Supplementary Material for: Copper-Zinc Superoxide Dismutase (SOD1) Is Released by Microglial Cells and Confers Neuroprotection against 6-OHDA Neurotoxicity

Microglial-neuronal interactions are essential for brain physiopathology. In this framework, recent data have changed the concept of microglia from essentially macrophagic cells to crucial elements in maintaining neuronal homeostasis and function through the release of neuroprotective molecules. Using proteomic analysis, here we identify copper-zinc superoxide dismutase (SOD1) as a protein produced and released by cultured rat primary microglia. Evidence for a neuroprotective role of microglia-derived SOD1 resulted from experiments in which primary cerebellar granule neurons (CGNs) were exposed to the dopaminergic toxin 6-hydroxydopamine (6-OHDA). Microglial conditioned medium, in which SOD1 had accumulated, protected CGNs from degeneration, and neuroprotection was abrogated by SOD1 inhibitors. These effects were replicated when exogenous SOD1 was added to a nonconditioned medium. SOD1 neuroprotective action was mediated by increased cell calcium from an external source. Further experiments demonstrated the specificity of SOD1 neuroprotection against 6-OHDA compared to other types of neurotoxic challenges. SOD1, constitutively produced and released by microglia through a lysosomal secretory pathway, is identified here for the first time as an essential component of neuroprotection mediated by microglia. This novel information is relevant to stimulating further studies of microglia-mediated neuroprotection in in vivo models of neurodegenerative diseases.小胶质细胞-神经元相互作用（Microglial-neuronal interactions）对于脑病理生理学（brain physiopathology）至关重要。在此研究框架下，近期研究数据已更新了对小胶质细胞的认知：不再将其单纯视为巨噬细胞样细胞，而是可通过释放神经保护分子维持神经元稳态与功能的关键调控因子。本研究通过蛋白质组学分析（proteomic analysis），鉴定出铜锌超氧化物歧化酶（copper-zinc superoxide dismutase, SOD1）是培养的大鼠原代小胶质细胞（cultured rat primary microglia）产生并释放的蛋白质。关于小胶质细胞来源SOD1的神经保护作用的相关证据，源自原代小脑颗粒神经元（primary cerebellar granule neurons, CGNs）暴露于多巴胺能毒素6-羟基多巴胺（dopaminergic toxin 6-hydroxydopamine, 6-OHDA）的实验：积累了SOD1的小胶质细胞条件培养基可保护CGNs免于变性坏死，且该神经保护效应可被SOD1抑制剂（SOD1 inhibitors）阻断；向无条件培养基（nonconditioned medium）中添加外源性SOD1（exogenous SOD1）时，上述保护效应得以重现。SOD1的神经保护作用由外源性钙离子内流所介导。进一步实验证实，相较于其他类型的神经毒性刺激，SOD1的神经保护作用对6-OHDA具有特异性。本研究首次鉴定出：小胶质细胞通过溶酶体分泌途径（lysosomal secretory pathway）组成型产生并释放的SOD1，是小胶质细胞介导的神经保护作用的核心组分。该创新性研究成果可为进一步探索神经退行性疾病（neurodegenerative diseases）体内模型中小胶质细胞介导的神经保护作用提供重要研究方向。