Two C-terminal sequence variations determine differential neurotoxicity between human and mouse α-synuclein
Two C-terminal sequence variations determine differential neurotoxicity between human and mouse α-synuclein
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DOI：
10.1186/s13024-020-00380-w
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发表时间：
2020-09-08
影响因子：
15.1
通讯作者：
Rochet, Jean-Christophe
中科院分区：
文献类型：
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作者：
Landeck, Natalie;Strathearn, Katherine E.;Rochet, Jean-Christophe
α-突触核蛋白（aSyn）聚集被认为在称为突触核蛋白病的神经退行性疾病（包括帕金森病（PD））中起核心作用。小鼠aSyn在位置53处含有苏氨酸残基，其模拟人类家族性PD取代A53 T，然而与A53 T患者相反，小鼠甚至在老化后也未显示aSyn神经病理学的证据。在这里，我们研究了人A53 T，小鼠aSyn和各种人-小鼠嵌合体在细胞和体内模型中的神经毒性，以及它们与aSyn病理生物学相关的生化特性。方法用aSyn腺病毒转导原代中脑培养物，免疫细胞化学分析，以确定相对多巴胺能神经元活力。从黑质内注射aSyn编码腺相关病毒的大鼠制备的脑切片进行化学分析，以确定黑质多巴胺能神经元活力和纹状体多巴胺能终末密度。重组aSyn变体通过测量硫磺素T荧光、经由电子显微镜和原子力显微镜的原纤维形态以及通过监测膜诱导的aSyn聚集和aSyn介导的囊泡破坏的蛋白质-脂质相互作用来表征。统计学检验由ANOVA，随后是Tukey多重比较事后检验和Kruskal-Wallis检验，随后是Dunn多重比较检验或双尾Mann-Whitney检验组成。结果在细胞培养物和大鼠中脑中，小鼠aSyn的神经毒性低于人aSyn A53 T，并且对于嵌合变体获得的数据表明，人对小鼠的取代D121 G和N122 S至少部分地导致神经毒性的这种降低。人aSyn A53 T和在位置121和122（分别）具有人残基D和N的嵌合变体显示出更大的经历膜诱导的聚集和引起囊泡破坏的倾向。人、小鼠和嵌合aSyn变体之间的神经毒性差异与纤维化速率或原纤维形态的差异弱相关。结论小鼠aSyn的神经毒性低于人A53 T变体，这是由于两个C端氨基酸取代对膜诱导的aSyn聚集和aSyn介导的囊泡透化的抑制作用。我们的研究结果强调了膜诱导的自组装在aSyn神经毒性中的重要性，并表明通过靶向C末端结构域来抑制该过程可以减缓PD和其他突触核蛋白病疾病中的神经变性。
Background alpha-Synuclein (aSyn) aggregation is thought to play a central role in neurodegenerative disorders termed synucleinopathies, including Parkinson's disease (PD). Mouse aSyn contains a threonine residue at position 53 that mimics the human familial PD substitution A53T, yet in contrast to A53T patients, mice show no evidence of aSyn neuropathology even after aging. Here, we studied the neurotoxicity of human A53T, mouse aSyn, and various human-mouse chimeras in cellular and in vivo models, as well as their biochemical properties relevant to aSyn pathobiology. Methods Primary midbrain cultures transduced with aSyn-encoding adenoviruses were analyzed immunocytochemically to determine relative dopaminergic neuron viability. Brain sections prepared from rats injected intranigrally with aSyn-encoding adeno-associated viruses were analyzed immunohistochemically to determine nigral dopaminergic neuron viability and striatal dopaminergic terminal density. Recombinant aSyn variants were characterized in terms of fibrillization rates by measuring thioflavin T fluorescence, fibril morphologies via electron microscopy and atomic force microscopy, and protein-lipid interactions by monitoring membrane-induced aSyn aggregation and aSyn-mediated vesicle disruption. Statistical tests consisted of ANOVA followed by Tukey's multiple comparisons post hoc test and the Kruskal-Wallis test followed by a Dunn's multiple comparisons test or a two-tailed Mann-Whitney test. Results Mouse aSyn was less neurotoxic than human aSyn A53T in cell culture and in rat midbrain, and data obtained for the chimeric variants indicated that the human-to-mouse substitutions D121G and N122S were at least partially responsible for this decrease in neurotoxicity. Human aSyn A53T and a chimeric variant with the human residues D and N at positions 121 and 122 (respectively) showed a greater propensity to undergo membrane-induced aggregation and to elicit vesicle disruption. Differences in neurotoxicity among the human, mouse, and chimeric aSyn variants correlated weakly with differences in fibrillization rate or fibril morphology. Conclusions Mouse aSyn is less neurotoxic than the human A53T variant as a result of inhibitory effects of two C-terminal amino acid substitutions on membrane-induced aSyn aggregation and aSyn-mediated vesicle permeabilization. Our findings highlight the importance of membrane-induced self-assembly in aSyn neurotoxicity and suggest that inhibiting this process by targeting the C-terminal domain could slow neurodegeneration in PD and other synucleinopathy disorders.