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 的错误定位导致类似 ALS 的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，因为缺少概括内源性 TDP-43 从细胞核到细胞质的错误定位的 TDP-43 动物模型。 CRISPR/Cas9 技术用于生成斑马鱼品系（称为 CytoTDP），该品系将内源性 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，通过全组织透明化对小胶质细胞进行免疫组织化学定位，并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 测序用于识别错误调控的途径，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、神经变性和肌肉萎缩。利用斑马鱼在受精后 5 天之前的胚胎发育完全依赖于卵黄的使用，我们证明下丘脑中的小胶质细胞增殖和激活与食物摄入无关。通过将 CytoTDP 与先前生成的 TDP-43 敲除系进行比较，转录组分析表明，内源性 TDP-43 的错误定位，而不是 TDP-43 核功能丧失，导致了早期发生的代谢功能障碍。新的 TDP-43 模型模仿了渐进性运动功能障碍的 ALS/FTLD 标志。我们的研究结果表明，下丘脑（代谢调节中心）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来识别疾病进展早期治疗干预的错误调节靶点。