Kim Sunghee, An Subin, Lee Jinwoo, Jeong Yideul, You Chang-Lim, Kim Hyebeen, Bae Ju-Hyeon, Yun Chae-Eun, Ryu Dongryul, Bae Gyu-Un, Kang Jong-Sun
Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, South Korea.
Single Cell Network Research Center, Sungkyunkwan University School of Medicine, Suwon, South Korea.
J Cachexia Sarcopenia Muscle. 2023 Oct;14(5):2239-2252. doi: 10.1002/jcsm.13308. Epub 2023 Aug 9.
The functional deterioration and loss of motor neurons are tightly associated with degenerative motor neuron diseases and aging-related muscle wasting. Motor neuron diseases or aging-related muscle wasting in turn contribute to increased risk of adverse health outcomes in the elderly. Cdon (cell adhesion molecule-downregulated oncogene) belongs to the immunoglobulin superfamily of cell adhesion molecule and plays essential roles in multiple signalling pathways, including sonic hedgehog (Shh), netrin, and cadherin-mediated signalling. Cdon as a Shh coreceptor plays a critical role in motor neuron specification during embryonic development. However, its role in adult motor neuron function is unknown.
Hb9-Cre recombinase-driven motor neuron-specific Cdon deficient mice (mnKO) and a compound mutant mice (mnKO::SOD1 ) were generated to investigate the role of Cdon in motor neuron degeneration. Motor neuron regeneration was examined by using a sciatic nerve crush injury model. To investigate the phenotype, physical activity, compound muscle action potential, immunostaining, and transmission electron microscopy were carried out. In the mechanism study, RNA sequencing and RNA/protein analyses were employed.
Mice lacking Cdon in motor neurons exhibited middle age onset lethality and aging-related decline in motor function. In the sciatic nerve crush injury model, mnKO mice exhibited an impairment in motor function recovery evident by prolonged compound muscle action potential duration (4.63 ± 0.35 vs. 3.93 ± 0.22 s for f/f, P < 0.01) and physical activity. Consistently, neuromuscular junctions of mnKO muscles were incompletely occupied (49.79 ± 5.74 vs. 79.39 ± 3.77% fully occupied neuromuscular junctions for f/f, P < 0.0001), suggesting an impaired reinnervation. The transmission electron microscopy analysis revealed that mnKO sciatic nerves had smaller axon diameter (0.88 ± 0.13 vs. 1.43 ± 0.48 μm for f/f, P < 0.0001) and myelination defects. RNA sequencing of mnKO lumbar spinal cords showed alteration in genes related to neurogenesis, inflammation and cell death. Among the altered genes, ErbB4 and FgfR expressions were significantly altered in mnKO as well as in Cdon-depleted NSC34 motor neuron cells. Consistently, Cdon-depleted NSC34 cells exhibited elevated levels of cleaved Caspase3 and γH2AX proteins, as well as Bax transcription. Cdon-depleted NSC34 cells also exhibited impaired activation of Akt in response to neuregulin-1 (NRG1) treatment.
Our current data demonstrate the functional importance of Cdon in motor neuron function and nerve repair. Cdon ablation causes alterations in neurotrophin signalling that leads to motor neuron degeneration.
运动神经元的功能退化和丧失与退行性运动神经元疾病以及与衰老相关的肌肉萎缩密切相关。运动神经元疾病或与衰老相关的肌肉萎缩反过来又会增加老年人不良健康结局的风险。Cdon（细胞粘附分子下调癌基因）属于细胞粘附分子免疫球蛋白超家族，在多种信号通路中发挥重要作用，包括音猬因子（Shh）、网蛋白和钙粘蛋白介导的信号通路。Cdon作为Shh共受体在胚胎发育过程中的运动神经元特化中起关键作用。然而，其在成年运动神经元功能中的作用尚不清楚。
构建Hb9-Cre重组酶驱动的运动神经元特异性Cdon缺陷小鼠（mnKO）和复合突变小鼠（mnKO::SOD1），以研究Cdon在运动神经元退化中的作用。使用坐骨神经挤压损伤模型检测运动神经元再生。为了研究表型，进行了身体活动、复合肌肉动作电位、免疫染色和透射电子显微镜检查。在机制研究中，采用了RNA测序和RNA/蛋白质分析。
运动神经元中缺乏Cdon的小鼠表现出中年发病的致死率和与衰老相关的运动功能下降。在坐骨神经挤压损伤模型中，mnKO小鼠表现出运动功能恢复受损，表现为复合肌肉动作电位持续时间延长（f/f组为4.63±0.35秒，mnKO组为3.93±0.22秒，P<0.01）和身体活动减少。一致地，mnKO肌肉的神经肌肉接头未被完全占据（f/f组完全占据的神经肌肉接头为79.39±3.77%，mnKO组为49.79±5.74%，P<0.0001），表明神经再支配受损。透射电子显微镜分析显示，mnKO坐骨神经的轴突直径较小（f/f组为1.43±0.48μm，mnKO组为0.88±0.13μm，P<0.0001）且存在髓鞘形成缺陷。mnKO腰脊髓的RNA测序显示与神经发生、炎症和细胞死亡相关的基因发生改变。在这些改变的基因中，ErbB4和FgfR的表达在mnKO以及Cdon缺失的NSC34运动神经元细胞中显著改变。一致地，Cdon缺失的NSC34细胞中裂解的Caspase3和γH2AX蛋白水平以及Bax转录水平升高。Cdon缺失的NSC34细胞在接受神经调节蛋白-1（NRG1）治疗后也表现出Akt激活受损。
我们目前的数据证明了Cdon在运动神经元功能和神经修复中的功能重要性。Cdon缺失导致神经营养因子信号改变，进而导致运动神经元退化。
