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.

中文翻译：

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N末端乙酰化突变体会影响α-突触核蛋白的稳定性，蛋白质水平和神经元毒性。

α-突触核蛋白（aSyn）蛋白水平足以驱动帕金森氏病（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，但对限制/控制aSyn水平的机制知之甚少。在这里，我们调查aSyn神经毒性的翻译后修饰，N末端乙酰化的作用。N末端乙酰化发生在所有aSyn分子中，并已被提议确定其脂质结合和聚集能力。但是，尚未评估其对aSyn稳定性/神经毒性的作用。我们生成了可改变或阻断野生型或病理突变E46K aSyn版本中生理aSyn N末端乙酰化的N末端突变体，并通过质谱法确认了N末端乙酰化状态。通过在活的原代神经元中进行光脉冲标记，我们记录了aSyn N末端突变体的半衰期缩短和积累。为了分析N末端乙酰化突变体在神经元毒性中的作用，我们利用了神经元模型，其中通过纵向生存分析对aSyn毒性进行评分。以前已经用这种方法研究了aSyn神经毒性的显着特征。在WT aSyn的情况下，较高的aSyn蛋白水平或通过蛋白水平和E46K突变传递的增强的神经毒性的联合作用，概括了aSyn依赖性神经元死亡。aSyn N末端突变可通过降低蛋白质水平，并且重要地，通过降低内在E46K aSyn毒性来降低E46K aSyn依赖性神经元死亡，D2P突变体毒性最低。一起，