoplasm is still poorly understood since TDP-43 animal models recapitulating mis-localization of endogenous TDP-43 from the nucleus to the cytoplasm are missing. CRISPR/Cas9 technology was used to generate a zebrafish line (called CytoTDP), that mis-locates endogenous TDP-43 from the nucleus to the cytoplasm. Phenotypic characterization of motor neurons and the neuromuscular junction was performed by immunostaining, microglia were immunohistochemically localized by whole-mount tissue clearing and muscle ultrastructure was analyzed by scanning electron microscopy. Behavior was investigated by video tracking and quantitative analysis of swimming parameters. RNA sequencing was used to identify mis-regulated pathways with validation by molecular analysis. CytoTDP fish have early larval phenotypes resembling clinical features of ALS such as progressive motor defects, neurodegeneration and muscle atrophy. Taking advantage of zebrafish’s embryonic development that solely relys on yolk usage until 5 days post fertilization, we demonstrated that microglia proliferation and activation in the hypothalamus is independent from food intake. By comparing CytoTDP to a previously generated TDP-43 knockout line, transcriptomic analyses revealed that mis-localization of endogenous TDP-43, rather than TDP-43 nuclear loss of function, leads to early onset metabolic dysfunction. The new TDP-43 model mimics the ALS/FTLD hallmark of progressive motor dysfunction. Our results suggest that functional deficits of the hypothalamus, the metabolic regulatory center, might be the primary cause of weight loss in ALS patients. Cytoplasmic gain of function of endogenous TDP-43 leads to metabolic dysfunction in vivo that are reminiscent of early ALS clinical non-motor metabolic alterations. Thus, the CytoTDP zebrafish model offers a unique opportunity to identify mis-regulated targets for therapeutic intervention early in disease progression.

中文翻译：

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内源性 TDP-43 的错误定位导致 ALS 样早期代谢功能障碍和进行性运动缺陷


ALS/FTLD 的关键病理特征是内源性 TDP-43 从细胞核到细胞质的错误定位。然而，TDP-43 在细胞质中的功能获得仍然知之甚少，因为缺少 TDP-43 动物模型概括内源性 TDP-43 从细胞核到细胞质的错误定位。CRISPR/Cas9 技术用于生成斑马鱼系（称为 CytoTDP），该系将内源性 TDP-43 从细胞核错误定位到细胞质。通过免疫染色进行运动神经元和神经肌肉接头的表型表征，通过整体组织透明化对小胶质细胞进行免疫组织化学定位，通过扫描电子显微镜分析肌肉超微结构。通过视频跟踪和游泳参数的定量分析来研究行为。RNA 测序用于识别失调的通路，并通过分子分析进行验证。CytoTDP 鱼具有类似于 ALS 临床特征的早期幼虫表型，例如进行性运动缺陷、神经退行性和肌肉萎缩。利用斑马鱼的胚胎发育在受精后 5 天之前完全依赖于蛋黄的使用，我们证明了下丘脑中小胶质细胞的增殖和激活与食物摄入无关。通过将 CytoTDP 与先前生成的 TDP-43 敲除细胞系进行比较，转录组学分析显示，内源性 TDP-43 的错误定位，而不是 TDP-43 核功能丧失，导致早发性代谢功能障碍。新的 TDP-43 模型模拟了进行性运动功能障碍的 ALS/FTLD 标志。我们的结果表明，代谢调节中心下丘脑的功能缺陷可能是 ALS 患者体重减轻的主要原因。 内源性 TDP-43 的细胞质功能获得导致体内代谢功能障碍，这让人想起早期 ALS 临床非运动代谢改变。因此，CytoTDP 斑马鱼模型提供了一个独特的机会，可以在疾病进展的早期识别错误调节的靶点以进行治疗干预。