Involvement of mTOR pathway in neurodegeneration in NSF-related developmental and epileptic encephalopathy

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 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

濾紙電気泳動法における泳動距離について(第7報) : MG値の検討(その2)

前報で吾々は濾紙面より蒸発を許す水平型濾紙電気泳動装置中の血清及びアミノ酸のMG値が、種々の泳動条件の影響を受けない恒数となることを数学的解析を用いて証明し、更に此等のMG値を実測によつて求めた。その後吾々はアミノ酸、血清蛋白について更に多くの実験を行い、統計的にMG値を求めると共に、前報で扱つた諸色素についても新しい定義によるMG値の解析及び実測を行い、良好な結果を得ることが出来た。之等をまとめて報告する次村である

濾紙電気泳動法における泳動距離について(第6報) : 色素類の泳動距離の検討(その1)

前報において、吾々は水平型濾紙電気泳動装置内でアミノ酸並びに血清蛋白質の泳動速度が時間的に対数減衰に極めて近似した減速を示す事実を述べ、相互の分離相対距離比MG値は多くの泳動条件の影響を受けない恒数となることを報告した。その後このような事実が他の物質にも見られるかどうかを検討する為、今回は食用色素を含む数十数の色素類について、毛管吸着分析を併用して実験を行つたところ、此等の泳動距離、MG値について更に新しい知見が得られたので報告する