Instituto Cajal, CSIC, Madrid, Spain.
CIBERNED, Madrid, Spain.
Mov Disord. 2017 Oct;32(10):1409-1422. doi: 10.1002/mds.27119. Epub 2017 Aug 5.
Heterozygous mutations in the GBA1 gene, which encodes the lysosomal enzyme β-glucocerebrosidase-1, increase the risk of developing Parkinson's disease, although the underlying mechanisms remain unclear. The aim of this study was to explore the impact of the N370S-GBA1 mutation on cellular homeostasis and vulnerability in a patient-specific cellular model of PD.
We isolated fibroblasts from 4 PD patients carrying the N370S/wild type GBA1 mutation and 6 controls to study the autophagy-lysosome pathway, endoplasmic reticulum stress, and Golgi apparatus structure by Western blot, immunofluorescence, LysoTracker and Filipin stainings, mRNA analysis, and electron microscopy. We evaluated cell vulnerability by apoptosis, reactive oxygen species and mitochondrial membrane potential with flow cytometry.
The N370S mutation produced a significant reduction in β-glucocerebrosidase-1 protein and enzyme activity and β-glucocerebrosidase-1 retention within the endoplasmic reticulum, which interrupted its traffic to the lysosome. This led to endoplasmic reticulum stress activation and triggered unfolded protein response and Golgi apparatus fragmentation. Furthermore, these alterations resulted in autophagosome and p62/SQSTM1 accumulation. This impaired autophagy was a result of dysfunctional lysosomes, indicated by multilamellar body accumulation probably caused by increased cholesterol, enlarged lysosomal mass, and reduced enzyme activity. This phenotype impaired the removal of damaged mitochondria and reactive oxygen species production and enhanced cell death.
Our results support a connection between the loss of β-glucocerebrosidase-1 function, cholesterol accumulation, and the disruption of cellular homeostasis in GBA1-PD. Our work reveals new insights into the cellular pathways underlying PD pathogenesis, providing evidence that GBA1-PD shares common features with lipid-storage diseases. © 2017 International Parkinson and Movement Disorder Society.
编码溶酶体酶β-葡糖苷脑苷脂酶-1 的 GBA1 基因的杂合突变增加了帕金森病的发病风险，尽管其潜在机制尚不清楚。本研究旨在探索 N370S-GBA1 突变对 PD 患者特异性细胞模型中细胞内稳态和易损性的影响。
我们从 4 名携带 N370S/野生型 GBA1 突变的 PD 患者和 6 名对照者中分离出成纤维细胞，通过 Western blot、免疫荧光、LysoTracker 和 Filipin 染色、mRNA 分析和电子显微镜观察来研究自噬溶酶体途径、内质网应激和高尔基体结构。我们通过流式细胞术评估细胞凋亡、活性氧和线粒体膜电位来评估细胞易损性。
N370S 突变导致β-葡糖苷脑苷脂酶-1 蛋白和酶活性显著降低，并使β-葡糖苷脑苷脂酶-1 在内质网中滞留，从而中断其向溶酶体的运输。这导致内质网应激激活，并引发未折叠蛋白反应和高尔基体片段化。此外，这些改变导致自噬体和 p62/SQSTM1 的积累。这种受损的自噬是溶酶体功能障碍的结果，这表现为可能由胆固醇增加引起的多层体积累、溶酶体质量增大和酶活性降低。这种表型会损害受损线粒体的清除、活性氧的产生，并增强细胞死亡。
我们的结果支持 GBA1-PD 中β-葡糖苷脑苷脂酶-1 功能丧失、胆固醇积累和细胞内稳态破坏之间的联系。我们的工作揭示了 PD 发病机制中细胞途径的新见解，为 GBA1-PD 与脂质贮积病具有共同特征提供了证据。
