Mizuta Naoki, Yanagida Kanta, Kodama Takashi, Tomonaga Takeshi, Takami Mako, Oyama Hiroshi, Kudo Takashi, Ikeda Manabu, Takeda Masatoshi, Tagami Shinji, Okochi Masayasu
Psychiatry, Department of Integrated Medicine, Division of Internal Medicine, Osaka University Graduate School of Medicine, Osaka, Japan.
Neurodegener Dis. 2017;17(2-3):103-109. doi: 10.1159/000453358. Epub 2017 Jan 20.
Amyloid-β (Aβ) degradation in brains of Alzheimer disease patients is a crucial focus for the clarification of disease pathogenesis. Nevertheless, the mechanisms underlying Aβ degradation in the human brain remain unclear.
This study aimed to quantify the levels of small C-terminal Aβ fragments generated upon Aβ degradation in human cerebrospinal fluid (CSF).
A fraction containing small peptides was isolated and purified from human CSF by high-pressure liquid chromatography. Degradation products of Aβ C termini were identified and measured by liquid chromatography-tandem mass spectrometry. The C-terminal fragments of Aβ in the conditioned medium of cultured cells transfected with the Swedish variant of βAPP (sw βAPP) were analyzed. These fragments in brains of PS1 I213T knock-in transgenic mice, overexpressing sw βAPP, were also analyzed.
The peptide fragments GGVV and GVV, produced by the cleavage of Aβ40, were identified in human CSF as well as in the brains of the transgenic mice and in the conditioned medium of the cultured cells. Relative to Aβ40 levels, GGVV and GVV levels were 7.6 ± 0.81 and 1.5 ± 0.18%, respectively, in human CSF. Levels of the GGVV fragment did not increase by the introduction of genes encoding neprilysin and insulin-degrading enzyme to the cultured cells.
Our results indicate that a substantial amount of Aβ40 in human brains is degraded via a neprilysin- or insulin-degrading enzyme-independent pathway.
阿尔茨海默病患者大脑中淀粉样β蛋白（Aβ）的降解是阐明疾病发病机制的关键研究重点。然而，人类大脑中Aβ降解的潜在机制仍不清楚。
本研究旨在量化人脑脊液（CSF）中Aβ降解后产生的小C末端Aβ片段的水平。
通过高压液相色谱从人CSF中分离并纯化含小肽的组分。采用液相色谱-串联质谱法鉴定并测定Aβ C末端的降解产物。分析用β淀粉样前体蛋白（βAPP）瑞典变异体（sw βAPP）转染的培养细胞条件培养基中Aβ的C末端片段。还分析了过表达sw βAPP的PS1 I213T基因敲入转基因小鼠大脑中的这些片段。
在人CSF、转基因小鼠大脑以及培养细胞的条件培养基中均鉴定出由Aβ40裂解产生的肽片段GGVV和GVV。相对于Aβ40水平，人CSF中GGVV和GVV水平分别为7.6±0.81%和1.5±0.18%。向培养细胞中引入编码中性内肽酶和胰岛素降解酶的基因后，GGVV片段水平未升高。
我们的结果表明，人脑中大量的Aβ40是通过一条不依赖中性内肽酶或胰岛素降解酶的途径降解的。
