张宝馥, 王楷栋, 张冰洋, 黄敏. 百草枯引起神经元α-突触核蛋白寡聚化并向细胞核易位的时间依赖性研究[J]. 环境与职业医学, 2022, 39(2): 193-199. DOI: 10.11836/JEOM21422
引用本文: 张宝馥, 王楷栋, 张冰洋, 黄敏. 百草枯引起神经元α-突触核蛋白寡聚化并向细胞核易位的时间依赖性研究[J]. 环境与职业医学, 2022, 39(2): 193-199. DOI: 10.11836/JEOM21422
ZHANG Baofu, WANG Kaidong, ZHANG Bingyang, HUANG Min. Temporal dependence of neuronal alpha-synuclein oligomerization and nuclear translocation induced by paraquat[J]. Journal of Environmental and Occupational Medicine, 2022, 39(2): 193-199. DOI: 10.11836/JEOM21422
Citation: ZHANG Baofu, WANG Kaidong, ZHANG Bingyang, HUANG Min. Temporal dependence of neuronal alpha-synuclein oligomerization and nuclear translocation induced by paraquat[J]. Journal of Environmental and Occupational Medicine, 2022, 39(2): 193-199. DOI: 10.11836/JEOM21422

百草枯引起神经元α-突触核蛋白寡聚化并向细胞核易位的时间依赖性研究

Temporal dependence of neuronal alpha-synuclein oligomerization and nuclear translocation induced by paraquat

  • 摘要: 背景 百草枯(PQ)作为散发性帕金森病(PD)的环境毒物之一可引起α-突触核蛋白(α-syn)异常聚集,但在其构象变化及亚细胞定位的研究有限。

    目的 探讨PQ对多巴胺能神经元中α-syn构象以及亚细胞定位的影响。

    方法 选取48只SPF级C57BL/6雄性小鼠随机分为对照组和模型组,模型组腹腔注射PQ(15 mg·kg−1),对照组腹腔注射等容量0.9%生理盐水,每周注射2次,连续8周以构建类PD样小鼠模型。观察各组小鼠神经行为学(旷场实验、爬杆实验)的变化,来评估小鼠运动能力的改变。染毒结束后取小鼠脑组织进行免疫组织化学染色(IHC),检测中脑多巴胺(DA)能神经元特异性标记物酪氨酸羟化酶(TH)以及α-syn的表达量;同时采用蛋白质印迹法(WB)检测小鼠中脑TH、α-syn的蛋白表达量。以人神经母细胞瘤SH-SY5Y细胞作为多巴胺能神经元的体外模型,使用固定浓度PQ(100 μmol·L−1)处理细胞不同时间(0、12、24、36、48 h)后,采用WB法分别检测全细胞、细胞质、细胞核α-syn的表达情况;酶联免疫吸附试验(ELISA)检测细胞外α-syn的表达水平;免疫荧光法(IFA)观察α-syn的位置变化。

    结果 神经行为学结果显示,与对照组比较,随染毒时间的增加PQ模型组小鼠旷场周边区域停留时间增加(P<0.05),中央区域停留时间和移动距离减少(P<0.05),爬杆时间增加(P<0.05);动物实验IHC结果显示,6周、8周模型组与对照组比较,中脑TH阳性细胞数减少(P<0.05),而α-syn阳性表达量在4、6和8周增多(P<0.05);WB结果同样显示TH相对表达量在PQ染毒6、8周明显减少(P<0.05),寡聚体α-syn相对表达量在4、6和8周增多(P<0.05)。细胞实验WB结果显示,细胞全蛋白寡聚体α-syn相对表达量随时间依赖性增多(R2=0.7440,P<0.05);细胞质寡聚体α-syn相对表达量随时间增加先增加后减少(P<0.05);细胞核寡聚体α-syn相对表达量随时间依赖性增加(R2=0.7913,P<0.05);IFA结果显示,寡聚化的α-syn表达增加并向细胞核易位(P<0.05);ELISA结果表明,细胞所释放的α-syn随PQ染毒时间增加呈现上升的趋势(P<0.05)。

    结论 PQ引起多巴胺能神经元内α-syn表达增加,结构发生寡聚化且向细胞核易位。

     

    Abstract: Background Paraquat (PQ), one of the environmental poisons associated with sporadic Parkinson's disease (PD), can cause abnormal aggregation of alpha-synuclein (α-syn), but the research on its conformational changes and subcellular localization is limited.

    Objective To investigate the effect of PQ on α-syn conformation and subcellular localization in dopaminergic neurons.

    Methods Forty-eight SPF C57BL/6 male mice were selected and randomly divided into a control group and a model group. The model group was intraperitoneally injected with PQ (15 mg·kg−1), and the control group was intraperitoneally injected with 0.9% normal saline, twice a week for eight weeks to construct a PD-like mouse model. The changes of neurobehavior (by open field test and pole climbing test) were observed to evaluate motor ability of mice. Immunohistochemical staining (IHC) was used to detect the expression levels of tyrosine hydroxylase (TH) and α-syn in the midbrain. Western blotting (WB) was used to measure the protein expression levels of TH and α-syn in midbrain. Human neuroblastoma SH-SY5Y cells were used as dopaminergic neuron in vitro models. After the cells were treated with PQ (100 μmol·L−1) for 0, 12, 24, 36 and 48 h, the expressions of α-syn in whole cell, cytoplasm, and nucleus were detected by WB; the expression level of extracellular α-syn was detected by enzyme-linked immunosorbent assay (ELISA); the change of α-syn location was observed by immunofluorescence assay (IFA).

    Results The neurobehavioral tests' results showed that compared with the control group, the residence time in peripheral area of mice in the PQ model group increased with the increase of exposure time (P<0.05), the residence time and moving distance in the central region decreased (P<0.05), and the pole climbing time increased (P<0.05). The mouse IHC results showed that compared with the control group, the number of TH positive cells in the midbrain decreased in the model group at week 6 and 8 (P<0.05), while the expression level of α-syn increased at week 4, 6, and 8 (P<0.05). The WB results of mouse showed that the relative expression of TH decreased significantly after 6 and 8 weeks of PQ exposure (P<0.05), and the relative expression of oligomer α-syn increased after 4, 6, and 8 weeks of PQ exposure (P<0.05). The WB ofin vitro models results showed that the relative expression of α-syn in cells increased with time (R2=0.7440, P<0.05); the relative expression of α-syn in cytoplasm increased firstly and then decreased with time (P<0.05); the relative expression of α-syn in nucleus increased with time (R2=0.7913, P<0.05). The IFA results ofin vitromodels showed that the expression of oligomerized α-syn increased and translocated to the nucleus (P<0.05). The ELISA results ofin vitromodels showed that α-syn increased with the increase of PQ exposure time (P<0.05).

    Conclusion PQ can increase the expression of α-syn in dopaminergic neurons, induce oligomerization and translocation to the nucleus.

     

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