Study On The Effect Of Aβ1-42 Oligomers And Recombinant Human α-synuclein On Synapses Of Primary Cultured Neurons

Background Dementia of Parkinson’s disease and dementia with Lewy body are common neurodegenerative diseases in clinical which pathological characteristic are abnormal accumulation of a-synuclein (α-syn) and β-Amyloid1-42 (Aβ1-42),ultimately they cause the cognitive function disorder, which severely affects the health and quality of life. Therefore, to explore pathological mechanisms lead to changes and the relationship between the pathogenesis and pathology is critical.Object (1)To investigate the effects of α-syn and Aβ1-42 oligomers on physiological functions of the axons of mouse cortical and hippocampal primary cell culture; (2) To validate a-syn and Aβ1-42 oligomers on synapses’ reuptake function and activity in primary cell culture, for illustrating the relationship between synaptic vesicle-associated protein which is cysteine-string protein a(cysteine-string protein a, CSPa) and synaptic function.Methods (1)Cortical and hippocampal neurons were prepared from the brains of fetuses of pregnant Kunming mouse (day 17), Neurons were plated at 2 ×105 cells/well in pre-coated plates,and divided into six groups including DMSO group, Aβ42-1 group, a-syn group, Aβ1-42 group, α-syn+Aβ42-1 group and α-syn+Aβ1-42 group, each group was detected for six wells. After 24 hours’ intervention, by using immunofluorescence to detect pathology of primary cells and changes in synapsin I,and also by using FM1-43 to detect the number and activity of synapse, while the expression of cysteine-string protein a(CSPa)were detected by using Western blot. (2) All data were presented as mean ±standard error of mean (SEM) and analyzed with GraphPad Prism. Differences between groups were compared by using a one-way analysis of variance (ANOVA). P<0.05 indicates statistically significant difference.Results (1)After 24 hours’ intervention in primary hippocampal and cortical neurons, the physical structure of the primary cells are affected, under a fluorescence microscope, when the primary cells are respectively interfere with a-synuclein, Aβ1-42 oligomers, the shape and the integrity of neuronal cell and axon are good, but axons are short compared to DMSO group, and there was statistically difference (P<0.05); being interfered with α-synuclein+neurons Aβ1-42 oligomers, we detect that the cell bodies began to swell and irregularly shaped, axons were shorter than the other five groups, with statistically difference (P<0.05); (2) The results of synapsin I in immunofluorescence:the Synapsin I positive signal points in Aβ1-42 group, α-syn group, α-syn+Aβ42-1 were decreased compared with control group with statistically difference (P<0.05); the content of synapsin I in Aβ1-42+α-syn group was decreased with most significant difference (P <0.01);(3)The ELISA results of Synaptophysin:the amount of Synaptophysin in Aβ1-42 group, a-syn group, α-syn+Aβ42-1 were decreased compared with control group with statistically difference (P<0.05); the amount of Synaptophysin in Aβ1-42+α-syn group was decreased with most significant difference (P<0.01); (4) The neuronal uptake of FM1-43:The dye uptake in α-syn group and Aβ1-42 group was less than the control group with statistically significant (P<0.05); while the uptake in α-syn+after Aβ1-42 group was significantly decreased with statistically significant (P<0.01); (5) The content of synapse-associated protein CSPa:detected by Western blot, The content of CSPa in Aβ1-42 group and the α-syn group were significantly lower compared with DMSO group and Aβ42-1 group, with statistical significance P<0.05; while The content of CSPa in α-syn+Aβ1-42 group reduced with the most statistical significant, P<0.01.Conclusion After 24 hours’ intervention in primary hippocampal and cortical neurons, α-syn, Aβ1-42 oligomer will respectively affect the physical structure of primary cells and the number of synapses as well as activity, and we found that the combined effect of the α-syn, Aβ1-42 oligomers is particularly significant, and also synapse-associated protein CSPa will significantly decreased, which indicates that the α-syn, Aβ1-42 oligomers will produce a synergistic effect when they both exist, accelerate the dysfunction of synapse-associated protein, and lead to synaptic dysfunction, then result in neuronal apoptosis.