Ramezani Meraj, Wagenknecht-Wiesner Alice, Wang Tong, Holowka David A, Eliezer David, Baird Barbara A
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853.
Department of Biochemistry, Weill Cornell Medicine, New York, NY 10065.
bioRxiv. 2023 Apr 24:2023.04.23.537965. doi: 10.1101/2023.04.23.537965.
Alpha synuclein (a-syn) is an intrinsically disordered protein prevalent in neurons, and aggregated forms are associated with synucleinopathies including Parkinson' disease (PD). Despite the biomedical importance and extensive studies, the physiological role of a-syn and its participation in etiology of PD remain uncertain. We showed previously in model RBL cells that a-syn colocalizes with mitochondrial membranes, depending on formation of N-terminal helices and increasing with mitochondrial stress. We have now characterized this colocalization and functional correlates in RBL, HEK293, and N2a cells. We find that expression of a-syn enhances stimulated mitochondrial uptake of Ca from the ER, depending on formation of its N-terminal helices but not on its disordered C-terminal tail. Our results are consistent with a-syn acting as a tether between mitochondria and ER, and we show increased contacts between these two organelles using structured illumination microscopy. We tested mitochondrial stress caused by toxins related to PD, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP/MPP+) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), and found that a-syn prevents recovery of stimulated mitochondrial Ca uptake. The C-terminal tail, and not N-terminal helices, is involved in this inhibitory activity, which is abrogated when phosphorylation site serine-129 is mutated (S129A). Correspondingly, we find that MPTP/MPP+ and CCCP stress is accompanied by both phosphorylation (pS129) and aggregation of a-syn. Overall, our results indicate that a-syn can participate as a tethering protein to modulate Ca flux between ER and mitochondria, with potential physiological significance. A-syn can also prevent cellular recovery from toxin-induced mitochondrial dysfunction, which may represent a pathological role of a-syn in the etiology of PD.
α-突触核蛋白（α-syn）是一种在神经元中普遍存在的内在无序蛋白，其聚集形式与包括帕金森病（PD）在内的突触核蛋白病有关。尽管具有重要的生物医学意义且已进行了广泛研究，但α-syn的生理作用及其在PD病因学中的参与情况仍不确定。我们之前在模型RBL细胞中表明，α-syn与线粒体膜共定位，这取决于N端螺旋的形成，并随线粒体应激而增加。我们现在已经在RBL、HEK293和N2a细胞中对这种共定位及其功能相关性进行了表征。我们发现α-syn的表达增强了内质网对Ca的刺激线粒体摄取，这取决于其N端螺旋的形成，而不取决于其无序的C端尾巴。我们的结果与α-syn作为线粒体和内质网之间的连接蛋白的作用一致，并且我们使用结构照明显微镜显示这两个细胞器之间的接触增加。我们测试了与PD相关的毒素1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP/MPP+）和羰基氰化物间氯苯腙（CCCP）引起的线粒体应激，发现α-syn可阻止刺激的线粒体Ca摄取的恢复。C端尾巴而非N端螺旋参与了这种抑制活性，当磷酸化位点丝氨酸-129发生突变（S129A）时，这种抑制活性被消除。相应地，我们发现MPTP/MPP+和CCCP应激伴随着α-syn的磷酸化（pS129）和聚集。总体而言，我们的结果表明，α-syn可以作为一种连接蛋白参与调节内质网和线粒体之间的Ca通量，具有潜在的生理意义。α-syn还可以阻止细胞从毒素诱导的线粒体功能障碍中恢复，这可能代表了α-syn在PD病因学中的病理作用。
