Robinson Edward J, Aguiar Sebastian P, Kouwenhoven Willemieke M, Starmans Dorinde S, von Oerthel Lars, Smidt Marten P, van der Heide Lars P
Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands.
Cell Death Discov. 2018 Nov 21;4:107. doi: 10.1038/s41420-018-0125-7. eCollection 2018.
Mitochondria-dependent apoptosis plays an important role in the embryonic development of the midbrain dopaminergic system as well as in Parkinson's disease. Central to mitochondria-dependent apoptosis is the Bcl2 family of apoptosis-regulating proteins. However, it was unclear which Bcl2 proteins are important for the survival of dopaminergic neurons. Here, we identify Mcl1 as a critical Bcl2 pro-survival factor in midbrain dopaminergic neurons. Using a chemical biology approach to inhibit various components of the apoptotic machinery in the dopaminergic MN9D cell line or the control neuroblastoma N2A cell line, we find that functional inhibition of Mcl1 with the high affinity small molecule inhibitor UMI-77 results in a rapid and dose-dependent loss of viability, selectively in dopaminergic cells. In-depth analysis of the apoptotic signaling pathway reveals that chemical inhibition of Mcl1 results in the activation of Bax, activation of cleaved caspase-3 and finally cell death. The dependence of mouse dopaminergic midbrain neurons on Mcl1 was confirmed using ex vivo slice cultures from Pitx3GFP/+ and wildtype mice. In mouse dopaminergic midbrain neurons positive for the midbrain dopaminergic marker Pitx3, or tyrosine hydroxylase, UMI-77 treatment caused a dramatic increase in cleaved caspase 3, indicating that Mcl1 activity is required for basal neuronal survival. Overall, our results suggest that Mcl1 is of critical importance to dopaminergic neurons and is a weak link in the chain controlling cellular survival. Boosting the pro-survival function of Mcl1 should be pursued as a therapeutic approach to augment the resilience of midbrain dopaminergic neurons to apoptotic stress in Parkinson's disease.
线粒体依赖性凋亡在中脑多巴胺能系统的胚胎发育以及帕金森病中发挥着重要作用。线粒体依赖性凋亡的核心是凋亡调节蛋白的Bcl2家族。然而，尚不清楚哪些Bcl2蛋白对多巴胺能神经元的存活至关重要。在此，我们确定Mcl1是中脑多巴胺能神经元中关键的Bcl2促存活因子。使用化学生物学方法抑制多巴胺能MN9D细胞系或对照神经母细胞瘤N2A细胞系中凋亡机制的各个组分，我们发现用高亲和力小分子抑制剂UMI-77对Mcl1进行功能抑制会导致多巴胺能细胞中活力迅速且剂量依赖性丧失。对凋亡信号通路的深入分析表明，对Mcl1的化学抑制导致Bax激活、裂解的caspase-3激活，最终导致细胞死亡。使用来自Pitx3GFP/+和野生型小鼠的离体脑片培养证实了小鼠多巴胺能中脑神经元对Mcl1的依赖性。在中脑多巴胺能标记物Pitx3或酪氨酸羟化酶呈阳性的小鼠多巴胺能中脑神经元中，UMI-77处理导致裂解的caspase 3显著增加，表明Mcl1活性是基础神经元存活所必需的。总体而言，我们的结果表明Mcl1对多巴胺能神经元至关重要，并且是控制细胞存活的链条中的薄弱环节。增强Mcl1的促存活功能应作为一种治疗方法来增强中脑多巴胺能神经元对帕金森病中凋亡应激的恢复力。
