Toulouse Institute for Infectious and Inflammatory Diseases (Infinity), Inserm U1291, CNRS U5051, University of Toulouse, Toulouse, France.
Necker Proteomics Platform, Structure Fédérative de Recherche Necker, INSERM US24/CNRS UAR3633, Université Paris Cité, 75015, Paris, France.
Mol Med. 2024 Nov 4;30(1):197. doi: 10.1186/s10020-024-00953-1.
In mucopolysaccharidosis type III (MPS III, also known as Sanfilippo syndrome), a pediatric neurodegenerative disorder, accumulation of abnormal glycosaminoglycans (GAGs) induces severe neuroinflammation by triggering the microglial pro-inflammatory cytokines production via a TLR4-dependent pathway. But the extent of the microglia contribution to the MPS III neuropathology remains unclear. Extracellular vesicles (EVs) mediate intercellular communication and are known to participate in the pathogenesis of adult neurodegenerative diseases. However, characterization of the molecular profiles of EVs released by MPS III microglia and their effects on neuronal functions have not been described.
Here, we isolated EVs secreted by the microglial cells after treatment with GAGs purified from urines of Sanfilippo patients (sfGAGs-EVs) or from age-matched healthy subjects (nGAGs-EVs) to explore the EVs' proteins and small RNA profiles using LC-MS/MS and RNA sequencing. We next performed a functional assay by immunofluorescence following nGAGs- or sfGAGs-EVs uptake by WT primary cortical neurons and analyzed their extensions metrics after staining of βIII-tubulin and MAP2 by confocal microscopy.
Functional enrichment analysis for both proteomics and RNA sequencing data from sfGAGs-EVs revealed a specific content involved in neuroinflammation and neurodevelopment pathways. Treatment of cortical neurons with sfGAGs-EVs induced a disease-associated phenotype demonstrated by a lower total neurite surface area, an impaired somatodendritic compartment, and a higher number of immature dendritic spines.
This study shows, for the first time, that GAGs from patients with Sanfilippo syndrome can induce microglial secretion of EVs that deliver a specific molecular message to recipient naive neurons, while promoting the neuroinflammation, and depriving neurons of neurodevelopmental factors. This work provides a framework for further studies of biomarkers to evaluate efficiency of emerging therapies.
黏多糖贮积症 III 型（MPS III，也称为 Sanfilippo 综合征）是一种儿科神经退行性疾病，异常糖胺聚糖（GAG）的积累通过 TLR4 依赖性途径触发小胶质细胞促炎细胞因子的产生，从而引起严重的神经炎症。但是，小胶质细胞对 MPS III 神经病理学的贡献程度尚不清楚。细胞外囊泡（EVs）介导细胞间通讯，已知它们参与成人神经退行性疾病的发病机制。然而，尚未描述由 MPS III 小胶质细胞释放的 EVs 的分子谱及其对神经元功能的影响。
在这里，我们分离了用从 Sanfilippo 患者尿液中纯化的 GAG（sfGAGs-EVs）或年龄匹配的健康受试者尿液中纯化的 GAG（nGAGs-EVs）处理后的小胶质细胞分泌的 EVs，以使用 LC-MS/MS 和 RNA 测序探索 EVs 的蛋白质和小 RNA 谱。接下来，我们通过免疫荧光法进行了功能测定，在 WT 原代皮质神经元摄取 nGAGs-EVs 或 sfGAGs-EVs 后，通过共聚焦显微镜分析了它们的延伸度测量值，并用 βIII-微管蛋白和 MAP2 染色。
对 sfGAGs-EVs 的蛋白质组学和 RNA 测序数据进行功能富集分析显示，存在与神经炎症和神经发育途径相关的特定内容。用 sfGAGs-EVs 处理皮质神经元会诱导疾病相关表型，表现为总神经突表面积降低，体树突区室受损，以及更多不成熟的树突棘。
这项研究首次表明，Sanfilippo 综合征患者的 GAG 可诱导小胶质细胞分泌 EVs，这些 EV 将特定的分子信息传递给未受刺激的神经元，同时促进神经炎症并剥夺神经元神经发育因子。这项工作为进一步研究生物标志物以评估新兴疗法的疗效提供了框架。
