University of Navarra, Center for Applied Medical Research (CIMA), Neuroscience Program, Pamplona 31008, Spain; University of Navarra, School of Medicine, Graduate Program on Neuroscience and Cognition, Pamplona 31008, Spain.
University of Navarra, Center for Applied Medical Research (CIMA), Neuroscience Program, Pamplona 31008, Spain.
Neurobiol Dis. 2020 Apr;137:104781. doi: 10.1016/j.nbd.2020.104781. Epub 2020 Jan 25.
Alpha-synuclein (aSyn) protein levels are sufficient to drive Parkinson's disease (PD) and other synucleinopathies. Despite the biomedical/therapeutic potential of aSyn protein regulation, little is known about mechanisms that limit/control aSyn levels. Here, we investigate the role of a post-translational modification, N-terminal acetylation, in aSyn neurotoxicity. N-terminal acetylation occurs in all aSyn molecules and has been proposed to determine its lipid binding and aggregation capacities; however, its effect in aSyn stability/neurotoxicity has not been evaluated. We generated N-terminal mutants that alter or block physiological aSyn N-terminal acetylation in wild-type or pathological mutant E46K aSyn versions and confirmed N-terminal acetylation status by mass spectrometry. By optical pulse-labeling in living primary neurons we documented a reduced half-life and accumulation of aSyn N-terminal mutants. To analyze the effect of N-terminal acetylation mutants in neuronal toxicity we took advantage of a neuronal model where aSyn toxicity was scored by longitudinal survival analysis. Salient features of aSyn neurotoxicity were previously investigated with this approach. aSyn-dependent neuronal death was recapitulated either by higher aSyn protein levels in the case of WT aSyn, or by the combined effect of protein levels and enhanced neurotoxicity conveyed by the E46K mutation. aSyn N-terminal mutations decreased E46K aSyn-dependent neuronal death both by reducing protein levels and, importantly, by reducing the intrinsic E46K aSyn toxicity, being the D2P mutant the least toxic. Together, our results illustrate that the N-terminus determines, most likely through its acetylation, aSyn protein levels and toxicity, identifying this modification as a potential therapeutic target.
α-突触核蛋白（aSyn）的蛋白水平足以引发帕金森病（PD）和其他突触核蛋白病。尽管 aSyn 蛋白调节具有生物医学/治疗潜力，但对于限制/控制 aSyn 水平的机制知之甚少。在这里，我们研究了一种翻译后修饰，即 N 端乙酰化，在 aSyn 神经毒性中的作用。N 端乙酰化发生在所有 aSyn 分子中，据推测它决定了其脂质结合和聚集能力；然而，其在 aSyn 稳定性/神经毒性中的作用尚未得到评估。我们生成了 N 端突变体，这些突变体改变或阻断了野生型或病理突变 E46K aSyn 版本中生理 aSyn N 端乙酰化，并用质谱法确认了 N 端乙酰化状态。通过在活原代神经元中的光脉冲标记，我们记录了 aSyn N 端突变体半衰期的缩短和积累。为了分析 N 端乙酰化突变体对神经元毒性的影响，我们利用神经元模型，通过纵向生存分析来评分 aSyn 毒性。以前使用这种方法研究了 aSyn 神经毒性的显著特征。在 WT aSyn 的情况下，通过增加 aSyn 蛋白水平，或通过 E46K 突变的蛋白水平和增强的神经毒性的共同作用，再现了 aSyn 依赖性神经元死亡。aSyn N 端突变通过降低蛋白水平，并且重要的是通过降低内在的 E46K aSyn 毒性，降低了 E46K aSyn 依赖性神经元死亡，其中 D2P 突变体毒性最小。总之，我们的结果表明，N 端通过其乙酰化决定了 aSyn 蛋白水平和毒性，这表明这种修饰可能是一种潜在的治疗靶点。
