| Objectives: To study the influence of over-expressing α-Synuclein(α-Syn) on related proteins’ expression and function in N2 A cells and transgenic mice brain. Methods:(1) Constructed in vitro cell model of PD: Dopaminergic cell line N2 A was transfected with recombinant plasmid α-Synuclein-pEGFP by transfection with 1% Lipofectamine2000 according to the manufacturer’s protocol. The stable clones were gained through screening of G418 and the stable expression of gene α-Synuclein-GFP was identified by means of Western blot and immunocytochemistry.(2)Observation the protein-expressed difference in α-Syn over-expressing N2 A cells: Using stably expressed α-Syn N2 A cells and no-transfected N2 A cells as the experimental group and the control group, respectively,detected the protein expression difference by Western blot.(3)Observing the cell functional impact of over-expression of α-Syn on N2A: detecting the difference of cell viability in different cell lines and after 6-OHDA, NH3CL/LEUPEPTIN, NMDA, AMP-Deoxynojirimycin and /or MK-801 treatment by means of MTT assay.(4) Measured the whole cell glucocerebrosidase(GCase) activity in different cell lines using the GCase activity assay;purified the lysosome and measured the lysosomal GCase activity; and detected the lysosomal GCase activity change after treatment with NMDA and\or MK-801.(5) Treated α-Syn-N2 A cells using NMDA or 6-OHDA, then blotted cells with α-Syn antibody and detected the α-Syn aggregation via immunocytochemistry.(6) By means of Western blot, detected the expression of related proteins and their phosphorylated proteins in striatum and substantia nigra(SN) of transgenic mice. Results:(1) Construction of in vitro cell model of PD: We successfully transfected cell line N2 A with plasmid α-Syn-pEGFP and gained positive clones through screening of 400 ug/ml G418. Outcomes of Western blot and immunocytochemistry manifested that the expression of fused protein α-Syn-GFP were significantly higher in α-Syn-N2 A cells than in N2 A cells.(2) The expression of Tyr1472-NR2 B, Ser896-NR1 and caveolin-1 were higher, while GCase was lower, in α-Syn-N2 A cells than in N2 A cells.(3) MTT assay showed that cell viability was higher in α-Syn-N2 A cells than in N2 A cells; the cell viability decreased with the increasing of 6-OHDA concentration, there was a significant viability difference between two groups at 30μM of 6-OHDA; α-Syn overexpression could intensify NMDA-induced cytotoxicity, and MK-801 could reverse NMDA-induced cytotoxicity; N2 A cells were more vulnerable to AMP-Deoxynojirimycin-combined toxicity than α-Syn-N2 A cells.(4) Lysosomal GCase activity was lower in experimental group than control group(about 50%); NMDA treatment could further decrease GCase activity.(5) There was more α-Syn aggregates in α-Syn-N2 A cells than in N2 A cells; 6-OHDA and NMDA treatment could further intensify α-Syn aggregates in α-Syn-N2 A cells.(6) There were higher α-Syn, Ser831-GluR1, Ser845-GluR1, Ser896-NR1, Ser897-NR1, Tyr1472-NR2 B, Ser1303-NR2 B, and lower GCase and Tyr125-α-Syn protein expression in striatum of α-Syn over-expression transgenic mice than wild type mice.(7)There were higher α-Syn, Ser87-α-Syn, Caveolin-1, Ser896-NR1, Ser897-NR1, Tyr1472-NR2 B, Ser1303-NR2 B and lower GCase, Tyr125-α-Syn protein expression in SN of transgenic mice than wild type mice. Conclusions:(1) Over-expression α-Syn enhanced the expression of phosphorylated NMDA receptor, cavoelin-1 and decreased the expression of GCase.(2) Over-expression α-Syn decreased the cell viability, intensified NMDA-induced cytotoxicity, while N2 A was more vulnerable to AMP-Deoxynojirimycin-combined treatment.(3) 6-OHDA and NMDA could increase α-Syn aggregates.(4) Over-expression α-Syn decreased the lysosomal GCase activity and NMDA treatment could further decreased lysosomal GCase activity.(5) There were increased phosphorylation of glutamate receptors and imbalance of different phosphorylation size of α-Syn in striatum and SN of transgenic mice. |
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