中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2018, Vol. 13 ›› Issue (11): 2005-2013.doi: 10.4103/1673-5374.239449

• 原著:退行性病与再生 • 上一篇    下一篇

SIRT1上调原代星形胶质细胞中溶酶体数量促进Aβ降解

  

  • 收稿日期:2018-07-25 出版日期:2018-11-15 发布日期:2018-11-15
  • 基金资助:

    国家自然科学基金（31670832，31470807，31270872）；国家重点研究开发项目（2016YFA0500301）；中国北京大学生命科学学院蛋白质和植物基因研究国家重点实验室的资助

SIRT1 facilitates amyloid beta peptide degradation by upregulating lysosome number in primary astrocytes

Min-Zhe Li1, Liang-Jun Zheng2, Jian Shen1, Xin-Ya Li2, Qi Zhang2, Xue Bai2, Qing-Song Wang2, Jian-Guo Ji2   

  1. 1 General Surgery Department, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China
    2 State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China
  • Received:2018-07-25 Online:2018-11-15 Published:2018-11-15
  • Contact: Min-Zhe Li, MD or Jian-Guo Ji, PhD, leeminzhe@hotmail.com or jijg@pku.edu.cn.
  • Supported by:

    This study was supported by the National Natural Science Foundation of China, No. 31670832, 31470807, 31270872; a grant from the National Key Research and Development Program of China, No. 2016YFA0500301; and a grant from the State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, China.

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摘要：

以往的研究表明，SIRT 1具有降低神经元Aβ的产生，抑制神经胶质的炎症反应，从而产生对抗阿尔茨海默病动物模型中Aβ神经毒性的作用，但SIRT 1对星形胶质细胞的保护作用尚在研究中。为此，实验体外建立原代星形胶质细胞吞噬降解Aβ的时间点模型，发现培养12 h可表征寡聚态Aβ吞噬时间点，培养36 h表征寡聚态Aβ有效降解时间点。(1) 实验利用免疫荧光技术检测胞内Aβ定位发现，原代星形胶质细胞降解Aβ过程依赖于溶酶体。实验利用SIRT1的激动剂和抑制剂以浓度梯度方式刺激细胞，然后向培养基中加入Aβ刺激36 h，Western blot检测不同处理条件下胞内剩余Aβ蛋白表达发现，SIRT1依赖其去乙酰化酶活性来促进胞内Aβ降解；(2) 实验进一步通过定量蛋白质组学技术筛选SIRT1促进原代星形胶质细胞溶酶体途径降解Aβ通路的潜在下游差异表达蛋白，发现多数检测出的差异蛋白与SIRT1的酶活功能更接近；(3)实验再通过免疫荧光染色观察发现，SIRT1可依赖其去乙酰化酶活性上调原代星形胶质细胞中的溶酶体数量；(4)上述数据说明，SIRT1依赖其去乙酰化酶活性可上调原代星形胶质细胞溶酶体数量，从而促进了寡聚态Aβ降解。

orcid:0000-0002-7168-6949(Min-Zhe Li)
        0000-0002-7863-4789(Jian-Guo Ji)

关键词: &beta, 淀粉样肽, 阿尔茨海默病, 神经退行性变, 星形胶质细胞, 胶质细胞, SIRT1蛋白, 定量蛋白质组学, 溶酶体, 时间点模型, 多肽降解, 神经再生

Abstract:

Previous studies have shown that sirtuin 1 (SIRT1) reduces the production of neuronal amyloid beta (Aβ) and inhibits the inflammatory response of glial cells, thereby generating a neuroprotective effect against Aβ neurotoxicity in animal models of Alzheimer’s disease. However, the protective effect of SIRT1 on astrocytes is still under investigation. This study established a time point model for the clearance of Aβ in primary astrocytes. Results showed that 12 hours of culture was sufficient for endocytosis of oligomeric Aβ, and 36 hours sufficient for effective degradation. Immunofluorescence demonstrated that Aβ degradation in primary astrocytes relies on lysosome function. Enzymatic agonists or SIRT1 inhibitors were used to stimulate cells over a concentration gradient. Aβ was co-cultured for 36 hours in medium. Western blot assay results under different conditions revealed that SIRT1 relies on its deacetylase activity to promote intracellular Aβ degradation. The experiment further screened SIRT1 using quantitative proteomics to investigate downstream, differentially expressed proteins in the Aβ degradation pathway and selected the ones related to enzyme activity of SIRT1. Most of the differentially expressed proteins detected are close to the primary astrocyte lysosomal pathway. Immunofluorescence staining demonstrated that SIRT1 relies on its deacetylase activity to upregulate lysosome number in primary astrocytes. Taken together, these findings confirm that SIRT1 relies on its deacetylase activity to upregulate lysosome number, thereby facilitating oligomeric Aβ degradation in primary astrocytes.

Key words: nerve regeneration, amyloid beta peptide, Alzheimer’s disease, neurodegeneration, astrocytes, gliocytes, sirtuin1, quantitative proteomics, lysosome, time point model, peptide degradation, neural regeneration