Lysosomal Health in Ageing, Hopwood Centre for Neurobiology, Lifelong Health Theme, South Australian Health and Medical Research Institute, Adelaide, Australia.
Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, Australia.
Autophagy. 2021 Oct;17(10):3238-3255. doi: 10.1080/15548627.2020.1846302. Epub 2020 Dec 11.
Autophagic flux is a critical cellular process that is vastly under-appreciated in terms of its importance to human health. Preclinical studies have demonstrated that reductions in autophagic flux cause cancer and exacerbate chronic diseases, including heart disease and the pathological hallmarks of dementia. Autophagic flux can be increased by targeting nutrition-related biochemical signaling. To date, translation of this knowledge has been hampered because there has been no way to directly measure autophagic flux in humans. In this study we detail a method whereby human macroautophagic/autophagic flux can be directly measured from human blood samples. We show that whole blood samples can be treated with the lysosomal inhibitor chloroquine, and peripheral blood mononuclear cells isolated from these samples could be used to measure autophagic machinery protein LC3B-II. Blocking of autophagic flux in cells while still in whole blood represents an important advance because it preserves genetic, nutritional, and signaling parameters inherent to the individual. We show this method was reproducible and defined LC3B-II as the best protein to measure autophagic flux in these cells. Finally, we show that this method is relevant to assess intra-individual variation induced by an intervention by manipulating nutrition signaling with an treatment of whole blood that comprised leucine and insulin. Significantly, this method will enable the identification of factors that alter autophagic flux in humans, and better aid their translation in the clinic. With further research, it could also be used as a novel biomarker for risk of age-related chronic disease. AMPK: AMP-activated protein kinase; ACTB: actin beta; ATG5: autophagy related 5; BAF: bafilomycin A; CQ: chloroquine; DMSO: dimethyl sulfoxide; DPBS: Dulbecco's phosphate-buffered saline; EDTA: ethylenediaminetetraacetic acid; KO: knockout; MAP1LC3A/LC3A: microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP1LC3C/LC3C: microtubule associated protein 1 light chain 3 gamma; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; PBMCs: peripheral blood mononuclear cells; PMNs: polymorphonuclear cells; RPMI: Roswell Park Memorial Institute; SQSTM1: sequestosome 1; TBST: Tris-buffered saline containing 0.1% (v:v) Tween 20; TEM: transmission electron microscopy.
自噬通量是一个重要的细胞过程，但其对人类健康的重要性在很大程度上被低估了。临床前研究表明，自噬通量的减少会导致癌症，并加剧慢性疾病，包括心脏病和痴呆症的病理特征。自噬通量可以通过靶向与营养相关的生化信号来增加。迄今为止，由于没有直接测量人类自噬通量的方法，这种知识的转化一直受到阻碍。在这项研究中，我们详细介绍了一种可以直接从人类血液样本中测量人类巨自噬/自噬通量的方法。我们表明，可以用溶酶体抑制剂氯喹处理全血样本，并从这些样本中分离出外周血单核细胞，用于测量自噬机制蛋白 LC3B-II。在全血中仍然阻断自噬通量是一个重要的进展，因为它保留了个体固有的遗传、营养和信号参数。我们表明，这种方法具有重现性，并将 LC3B-II 定义为测量这些细胞中自噬通量的最佳蛋白。最后，我们表明，通过用包含亮氨酸和胰岛素的全血处理来操纵营养信号，这种方法可以评估个体内的变化，这是一种重要的进展。值得注意的是，这种方法将能够识别改变人类自噬通量的因素，并更好地帮助它们在临床上的转化。随着进一步的研究，它也可以作为一种与年龄相关的慢性疾病风险的新型生物标志物。AMPK：AMP 激活的蛋白激酶；ACTB：肌动蛋白 β；ATG5：自噬相关 5；BAF：巴弗霉素 A；CQ：氯喹；DMSO：二甲基亚砜；DPBS：杜尔贝科磷酸盐缓冲盐水；EDTA：乙二胺四乙酸；KO：敲除；MAP1LC3A/LC3A：微管相关蛋白 1 轻链 3α；MAP1LC3B/LC3B：微管相关蛋白 1 轻链 3β；MAP1LC3C/LC3C：微管相关蛋白 1 轻链 3γ；MTOR：雷帕霉素靶蛋白激酶；NBR1：NBR1 自噬货物受体；PBMCs：外周血单核细胞；PMN：多形核细胞；RPMI：罗塞勒帕克纪念研究所；SQSTM1：自噬体 1；TBST：含 0.1%（v:v）吐温 20 的 Tris 缓冲盐水；TEM：透射电子显微镜。
