Department of Biophysics, National Institute of Mental Health and Neurosciences, Institute of National Importance, Bengaluru, 560029, Karnataka, India.
Department of Neuropathology, National Institute of Mental Health and Neurosciences, Institute of National Importance, Bengaluru, 560029, Karnataka, India.
Mol Neurobiol. 2023 Sep;60(9):5309-5329. doi: 10.1007/s12035-023-03400-0. Epub 2023 Jun 8.
α-Synuclein has a critical role in Parkinson's disease, but the mechanism of how extracellular α-synuclein aggregates lead to astrocytic degeneration remains unknown. Our recent study in astrocytes highlighted that α-synuclein aggregates undergo lower endocytosis than the monomeric-form, even while displaying a higher impact on glutathione-machinery and glutamate-metabolism under sublethal conditions. As optimal intracellular calcium levels are essential for these functions, we aimed to study the effect of extracellular α-synuclein aggregates on ER calcium entry. We assessed the association of extracellular aggregated-α-synuclein (WT and A30P/A53T double-mutant) with the astrocytic membrane (lipid rafts) and studied its effects on membrane fluidity, ER stress, and ER calcium refilling in three systems-purified rat primary midbrain astrocyte culture, human iPSC-derived astrocytes, and U87 cells. The corresponding timeline effect on mitochondrial membrane potential was also evaluated. Post-24 h exposure to extracellular WT and mutant α-synuclein aggregates, fluorescence-based studies showed a significant increase in astrocyte membrane rigidity over control, with membrane association being significantly higher for the double mutant aggregates. α-Synuclein aggregates also showed preferentially higher association with lipid rafts of astrocytic membrane. A simultaneous increase in ER stress markers (phosphorylated PERK and CHOP) with significantly higher SOCE was also observed in aggregate-treated astrocytes, with higher levels for double mutant variant. These observations correlate with increased expression of SOCE markers, especially Orai3, on plasma membrane. Alterations in mitochondrial membrane potential were only noted post-48 h of exposure to α-synuclein aggregates. We therefore suggest that in astrocytes, α-synuclein-aggregates preferentially associate with lipid rafts of membrane, altering membrane fluidity and consequently inducing ER stress mediated by interaction with membrane SOCE proteins, resulting in higher Ca entry. A distinct cascade of events of sequential impairment of ER followed by mitochondrial alteration is observed. The study provides novel evidence elucidating relationships between extracellular α-synuclein aggregates and organellar stress in astrocytes and indicates the therapeutic potential in targeting the association of α-synuclein aggregates with astrocytic membrane.
α-突触核蛋白在帕金森病中起着关键作用，但细胞外α-突触核蛋白聚集体如何导致星形胶质细胞变性的机制尚不清楚。我们最近在星形胶质细胞中的研究强调，与单体形式相比，α-突触核蛋白聚集体的内吞作用更低，即使在亚致死条件下对谷胱甘肽机制和谷氨酸代谢的影响更高。由于最佳细胞内钙水平对于这些功能至关重要，我们旨在研究细胞外α-突触核蛋白聚集体对 ER 钙内流的影响。我们评估了细胞外聚集的α-突触核蛋白（WT 和 A30P/A53T 双突变体）与星形胶质细胞膜（脂筏）的关联，并研究了其对三种系统中的膜流动性、内质网应激和 ER 钙再填充的影响-纯化的大鼠原代中脑星形胶质细胞培养物、人 iPSC 衍生的星形胶质细胞和 U87 细胞。还评估了相应的线粒体膜电位的时间效应。暴露于细胞外 WT 和突变型α-突触核蛋白聚集体 24 小时后，基于荧光的研究表明星形胶质细胞膜刚性相对于对照显著增加，并且双突变体聚集体的膜结合明显更高。α-突触核蛋白聚集体也显示出与星形胶质细胞膜脂筏的优先更高的关联。在聚集体处理的星形胶质细胞中还观察到内质网应激标志物（磷酸化 PERK 和 CHOP）的同时增加，并且双突变体变体的水平更高。这些观察结果与质膜上 SOCE 标志物（尤其是 Orai3）的表达增加相关。仅在暴露于α-突触核蛋白聚集体 48 小时后才注意到线粒体膜电位的改变。因此，我们认为在星形胶质细胞中，α-突触核蛋白聚集体优先与膜脂筏结合，改变膜流动性，并因此通过与膜 SOCE 蛋白相互作用诱导内质网应激，导致更高的钙内流。观察到内质网随后依次受损，然后是线粒体改变的独特级联事件。该研究提供了新的证据，阐明了细胞外α-突触核蛋白聚集体与星形胶质细胞中细胞器应激之间的关系，并表明在靶向α-突触核蛋白聚集体与星形胶质细胞膜的关联方面具有治疗潜力。
