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^G93A). 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^G93A mice.

肌萎缩性侧索硬化症（ALS）是一种神经退行性疾病，其特征在于骨骼肌的最初神经支配失调和随后的运动神经元死亡。ALS的垂死模式表明神经肌肉连接功能障碍的关键作用。在本研究中，使用微电极记录突触后电流并光学检测突触囊泡流量（FM1-43染料）和细胞内NO水平（DAF-FM DA）来检查主要的脑源性胆固醇代谢产物24S-羟基胆固醇的作用（24S-HC，0.4μM）对携带突变型超氧化物歧化酶1（SOD ^G93A）的转基因小鼠的diaphragm肌神经肌肉传递的影响）。我们发现24S-HC抑制高频刺激过程中自发性神经递质的释放和神经递质的胞吐作用。后者伴随着突触小泡再循环速率的降低和NO产生的活性依赖性增强。L-NAME对NO合酶的抑制作用还减弱了高频刺激过程中突触小泡的胞吐作用，并完全废除了24S-HC本身的作用。值得注意的是，24S-HC增强了霍乱毒素B亚基的突触膜标记，表明脂质有序性增加。脂质筏破坏剂（甲基-β-环糊精，鞘磷脂酶）阻止了24S-HC对脂质筏标记物标记和NO合成的作用。一起，^G93A小鼠。