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.