Kuznetsova Eva A, Zakirjanova Guzalia F, Tsentsevitsky Andrei N, Petrov Alexey M
Laboratory of Biophysics of Synaptic Processes, Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center of RAS, 2/31 Lobachevsky St, Kazan, 420111, RT, Russia.
Department of Human and Animal Physiology, Lomonosov Moscow State University, Leninskiye Gory, 1, 12, Moscow, 119234, Russia.
Pflugers Arch. 2025 Mar;477(3):421-439. doi: 10.1007/s00424-024-03058-0. Epub 2025 Jan 9.
Many synaptic vesicles undergo exocytosis in motor nerve terminals during neuromuscular communication. Endocytosis then recovers the synaptic vesicle pool and presynaptic membrane area. The kinetics of endocytosis may shape neuromuscular transmission, determining its long-term reliability. Here, using fluorescent dyes, the time course of endocytosis induced by intense activity of the phrenic nerve was studied at the mouse diaphragm neuromuscular junction. It was found that a significant portion of endocytic events occurs after the end of tetanic stimulation. Pitstop 2, clathrin inhibitor, and more profoundly dynole 34-2, dynamin antagonist, suppressed endocytic FM1-43 dye uptake both during and after tetanus. Furthermore, synaptic vesicles formed in the presence of the endocytic blockers released FM-dye during subsequent evoked exocytosis at a lower rate. 25-Hydroxycholesterol (25HC) is an oxysterol, ubiquitously synthetized from excessive cholesterol. In addition, its production greatly increases by activated macrophages. 25HC accelerated FM-dye endocytosis and its sequential evoked exocytosis, and dynole (but not pitstop) prevented 25HC-mediated enhancement of endocytic FM-dye uptake. The positive effects of 25HC were interfered with chelation of cytosolic Ca with a slow Ca buffer EGTA-AM, Ca antagonist TMB8, and sphingomyelin-hydrolyzing enzyme. In contrast to amphiphilic FM1-43 dye capture, 25HC reduced uptake of hydrophilic high molecular weight markers (labeled dextrans and toxin), which utilize bulk endocytosis to enter into nerve terminals. Thus, synaptic vesicle endocytosis had a relatively slow kinetics following the tetanic activity and can be accelerated by 25HC. The positive effect of 25HC on endocytosis engages a dynamin-dependent pathway, interconnected with cytoplasmic Ca and sphingomyelin integrity.
在神经肌肉通讯过程中，许多突触小泡在运动神经末梢经历胞吐作用。然后内吞作用恢复突触小泡池和突触前膜面积。内吞作用的动力学可能影响神经肌肉传递，决定其长期可靠性。在此，利用荧光染料，在小鼠膈神经肌肉接头处研究了膈神经强烈活动诱导的内吞作用的时间进程。结果发现，很大一部分内吞事件发生在强直刺激结束后。网格蛋白抑制剂Pitstop 2，以及更显著的发动蛋白拮抗剂dynole 34-2，在破伤风期间和之后均抑制了内吞性FM1-43染料摄取。此外，在内吞阻滞剂存在下形成的突触小泡在随后诱发的胞吐作用期间以较低速率释放FM染料。25-羟基胆固醇（25HC）是一种氧甾醇，由过量胆固醇普遍合成。此外，活化的巨噬细胞会使其产量大幅增加。25HC加速了FM染料内吞作用及其随后诱发的胞吐作用，并且dynole（而非Pitstop）阻止了25HC介导的内吞性FM染料摄取增强。25HC的积极作用受到用慢钙缓冲剂EGTA-AM螯合胞质钙、钙拮抗剂TMB8和鞘磷脂水解酶的干扰。与两亲性FM1-43染料捕获相反，25HC减少了亲水性高分子量标记物（标记葡聚糖和毒素）的摄取，这些标记物利用批量内吞作用进入神经末梢。因此，强直活动后突触小泡内吞作用具有相对较慢的动力学，并且可以被25HC加速。25HC对内吞作用的积极作用涉及一条发动蛋白依赖性途径，该途径与细胞质钙和鞘磷脂完整性相互关联。
