Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan.
Thyas Co. Ltd, Kyoto 606-8501, Japan.
Hum Mol Genet. 2023 May 5;32(10):1683-1697. doi: 10.1093/hmg/ddad008.
Membrane fusion is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. During neurotransmitter exocytosis, SNARE proteins on a synaptic vesicle and the target membrane form a complex, resulting in neurotransmitter release. N-ethylmaleimide-sensitive factor (NSF), a homohexameric ATPase, disassembles the complex, allowing individual SNARE proteins to be recycled. Recently, the association between pathogenic NSF variants and developmental and epileptic encephalopathy (DEE) was reported; however, the molecular pathomechanism of NSF-related DEE remains unclear. Here, three patients with de novo heterozygous NSF variants were presented, of which two were associated with DEE and one with a very mild phenotype. One of the DEE patients also had hypocalcemia from parathyroid hormone deficiency and neuromuscular junction impairment. Using PC12 cells, a neurosecretion model, we show that NSF with DEE-associated variants impaired the recycling of vesicular membrane proteins and vesicle enlargement in response to exocytotic stimulation. In addition, DEE-associated variants caused neurodegenerative change and defective autophagy through overactivation of the mammalian/mechanistic target of rapamycin (mTOR) pathway. Treatment with rapamycin, an mTOR inhibitor or overexpression of wild-type NSF ameliorated these phenotypes. Furthermore, neurons differentiated from patient-derived induced pluripotent stem cells showed neurite degeneration, which was also alleviated by rapamycin treatment or gene correction using genome editing. Protein structure analysis of NSF revealed that DEE-associated variants might disrupt the transmission of the conformational change of NSF monomers and consequently halt the rotation of ATP hydrolysis, indicating a dominant negative mechanism. In conclusion, this study elucidates the pathomechanism underlying NSF-related DEE and identifies a potential therapeutic approach.
膜融合由可溶性 N-乙基马来酰亚胺敏感因子附着蛋白受体 (SNARE) 蛋白介导。在神经递质胞吐过程中，突触囊泡和靶膜上的 SNARE 蛋白形成复合物，导致神经递质释放。N-乙基马来酰亚胺敏感因子 (NSF) 是一种同六聚体 ATP 酶，可拆分复合物，使单个 SNARE 蛋白得以回收。最近，报道了致病性 NSF 变体与发育性和癫痫性脑病 (DEE) 之间的关联；然而，NSF 相关 DEE 的分子病理机制尚不清楚。在此，介绍了 3 名新诊断为杂合性 NSF 变体的患者，其中 2 名与 DEE 相关，1 名表现为非常轻微的表型。1 名 DEE 患者还伴有甲状旁腺激素缺乏和神经肌肉接头损伤引起的低钙血症。使用 PC12 细胞，一种神经分泌模型，我们表明与 DEE 相关的 NSF 变体变体损害了囊泡膜蛋白的循环和囊泡对胞吐刺激的放大。此外，DEE 相关变体通过过度激活哺乳动物/雷帕霉素靶蛋白 (mTOR) 途径引起神经退行性变化和功能失调的自噬。使用雷帕霉素（一种 mTOR 抑制剂）或过表达野生型 NSF 治疗可改善这些表型。此外，来自患者来源的诱导多能干细胞分化的神经元表现出轴突退化，雷帕霉素治疗或使用基因组编辑进行基因校正也可减轻这种表型。NSF 的蛋白结构分析表明，DEE 相关变体可能破坏 NSF 单体构象变化的传递，从而阻止 ATP 水解的旋转，表明存在显性负性机制。总之，本研究阐明了 NSF 相关 DEE 的发病机制，并确定了一种潜在的治疗方法。
