Institut Curie, PSL Research University, INSERM U934, CNRS UMR3215, Sorbonne Université, F-75005, Paris, France.
Present Address: VIB-KU Leuven, Center for Brain & Disease Research, Leuven, Belgium.
Mol Neurodegener. 2019 Jul 10;14(1):27. doi: 10.1186/s13024-019-0327-3.
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.
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.
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.
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.
动力蛋白激活因子 dynactin 亚基 1 是 dynactin 复合物的最大亚基，dynactin 复合物是分子马达蛋白复合物 dynein 的激活因子。在散发性肌萎缩侧索硬化症（ALS）患者中发现 DCTN1 mRNA 和蛋白质水平降低，并且突变与疾病相关，但该蛋白质在疾病发病机制中的作用仍不清楚。
我们在斑马鱼胚胎中对 Dynactin1a 缺失模型进行了表征，并将原发性运动神经元发育的体内分子分析与单细胞中的活体内轴突运输测定相结合，以研究与 ALS 相关的缺陷。为了探究神经肌肉接点（NMJ）的功能和组织，我们在活体、完整的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了突触结构。
在这里，我们表明 Dynactin1a 的缺失足以诱导脊髓运动神经元发育和 NMJ 功能的缺陷。我们观察到突触不稳定、原发性运动神经元生长受损以及 NMJ 处动作电位的失败率增加。此外，胚胎还表现出与 NMJ 功能障碍一致的运动缺陷。野生型人 DCTN1-GFP 的过表达可挽救观察到的表型，表明这是一种细胞自主机制。DCTN1-GFP 的突触积累以及 NMJ 突触的超微结构分析显示，Dynactin1a 在突触功能中具有局部作用。此外，对原发性运动神经元的活体内轴突运输和细胞骨架动力学的分析表明，这里报道的表型独立于这些过程的调节。
我们的研究揭示了 Dynactin1 在 ALS 发病机制中的新作用，它在细胞自主的基础上独立于 dynein 介导的轴突运输来促进运动神经元突触的稳定性。
