Extracellular secretion is an essential mechanism for α-synuclein (α-syn) proteostasis. Although it has been reported that neuronal activity affects α-syn secretion, the underlying mechanisms remain unclear. Here, we investigated the autophagic processes that regulate the physiological release of α-syn in mouse primary cortical neurons and SH-SY5Y cells. Stimulating neuronal activity with glutamate or depolarization with high KCl enhanced α-syn secretion. This glutamate-induced α-syn secretion was blocked by a mixture of NMDA receptor antagonist AP5 and AMPA receptor antagonist NBQX, as well as by cytosolic Ca chelator BAPTA-AM. Additionally, mTOR inhibitor rapamycin increased α-syn and p62/SQSTM1 (p62) secretion, and this effect of rapamycin was reduced in primary cortical neurons deficient in the autophagy regulator beclin 1 (derived from mice). Glutamate-induced α-syn and p62 secretion was suppressed by the knockdown of , which is required for autophagosome formation. Glutamate increased LC3-II generation and decreased intracellular p62 levels, and the increase in LC3-II levels was blocked by BAPTA-AM. Moreover, glutamate promoted co-localization of α-syn with LC3-positive puncta, but not with LAMP1-positive structures in the neuronal somas. Glutamate-induced α-syn and p62 secretion were also reduced by the knockdown of , which is required for autophagosome fusion with the plasma membrane. Collectively, these findings suggest that stimulating neuronal activity mediates autophagic α-syn secretion in a cytosolic Ca-dependent manner, and autophagosomes may participate in autophagic secretion by functioning as α-syn carriers.

中文翻译：

---

神经元活动促进分泌性自噬，将 α-突触核蛋白释放到细胞外


细胞外分泌是 α-突触核蛋白 (α-syn) 蛋白质稳态的重要机制。尽管有报道称神经元活动影响α-syn分泌，但其潜在机制仍不清楚。在这里，我们研究了调节小鼠原代皮质神经元和 SH-SY5Y 细胞中 α-syn 生理释放的自噬过程。用谷氨酸刺激神经元活动或用高 KCl 去极化增强 α-syn 分泌。这种谷氨酸诱导的 α-syn 分泌被 NMDA 受体拮抗剂 AP5 和 AMPA 受体拮抗剂 NBQX 的混合物以及胞质 Ca 螯合剂 BAPTA-AM 阻断。此外，mTOR 抑制剂雷帕霉素可增加 α-syn 和 p62/SQSTM1 (p62) 的分泌，而雷帕霉素的这种作用在自噬调节因子 beclin 1（源自小鼠）缺陷的原代皮层神经元中减弱。谷氨酸诱导的 α-syn 和 p62 分泌被 的敲低所抑制，这是自噬体形成所必需的。谷氨酸增加了 LC3-II 的生成并降低了细胞内 p62 水平，并且 BAPTA-AM 阻断了 LC3-II 水平的增加。此外，谷氨酸促进 α-syn 与 LC3 阳性点的共定位，但不促进 α-syn 与神经元胞体中 LAMP1 阳性结构的共定位。谷氨酸诱导的 α-syn 和 p62 分泌也因 的敲低而减少，这是自噬体与质膜融合所必需的。总的来说，这些发现表明刺激神经元活动以胞质 Ca 依赖性方式介导自噬 α-syn 分泌，自噬体可能通过充当 α-syn 载体参与自噬分泌。