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Abstract：
BACKGROUND: Extracellular vesicles (EVs) hold the potential for elucidating the pathogenesis of amyotrophic lateral sclerosis (ALS) and serve as biomarkers. Notably, the comparative and longitudinal alterations in the protein profiles of EVs in serum (sEVs) and cerebrospinal fluid (CSF; cEVs) of sporadic ALS (SALS) patients remain uncharted. Ropinirole hydrochloride (ROPI; dopamine D2 receptor [D2R] agonist), a new anti-ALS drug candidate identified through induced pluripotent stem cell (iPSC)-based drug discovery, has been suggested to inhibit ALS disease progression in the Ropinirole Hydrochloride Remedy for Amyotrophic Lateral Sclerosis (ROPALS) trial, but its mechanism of action is not well understood. Therefore, we tried to reveal longitudinal changes with disease progression and the effects of ROPI on protein profiles of EVs.
METHODS: We collected serum and CSF at fixed intervals from ten controls and from 20 SALS patients participating in the ROPALS trial. Comprehensive proteomic analysis of EVs, extracted from these samples, was conducted using liquid chromatography/mass spectrometer (LC/MS). Furthermore, we generated iPSC-derived astrocytes (iPasts) and performed RNA sequencing on astrocytes with or without ROPI treatment.
RESULTS: The findings revealed notable disparities yet high congruity in sEVs and cEVs protein profiles concerning disease status, time and ROPI administration. In SALS, both sEVs and cEVs presented elevated levels of inflammation-related proteins but reduced levels associated with unfolded protein response (UPR). These results mirrored the longitudinal changes after disease onset and correlated with the revised ALS Functional Rating Scale (ALSFRS-R) at sampling time, suggesting a link to the onset and progression of SALS. ROPI appeared to counteract these changes, attenuating inflammation-related protein levels and boosting those tied to UPR in SALS, proposing an anti-ALS impact on EV protein profiles. Reverse translational research using iPasts indicated that these changes may partly reflect the DRD2-dependent neuroinflammatory inhibitory effects of ROPI. We have also identified biomarkers that predict diagnosis and disease progression by machine learning-driven biomarker search.
CONCLUSIONS: Despite the limited sample size, this study pioneers in reporting time-series proteomic alterations in serum and CSF EVs from SALS patients, offering comprehensive insights into SALS pathogenesis, ROPI-induced changes, and potential prognostic and diagnostic biomarkers.
METHODS: We collected serum and CSF at fixed intervals from ten controls and from 20 SALS patients participating in the ROPALS trial. Comprehensive proteomic analysis of EVs, extracted from these samples, was conducted using liquid chromatography/mass spectrometer (LC/MS). Furthermore, we generated iPSC-derived astrocytes (iPasts) and performed RNA sequencing on astrocytes with or without ROPI treatment.
RESULTS: The findings revealed notable disparities yet high congruity in sEVs and cEVs protein profiles concerning disease status, time and ROPI administration. In SALS, both sEVs and cEVs presented elevated levels of inflammation-related proteins but reduced levels associated with unfolded protein response (UPR). These results mirrored the longitudinal changes after disease onset and correlated with the revised ALS Functional Rating Scale (ALSFRS-R) at sampling time, suggesting a link to the onset and progression of SALS. ROPI appeared to counteract these changes, attenuating inflammation-related protein levels and boosting those tied to UPR in SALS, proposing an anti-ALS impact on EV protein profiles. Reverse translational research using iPasts indicated that these changes may partly reflect the DRD2-dependent neuroinflammatory inhibitory effects of ROPI. We have also identified biomarkers that predict diagnosis and disease progression by machine learning-driven biomarker search.
CONCLUSIONS: Despite the limited sample size, this study pioneers in reporting time-series proteomic alterations in serum and CSF EVs from SALS patients, offering comprehensive insights into SALS pathogenesis, ROPI-induced changes, and potential prognostic and diagnostic biomarkers.
摘要：
背景：细胞外囊泡（EV）具有阐明肌萎缩侧索硬化症（ALS）发病机理的潜力，并可作为生物标志物。值得注意的是,散发性ALS（SALS）患者的血清（sEVs）和脑脊液（CSF；cEVs）中EV蛋白谱的比较和纵向变化仍然未知。盐酸罗匹尼罗（ROPI；多巴胺D2受体[D2R]激动剂），通过基于诱导多能干细胞（iPSC）的药物发现确定了一种新的抗ALS药物候选物，在肌萎缩性侧索硬化（ROPALS）试验中，盐酸罗匹尼罗补救措施被建议抑制ALS疾病的进展，但是它的作用机制还不清楚。因此,我们试图揭示疾病进展的纵向变化以及ROPI对EV蛋白谱的影响.
方法：我们以固定间隔从10名对照和20名参与ROPALS试验的SALS患者中收集血清和CSF。电动汽车的综合蛋白质组学分析，从这些样本中提取，使用液相色谱/质谱仪(LC/MS)进行。此外，我们产生了iPSC来源的星形胶质细胞(iPasts),并在有或没有ROPI治疗的情况下对星形胶质细胞进行了RNA测序.
结果：研究结果揭示了与疾病状态有关的sEV和cEV蛋白谱的显著差异和高度一致性，时间和ROPI管理。在Sals,sEV和cEV均呈现炎症相关蛋白水平升高,但与未折叠蛋白反应(UPR)相关的水平降低.这些结果反映了疾病发作后的纵向变化，并与采样时修订的ALS功能评定量表（ALSFRS-R）相关。提示与SALS的发病和进展有关。ROPI似乎抵消了这些变化，在SALS中降低炎症相关蛋白水平并提高与UPR相关的蛋白水平，提出了抗ALS对EV蛋白谱的影响。使用iPast的反向翻译研究表明，这些变化可能部分反映了ROPI的DRD2依赖性神经炎症抑制作用。我们还确定了通过机器学习驱动的生物标志物搜索来预测诊断和疾病进展的生物标志物。
结论：尽管样本量有限，这项研究是报告SALS患者血清和CSFEV时间序列蛋白质组学改变的先驱，提供对SALS发病机制的全面见解，ROPI引起的变化，以及潜在的预后和诊断生物标志物。
方法：我们以固定间隔从10名对照和20名参与ROPALS试验的SALS患者中收集血清和CSF。电动汽车的综合蛋白质组学分析，从这些样本中提取，使用液相色谱/质谱仪(LC/MS)进行。此外，我们产生了iPSC来源的星形胶质细胞(iPasts),并在有或没有ROPI治疗的情况下对星形胶质细胞进行了RNA测序.
结果：研究结果揭示了与疾病状态有关的sEV和cEV蛋白谱的显著差异和高度一致性，时间和ROPI管理。在Sals,sEV和cEV均呈现炎症相关蛋白水平升高,但与未折叠蛋白反应(UPR)相关的水平降低.这些结果反映了疾病发作后的纵向变化，并与采样时修订的ALS功能评定量表（ALSFRS-R）相关。提示与SALS的发病和进展有关。ROPI似乎抵消了这些变化，在SALS中降低炎症相关蛋白水平并提高与UPR相关的蛋白水平，提出了抗ALS对EV蛋白谱的影响。使用iPast的反向翻译研究表明，这些变化可能部分反映了ROPI的DRD2依赖性神经炎症抑制作用。我们还确定了通过机器学习驱动的生物标志物搜索来预测诊断和疾病进展的生物标志物。
结论：尽管样本量有限，这项研究是报告SALS患者血清和CSFEV时间序列蛋白质组学改变的先驱，提供对SALS发病机制的全面见解，ROPI引起的变化，以及潜在的预后和诊断生物标志物。
