背景: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,因为缺少再现内源性TDP-43从细胞核到细胞质的错误定位的TDP-43动物模型。
方法:CRISPR/Cas9技术用于产生斑马鱼品系(称为CytoTDP),错误地将内源性TDP-43从细胞核定位到细胞质。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,通过整体组织清除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测序用于鉴定通过分子分析验证的错误调节的途径。
结果:CytoTDP鱼具有早期幼体表型,类似于ALS的临床特征,例如进行性运动缺陷,神经变性和肌肉萎缩。利用斑马鱼的胚胎发育,只依赖于卵黄的使用,直到受精后5天,我们证明了下丘脑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,导致早发代谢功能障碍。
结论:新的TDP-43模型模拟进行性运动功能障碍的ALS/FTLD标志。我们的结果表明,下丘脑的功能缺陷,代谢调节中心,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,可以识别疾病进展早期治疗干预的失调靶点.
BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.