Institute of Infection, Immunity, and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.
Institute of Infection, Immunity, and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.
Exp Neurol. 2022 Sep;355:114127. doi: 10.1016/j.expneurol.2022.114127. Epub 2022 May 29.
The acute motor axonal variant of Guillain-Barré syndrome is associated with the attack of motor axons by anti-ganglioside antibodies which activate complement on the axonal plasma membrane. Animal models have indirectly implicated complement pore-mediated calcium influx as a trigger of axonal damage, through the activation of the protease calpain. However, this calcium influx has never been imaged directly. Herein we describe a method to detect changes in intra-axonal calcium in an ex vivo mouse model of axonal Guillain-Barré syndrome and describe the influence of calcium on axonal injury and the effects of calpain inhibition on axonal outcome. Using ex vivo nerve-muscle explants from Thy1-TNXXL mice which axonally express a genetically encoded calcium indicator, we studied the effect of the binding and activation of complement by an anti-GD1b ganglioside antibody which targets the motor axon. Using live multiphoton imaging, we found that a wave of calcium influx extends retrogradely from the motor nerve terminal as far back as the large bundles within the muscle explant. Despite terminal complement pores being detectable only at the motor nerve terminal and, to a lesser degree, the most distal node of Ranvier, disruption of axonal proteins occurred at more proximal sites implicating the intra-axonal calcium wave. Morphological analysis indicated two different types of calcium-induced changes: acutely, distal axons showed swelling and breakdown at sites where complement pores were present. Distally, in areas of raised calcium which lacked detectable complement pores, axons developed a spindly, vacuolated appearance suggestive of early signs of degeneration. All morphological changes were prevented with treatment with a calpain inhibitor. This is the first investigation of axonal calcium dynamics in a mouse model of Guillain-Barré syndrome and demonstrates the proximal reach of calcium influx following an injury which is confined to the most distal parts of the motor axon. We also demonstrate that calpain inhibition remains a promising candidate for both acute and sub-acute consequences of calcium-induced calpain activation.
急性运动轴索性吉兰-巴雷综合征与抗神经节苷脂抗体攻击运动轴突有关，这些抗体激活轴突质膜上的补体。动物模型间接表明，补体孔介导的钙内流通过激活蛋白酶钙蛋白酶，成为轴突损伤的触发因素。然而，这种钙内流从未被直接成像。本文描述了一种在轴突吉兰-巴雷综合征的体外小鼠模型中检测轴内钙变化的方法，并描述了钙对轴突损伤的影响以及钙蛋白酶抑制对轴突结果的影响。我们使用在体外神经-肌肉外植体中，从 Thy1-TNXXL 小鼠中，其轴突表达基因编码的钙指示剂，研究了靶向运动轴突的抗 GD1b 神经节苷脂抗体结合和激活补体对轴突的影响。使用活多光子成像，我们发现钙内流波从运动神经末梢逆行扩展，远至肌肉外植体中的大束。尽管终端补体孔仅在运动神经末梢和程度较轻的最远端郎飞氏结处可检测到，但轴突蛋白的破坏发生在更靠近近端的部位，这表明存在轴内钙波。形态分析表明两种不同类型的钙诱导变化：急性时，在存在补体孔的部位，远端轴突显示肿胀和破裂。在远端，在钙升高但缺乏可检测补体孔的区域，轴突呈现出纺锤形、空泡样外观，提示早期退变迹象。所有形态变化都可以通过使用钙蛋白酶抑制剂来预防。这是首次在吉兰-巴雷综合征的小鼠模型中研究轴突钙动力学，并证明了钙内流的近端范围，而损伤仅限于运动轴突的最远端部分。我们还证明钙蛋白酶抑制仍然是钙诱导钙蛋白酶激活的急性和亚急性后果的有希望的候选药物。
