Magalhães-Gomes Matheus P S, Camargos Wallace, Valadão Priscila A C, Garcias Rubens S, Rodrigues Hermann A, Andrade Jéssica N, Teixeira Vanessa P, Naves Lígia A, Cavalcante Walter L G, Gallaci Marcia, Guatimosim Silvia, Prado Vânia F, Prado Marco A M, Guatimosim Cristina
Departamento de Morfologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil; Departamento de Medicina, Faculdade Ciências Médicas de Minas Gerais, FCMMG, Belo Horizonte, MG, Brazil.
Departamento de Fisiologia e Biofísica, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
Neuroscience. 2021 Apr 15;460:31-42. doi: 10.1016/j.neuroscience.2020.12.025. Epub 2021 Feb 4.
In vertebrates, muscle activity is dependent on acetylcholine (ACh) released from neuromuscular junctions (NMJs), and changes in cholinergic neurotransmission are linked to a variety of neuromuscular diseases, including congenital myasthenic syndromes (CMS). The storage and release of ACh depends on the activity of the Vesicular Acetylcholine Transporter (VAChT), a rate-limiting step for cholinergic neurotransmission whose loss of function mutations was shown to cause human congenital myasthenia. However, we know much less about increased VAChT activity, due to copy number variations, for example. Therefore, here we investigated the impact of increased VAChT expression and consequently ACh levels at the synaptic cleft of the diaphragm NMJs. We analyzed structure and function of nerve and muscles from a mouse model of cholinergic hyperfunction (ChAT-ChR2-EYFP) with increased expression of VAChT. Our results showed a significant increase of ACh released under evoked stimuli. However, we observed deleterious changes in synaptic vesicles cycle (impaired endocytosis and decrease in vesicles number), together with structural alterations of NMJs. Interestingly, ultrastructure analyses showed that synaptic vesicles from ChAT-ChR2-EYFP mice NMJs were larger, which might be related to increased ACh load. We also observed that these larger synaptic vesicles were less rounded in comparison with control. Finally, we showed that ChAT-ChR2-EYFP mice NMJs have compromised safety factor, possible due to the structural alterations we described. These findings reveal that physiological cholinergic activity is important to maintain the structure and function of the neuromuscular system and help to understand some of the neuromuscular adverse effects experienced by chronically increased NMJ neurotransmission, such as individuals treated with cholinesterase inhibitors.
在脊椎动物中，肌肉活动依赖于神经肌肉接头（NMJ）释放的乙酰胆碱（ACh），胆碱能神经传递的变化与多种神经肌肉疾病相关，包括先天性肌无力综合征（CMS）。ACh的储存和释放取决于囊泡型乙酰胆碱转运体（VAChT）的活性，这是胆碱能神经传递的限速步骤，其功能丧失突变已被证明会导致人类先天性肌无力。然而，例如由于拷贝数变异导致的VAChT活性增加，我们了解得要少得多。因此，在这里我们研究了VAChT表达增加以及随之而来的膈肌神经肌肉接头突触间隙中ACh水平升高的影响。我们分析了胆碱能功能亢进小鼠模型（ChAT-ChR2-EYFP）中神经和肌肉的结构与功能，该模型中VAChT表达增加。我们的结果显示，在诱发刺激下释放的ACh显著增加。然而，我们观察到突触小泡循环出现有害变化（内吞作用受损和小泡数量减少），同时神经肌肉接头出现结构改变。有趣的是，超微结构分析表明，ChAT-ChR2-EYFP小鼠神经肌肉接头的突触小泡更大，这可能与ACh负载增加有关。我们还观察到，与对照组相比，这些更大的突触小泡形状更不圆润。最后，我们表明ChAT-ChR2-EYFP小鼠的神经肌肉接头安全系数受损，可能是由于我们所描述的结构改变。这些发现揭示了生理性胆碱能活动对于维持神经肌肉系统的结构和功能很重要，并有助于理解神经肌肉接头神经传递长期增加所经历的一些神经肌肉不良反应，例如接受胆碱酯酶抑制剂治疗的个体。
