BACKGROUND Dynactin subunit 1 is the largest subunit of the dynactin complex, an activator of the molecular motor protein complex dynein. Reduced levels of DCTN1 mRNA and protein have been found in sporadic amyotrophic lateral sclerosis (ALS) patients, and mutations have been associated with disease, but the role of this protein in disease pathogenesis is still unknown. METHODS We characterized a Dynactin1a depletion model in the zebrafish embryo and combined in vivo molecular analysis of primary motor neuron development with live in vivo axonal transport assays in single cells to investigate ALS-related defects. To probe neuromuscular junction (NMJ) function and organization we performed paired motor neuron-muscle electrophysiological recordings and GCaMP calcium imaging in live, intact larvae, and the synapse structure was investigated by electron microscopy. RESULTS Here we show that Dynactin1a depletion is sufficient to induce defects in the development of spinal cord motor neurons and in the function of the NMJ. We observe synapse instability, impaired growth of primary motor neurons, and higher failure rates of action potentials at the NMJ. In addition, the embryos display locomotion defects consistent with NMJ dysfunction. Rescue of the observed phenotype by overexpression of wild-type human DCTN1-GFP indicates a cell-autonomous mechanism. Synaptic accumulation of DCTN1-GFP, as well as ultrastructural analysis of NMJ synapses exhibiting wider synaptic clefts, support a local role for Dynactin1a in synaptic function. Furthermore, live in vivo analysis of axonal transport and cytoskeleton dynamics in primary motor neurons show that the phenotype reported here is independent of modulation of these processes. CONCLUSIONS Our study reveals a novel role for Dynactin1 in ALS pathogenesis, where it acts cell-autonomously to promote motor neuron synapse stability independently of dynein-mediated axonal transport.

中文翻译：

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Dynactin1耗竭导致神经肌肉突触不稳定和功能异常。

背景技术动态肌动蛋白亚基1是动态肌动蛋白复合物的最大亚基，动态肌动蛋白复合物是分子运动蛋白复合物动力蛋白的激活剂。在散发性肌萎缩性侧索硬化症（ALS）患者中发现DCTN1 mRNA和蛋白水平降低，并且突变与疾病相关，但该蛋白在疾病发病机理中的作用仍未知。方法我们对斑马鱼胚胎中的Dynactin1a耗竭模型进行了表征，并在单个细胞中结合了原发性运动神经元发育的体内分子分析与活体内轴突运输测定，以研究ALS相关缺陷。为了探查神经肌肉接头（NMJ）的功能和组织，我们在完整的活幼虫中进行了配对的运动神经元-肌肉电生理记录和GCaMP钙成像，并通过电子显微镜研究突触的结构。结果在这里，我们显示Dynactin1a耗竭足以诱导脊髓运动神经元的发育和NMJ的功能中的缺陷。我们观察到突触不稳定，主要运动神经元的生长受损，以及NMJ动作电位的更高失败率。另外，胚胎显示出与NMJ功能障碍一致的运动缺陷。通过野生型人DCTN1-GFP的过表达抢救观察到的表型表明细胞自主机制。DCTN1-GFP的突触积累，以及表现出较宽突触间隙的NMJ突触的超微结构分析，都支持Dynactin1a在突触功能中的局部作用。此外，对原代运动神经元中轴突运输和细胞骨架动力学的活体内分析表明，此处报道的表型与这些过程的调节无关。结论我们的研究揭示了Dynactin1在ALS发病机制中的新作用，其中它独立于动力蛋白介导的轴突运输，在细胞中自主发挥作用来促进运动神经元突触的稳定性。