Neuroinflammation and chronic activation of microglial cells are the prominent features of amyotrophic lateral sclerosis (ALS) pathology. While alterations in the mRNA profile of diseased microglia have been well documented, the actual microglia proteome remains poorly characterized. Here we performed a functional characterization together with proteome analyses of microglial cells at different stages of disease in the SOD1‐G93A model of ALS. Functional analyses of microglia derived from the lumbar spinal cord of symptomatic mice revealed: (i) remarkably high mitotic index (close to 100% cells are Ki67+) (ii) significant decrease in phagocytic capacity when compared to age‐matched control microglia, and (iii) diminished response to innate immune challenges in vitro and in vivo. Proteome analysis revealed a development of two distinct molecular signatures at early and advanced stages of disease. While at early stages of disease, we identified several proteins implicated in microglia immune functions such as GPNMB, HMBOX1, at advanced stages of disease microglia signature at protein level was characterized with a robust upregulation of several unconventional proteins including rootletin, major vaults proteins and STK38. Upregulation of GPNMB and rootletin has been also found in the spinal cord samples of sporadic ALS. Remarkably, the top biological functions of microglia, in particular in the advanced disease, were not related to immunity/immune response, but were highly enriched in terms linked to RNA metabolism. Together, our results suggest that, over the course of disease, chronically activated microglia develop unconventional protein signatures and gradually lose their immune identity ultimately turning into functionally inefficient immune cells.

中文翻译：

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ALS 中慢性激活的小胶质细胞逐渐失去免疫功能并形成非常规的蛋白质组

神经炎症和小胶质细胞的慢性激活是肌萎缩侧索硬化症（ALS）病理学的突出特征。虽然患病小胶质细胞 mRNA 谱的改变已得到充分记录，但实际的小胶质细胞蛋白质组仍知之甚少。在这里，我们对 ALS 的 SOD1-G93A 模型中不同疾病阶段的小胶质细胞进行了功能表征和蛋白质组分析。对来自有症状小鼠腰脊髓的小胶质细胞的功能分析显示：(i) 有丝分裂指数非常高（接近 100% 的细胞为 Ki67+）(ii) 与年龄匹配的对照小胶质细胞相比，吞噬能力显着下降，并且（ iii) 对体外和体内先天免疫挑战的反应减弱。蛋白质组分析揭示了疾病早期和晚期阶段两种不同分子特征的发展。在疾病的早期阶段，我们鉴定了几种与小胶质细胞免疫功能有关的蛋白质，例如 GPNMB、HMBOX1，在疾病的晚期阶段，小胶质细胞在蛋白质水平上的特征表现为几种非常规蛋白质的强烈上调，包括 rootletin、主要穹窿蛋白和 STK38 。在散发性 ALS 的脊髓样本中也发现了 GPNMB 和 rootletin 的上调。值得注意的是，小胶质细胞的主要生物学功能，特别是在晚期疾病中，与免疫/免疫反应无关，但在与 RNA 代谢相关的方面高度丰富。总之，我们的结果表明，在疾病过程中，长期激活的小胶质细胞会产生非常规的蛋白质特征，并逐渐失去其免疫特性，最终变成功能低下的免疫细胞。