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,突触小泡和靶膜上的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),一种同聚ATPase,分解复合体,允许单个SNARE蛋白质被回收。最近,报道了致病性NSF变异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.这里,介绍了三名具有从头杂合NSF变异的患者,其中两个与DEE有关,一个与非常轻度的表型有关。其中一名DEE患者还因甲状旁腺激素缺乏和神经肌肉接头受损而出现低钙血症。使用PC12单元格,神经分泌模型,我们表明,带有DEE相关变体的NSF在胞吐刺激下损害了囊泡膜蛋白的再循环和囊泡扩大。此外,DEE相关变异体通过mTOR通路过度激活引起神经退行性改变和自噬缺陷.用雷帕霉素治疗,一种mTOR抑制剂,或野生型NSF的过表达改善了这些表型。此外,从患者来源的诱导多能干细胞分化的神经元显示神经突变性,雷帕霉素治疗或使用基因组编辑的基因校正也缓解了这种情况。NSF的蛋白质结构分析显示,DEE相关变体可能会破坏NSF单体构象变化的传递,从而停止ATP水解的旋转,表明了一种主导的消极机制。总之,本研究阐明了NSF相关DEE的病理机制,并确定了一种潜在的治疗方法.
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.