Supplementary Material for: Targeting Voltage-Dependent Calcium Channels with Pregabalin Exerts a Direct Neuroprotective Effect in an Animal Model of Multiple Sclerosis

Background/Aims: Multiple sclerosis (MS) is a prototypical autoimmune central nervous system (CNS) disease. Particularly progressive forms of MS (PMS) show significant neuroaxonal damage as consequence of demyelination and neuronal hyperexcitation. Immuno-modulatory treatment strategies are beneficial in relapsing MS (RMS), but mostly fail in PMS. Pregabalin (Lyrica®) is prescribed to MS patients to treat neuropathic pain. Mechanistically, it targets voltage-dependent Ca2+ channels and reduces harmful neuronal hyperexcitation in mouse epilepsy models. Studies suggest that GABA analogues like pregabalin exert neuroprotective effects in animal models of ischemia and trauma. Methods: We tested the impact of pregabalin in a mouse model of MS (experimental autoimmune encephalomyelitis, EAE) and performed histological and immunological evaluations as well as intravital two-photon-microscopy of brainstem EAE lesions. Results: Both prophylactic and therapeutic treatments ameliorated the clinical symptoms of EAE and reduced immune cell infiltration into the CNS. On neuronal level, pregabalin reduced long-term potentiation in hippocampal brain slices indicating an impact on mechanisms of learning and memory. In contrast, T cells, microglia and brain endothelial cells were unaffected by pregabalin. However, we found a direct impact of pregabalin on neurons during CNS inflammation as it reversed the pathological elevation of neuronal intracellular Ca2+ levels in EAE lesions. Conclusion: The presented data suggest that pregabalin primarily acts on neuronal Ca2+ channel trafficking thereby reducing Ca2+-mediated cytotoxicity and neuronal damage in an animal model of MS. Future clinical trials need to assess the benefit for neuronal survival by expanding the indication for pregabalin administration to MS patients in further disease phases.背景与目的：多发性硬化（Multiple sclerosis）是典型的自身免疫性中枢神经系统（central nervous system）疾病。其中进展型多发性硬化（progressive forms of MS，PMS）以脱髓鞘与神经元过度兴奋引发的显著神经轴突损伤为主要特征。免疫调节治疗策略对复发型多发性硬化（relapsing MS，RMS）具有临床获益，但在PMS中大多无效。普瑞巴林（Lyrica®）常被用于治疗多发性硬化患者的神经性疼痛。从机制上讲，普瑞巴林可靶向作用于电压门控钙离子通道（voltage-dependent Ca2+ channels），在小鼠癫痫模型中可抑制有害的神经元过度兴奋。已有研究表明，诸如普瑞巴林这类γ-氨基丁酸（gamma-aminobutyric acid，GABA）类似物，在缺血和创伤的动物模型中可发挥神经保护作用。 方法：本研究在多发性硬化小鼠模型——实验性自身免疫性脑脊髓炎（experimental autoimmune encephalomyelitis，EAE）中评估普瑞巴林的干预效果，并通过组织学、免疫学检测以及脑干EAE病灶的活体双光子显微镜成像（intravital two-photon-microscopy）开展相关分析。 结果：预防性与治疗性给药均能改善EAE的临床症状，并减少免疫细胞向中枢神经系统的浸润。在神经元层面，普瑞巴林可降低海马脑片（hippocampal brain slices）的长时程增强效应（long-term potentiation），提示其对学习记忆机制存在影响。与之相反，普瑞巴林对T细胞、小胶质细胞（microglia）及脑内皮细胞（brain endothelial cells）并无显著作用。但本研究发现，在中枢神经系统炎症状态下，普瑞巴林可直接作用于神经元：其能够逆转EAE病灶中神经元细胞内钙离子水平的病理性升高。 结论：本研究数据表明，普瑞巴林主要通过调控神经元钙离子通道转运，从而降低钙离子介导的细胞毒性，在多发性硬化动物模型中减轻神经元损伤。未来需开展临床试验，拓展普瑞巴林在多发性硬化不同疾病阶段的适用指征，以评估其对神经元存活的获益价值。