Institute of Neuroscience, Kazan State Medial University, Butlerova st. 49, Kazan 420012, Russia.
Institute of Neuroscience, Kazan State Medial University, Butlerova st. 49, Kazan 420012, Russia; Laboratory of Biophysics of Synaptic Processes, Kazan Institute of Biochemistry and Biophysics, Federal Research Center "Kazan Scientific Center of RAS", P. O. Box 30, Lobachevsky St., 2/31, Kazan 420111, Russia.
Mol Cell Neurosci. 2018 Apr;88:308-318. doi: 10.1016/j.mcn.2018.03.006. Epub 2018 Mar 14.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the initial denervation of skeletal muscle and subsequent death of motor neurons. A dying-back pattern of ALS suggests a crucial role for neuromuscular junction dysfunction. In the present study, microelectrode recording of postsynaptic currents and optical detection of synaptic vesicle traffic (FM1-43 dye) and intracellular NO levels (DAF-FM DA) were used to examine the effect of the major brain-derived cholesterol metabolite 24S-hydroxycholesterol (24S-HC, 0.4 μM) on neuromuscular transmission in the diaphragm of transgenic mice carrying a mutant superoxide dismutase 1 (SOD). We found that 24S-HC suppressed spontaneous neurotransmitter release and neurotransmitter exocytosis during high-frequency stimulation. The latter was accompanied by a decrease in both the rate of synaptic vesicle recycling and activity-dependent enhancement of NO production. Inhibition of NO synthase with L-NAME also attenuated synaptic vesicle exocytosis during high-frequency stimulation and completely abolished the effect of 24S-HC itself. Of note, 24S-HC enhanced the labeling of synaptic membranes with B-subunit of cholera toxin, suggesting an increase in lipid ordering. Lipid raft-disrupting agents (methyl-β-cyclodextrin, sphingomyelinase) prevented the action of 24S-HC on both lipid raft marker labeling and NO synthesis. Together, these experiments indicate that 24S-HC is able to suppress the exocytotic release of neurotransmitter in response to intense activity via a NO/lipid raft-dependent pathway in the neuromuscular junctions of SOD mice.
肌萎缩侧索硬化症（ALS）是一种神经退行性疾病，其特征是骨骼肌最初去神经支配，随后运动神经元死亡。ALS 的退行性变模式表明神经肌肉接头功能障碍起着关键作用。在本研究中，我们使用微电极记录突触后电流和突触小泡运输的光学检测（FM1-43 染料）以及细胞内 NO 水平（DAF-FM DA）来研究主要脑源性胆固醇代谢产物 24S-羟基胆固醇（24S-HC，0.4 μM）对携带突变超氧化物歧化酶 1（SOD）的转基因小鼠膈肌神经肌肉传递的影响。我们发现 24S-HC 抑制自发性神经递质释放和高频刺激时的神经递质胞吐作用。后者伴随着突触小泡再循环率降低和活性依赖性增强的 NO 产生。用 L-NAME 抑制一氧化氮合酶也会减弱高频刺激时的突触小泡胞吐作用，并完全消除 24S-HC 本身的作用。值得注意的是，24S-HC 增强了 B 亚单位霍乱毒素与突触膜的标记，表明脂质有序性增加。脂筏破坏剂（甲基-β-环糊精、鞘磷脂酶）可防止 24S-HC 对脂筏标记和 NO 合成的作用。总之，这些实验表明 24S-HC 能够通过 SOD 小鼠神经肌肉接头中的 NO/脂筏依赖性途径抑制高强度活动引起的神经递质的胞吐释放。
