Study On The Toxic Effects Of Aβ_1-42 Oligomers And Human A53T Mutant α-synuclein In Vitro Cell Model Of Parkinson’s Disease

BACKGROUND & OBJECTIVE: Parkinson’s disease（PD）is a common chronic central nervous system degenerative disease,which is common in middle-aged and elderly people.The main pathological feature of Parkinson’s disease is the progressive degeneration of dopamine（DA）neurons in the substantia nigra pars compacta and the decrease of DA in striatum.Abnormal Lewy Body inclusion was found in the residual neurons.The study found that abnormal beta amyloid deposition can be detected in brain tissue of Parkinson’s disease patients,except for typical alpha-synucleinopathy.In Parkinson’s disease patients,β amyloid load is positively correlated with the severity of posture gait disorder.The level of Aβ_1-42 oligomer in cerebrospinal fluid of patients with Parkinson’s disease is negatively correlated with patient’s cognition.The lower the Aβ_1-42,the higher risk of developing of Parkinson’s disease dementia.The mechanism of action of alpha synuclein and Aβ 1-42 oligomers in Parkinson’s disease has not been elucidated.In this study,the cell model of Parkinson’s disease in vitro was constructed based on lentiviral transfection technology,and Aβ_1-42 oligomers were treated to investigate the cytotoxic effects and synergistic mechanisms of α-synuclein and Aβ_1-42 oligomers.It provides an objective basis for the pathogenesis of Parkinson’s disease.Methods: The cell model of SHSY5 Y Parkinson’s disease in vitro was constructed by lentiviral transfection technique,carrying the Parkinson’s disease-causing mutation gene SCANA A53 T.The transcription level of the target gene SNCA m RNA was identified by RT-QPCR.Western blotting was used to detect the expression level of the target protein alpha synuclein.The CCK-8 method was used to assess the viability of the cell model.The Aβ_1-42 oligomers of the experimental group and the control group were separately treated.Flow cytometry detects the level of apoptosis in cells.Cellular immunofluorescence assay detects intracellular tyrosine hydroxylase expression and phosphorylation of alpha synuclein and assesses cell damage.Further,Western blotting was used to detect the expression of autophagy protein and the expression of the pathway protein in the upstream of autophagy.Statistical analysis was performed using two-way analysis of variance for repeated measures.Results:（1）In the Parkinson’s disease cell model overexpressing human A53 T mutant α-synuclein,the expression of α-synuclein m RNA was 41-fold higher than that of the control group,and the expression of α-synuclein protein was significantly higher than that of the control group.There was no statistical difference in the proliferation of cell model compared with the control group;（2）The cells in the control group and the Parkinson’s disease model group were treated with Aβ_1-42 oligomers.The apoptosis level of the two groups increased and the level of α-synuclein phosphorylation increased significantly（F=53.48,P<0.001）while the tyrosine hydroxylase expression decreased（F = 22.88,P < 0.001）.After treated with Aβ_1-42 oligomer,the phosphorylation level of α-synuclein in the Parkinson’s disease cell model group was significantly higher than that in the control group,and the difference was statistically significant（F=9.73,P<0.05）.（3）Western blotting was used to detect the level of autophagy in each group.The expression of LC3-II/I and Beclin-1 in Parkinson’s disease cell model group was lower than that in the control group（P<0.05）,and the expression of p62/SQSTM1 was slightly increased.The expression of LC3-II/I and Beclin-1 in the two groups was slightly increased after treatment with Aβ_1-42 oligomer,which was still lower than that of the control group,accompanied by increased phosphorylation of ERK1/2/m TOR/4EBP-1.Conclusions:（1）Successfully constructed a Parkinson’s disease cell model that overexpresses human A53 T mutant α synuclein,and the cell growth and proliferation is normal;（2）Aβ_1-42 oligomer promotes apoptosis and promotes the phosphorylation level of α-synuclein,disrupts the expression of tyrosine hydroxylase,and inhibition of tyrosine hydroxylase is significantly greater in the Parkinson’s disease model than in the control group.（3）Overexpressing of human A53 T mutant alpha-synuclein,the autophagy function of the cells is inhibited,without causing significantly damage to the cells.Aβ_1-42 oligomers stimulate the activation of ERK1/2/m TOR/4EBP-1 signaling pathway,autophagy function of cells,and aggravate cell death.