Department of Clinical Genetics, Amsterdam University Medical Centers Location VUmc, Amsterdam, The Netherlands.
Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Vrije Universiteit (VU), Amsterdam, The Netherlands.
J Neurochem. 2020 Jan;152(2):208-220. doi: 10.1111/jnc.14856. Epub 2019 Oct 21.
The unfolded protein response (UPR) is one of the major cell-autonomous proteostatic stress responses. The UPR has been implicated in the pathogenesis of neurodegenerative diseases and is therefore actively investigated as therapeutic target. In this respect, cell non-autonomous effects of the UPR including the reported cell-to-cell transmission of UPR activity may be highly important. A pharmaca-based UPR induction was employed to generate conditioned media (CM) from CM-donating neuronal ('donor') cells (SK-N-SH and primary mouse neurons). As previously reported, upon subsequent transfer of CM to naive neuronal 'acceptor' cells, we confirmed UPR target mRNA and protein expression by qPCR and automated microscopy. However, UPR target gene expression was also induced in the absence of donor cells, indicating carry-over of pharmaca. Genetic induction of single pathways of the UPR in donor cells did not result in UPR transmission to acceptor cells. Moreover, no transmission was detected upon full UPR activation by nutrient deprivation or inducible expression of the heavy chain of immunoglobulin M in donor HeLa cells. In addition, in direct co-culture of donor cells expressing the immunoglobulin M heavy chain and fluorescent UPR reporter acceptor HeLa cells, UPR transmission was not observed. In conclusion, carry-over of pharmaca is a major confounding factor in pharmaca-based UPR transmission protocols that are therefore unsuitable to study cell-to-cell UPR transmission. In addition, the absence of UPR transmission in non-pharmaca-based models of UPR activation indicates that cell-to-cell UPR transmission does not occur in cell culture.
未折叠蛋白反应 (UPR) 是主要的细胞自主蛋白稳态应激反应之一。UPR 与神经退行性疾病的发病机制有关，因此被积极研究作为治疗靶点。在这方面，UPR 的细胞非自主性效应，包括报道的 UPR 活性的细胞间传递，可能非常重要。采用基于药物的 UPR 诱导方法从 CM 供体细胞（SK-N-SH 和原代小鼠神经元）产生条件培养基 (CM)。如前所述，在随后将 CM 转移到幼稚的神经元“接受者”细胞后，我们通过 qPCR 和自动显微镜确认了 UPR 靶 mRNA 和蛋白的表达。然而，即使没有供体细胞，UPR 靶基因的表达也被诱导，这表明药物的残留。在供体细胞中遗传诱导 UPR 的单一途径不会导致 UPR 向接受细胞传递。此外，通过营养剥夺或在诱导表达免疫球蛋白 M 重链的供体细胞中完全激活 UPR 时，未检测到传递。此外，在表达免疫球蛋白 M 重链的供体细胞与荧光 UPR 报告接受者 HeLa 细胞的直接共培养中，未观察到 UPR 传递。总之，药物的残留是基于药物的 UPR 传递方案中的一个主要混杂因素，因此不适合研究细胞间 UPR 传递。此外，在非基于药物的 UPR 激活模型中没有 UPR 传递表明，细胞间 UPR 传递不会在细胞培养中发生。
