Temporal dependence of neuronal alpha-synuclein oligomerization and nuclear translocation induced by paraquat / 环境与职业医学

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 of in 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 of in vitro models showed that the expression of oligomerized α-syn increased and translocated to the nucleus (P<0.05). The ELISA results of in vitro models 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.