Petersburg Nuclear Physics Institute Named By B.P. Konstantinov of National Research Center «Kurchatov Institute», Gatchina, Russia.
Petersburg Nuclear Physics Institute Named By B.P. Konstantinov of National Research Center «Kurchatov Institute», Gatchina, Russia; Peter the Great St.Petersburg Polytechnic University, Saint-Petersburg, Russia; Kurchatov Genome Center - PNPI, Gatchina, Russia.
Parkinsonism Relat Disord. 2021 Mar;84:112-121. doi: 10.1016/j.parkreldis.2021.02.003. Epub 2021 Feb 10.
Mutations in the glucocerebrosidase gene (GBA) encoding the lysosomal enzyme glucocerebrosidase (GCase) cause Gaucher disease (GD) and are the most commonly known genetic risk factor for Parkinson disease (PD). Ambroxol is one of the most effective pharmacological chaperones of GCase. Fourteen GD patients, six PD patients with mutations in the GBA gene (GBA-PD), and thirty controls were enrolled. GCase activity and hexosylsphingosine (HexSph) concentration were measured in dried blood and macrophage spots using liquid chromatography coupled with tandem mass spectrometry. The effect of ambroxol on GCase translocation to lysosomes was assessed using confocal microscopy. The results showed that ambroxol treatment significantly increased GCase activity in cultured macrophages derived from patient blood monocytic cell (PBMC) of GD (by 3.3-fold) and GBA-PD patients (by 3.5-fold) compared to untreated cells (p < 0.0001 and p < 0.0001, respectively) four days after cultivation. Ambroxol treatment significantly reduced HexSph concentration in GD (by 2.1-fold) and GBA-PD patients (by 1.6-fold) (p < 0.0001 and p < 0.0001, respectively). GD macrophage treatment resulted in increased GCase level and increased enzyme colocalization with the lysosomal marker LAMP2. The possible binding modes of ambroxol to mutant GCase carrying N370S amino acid substitution at pH 4.7 were examined using molecular docking and molecular dynamics simulations. The ambroxol position characterized by minimal binding free energy was observed in close vicinity to the residue, at position 370. Taken together, these data showed that PBMC-derived macrophages could be used for assessing ambroxol therapy response for GD patients and also for GBA-PD patients.
突变的葡萄糖脑苷脂酶基因（GBA）编码溶酶体酶葡萄糖脑苷脂酶（GCase）导致戈谢病（GD），是帕金森病（PD）最常见的遗传风险因素。氨溴索是 GCase 的最有效的药理学伴侣之一。研究纳入了 14 名 GD 患者、6 名携带 GBA 基因突变的 PD 患者（GBA-PD）和 30 名对照者。采用液相色谱-串联质谱法在干血斑和巨噬细胞斑中测量葡萄糖脑苷脂酶活性和己糖神经酰胺（HexSph）浓度。使用共聚焦显微镜评估氨溴索对 GCase 向溶酶体易位的影响。结果表明，与未处理的细胞相比，氨溴索处理后，GD（培养 4 天后活性增加 3.3 倍）和 GBA-PD 患者（培养 4 天后活性增加 3.5 倍）的原代培养巨噬细胞来源的 PBMC 中 GCase 活性显著增加（p<0.0001 和 p<0.0001）。氨溴索处理还显著降低了 GD（HexSph 浓度降低 2.1 倍）和 GBA-PD 患者（HexSph 浓度降低 1.6 倍）（p<0.0001 和 p<0.0001）的 HexSph 浓度。GD 巨噬细胞治疗导致 GCase 水平升高，并增加了与溶酶体标志物 LAMP2 的酶共定位。在 pH 4.7 下，使用分子对接和分子动力学模拟检查了氨溴索与携带 N370S 氨基酸取代的突变型 GCase 的可能结合模式。观察到结合自由能最小的氨溴索位置靠近残基 370。综上所述，这些数据表明，PBMC 来源的巨噬细胞可用于评估 GD 患者氨溴索治疗反应，也可用于 GBA-PD 患者。
