The Toxic Effects Of MPP+ On PC12 Cells Over-expressing WT, A30P Or A53T α-synuclein Isoforms And Its Influence On Membrane-expressing DAT

PartⅠConstruction of recombinant vector containing wild type SNCA synonymous mutation or its Ala30Pro and Ala53Thr pathogenic mutationsObjective To construct recombinant vector containing wild type SNCA synonymous mutation or its Ala30Pro and Ala53Thr pathogenic mutationsMethods Human wild type SNCA gene was cloned from fetus brain by using RT-PCR.The recombinant pEGFP-C3-SNCA vectors containing wild type SNCA synonymous mutations involving EcoRⅠa nd BamHⅠsites within code region of SNCA gene were constructed by site-directed mutagenesis using primer variance in mononucleotide, respectively. Based on these synonymous mutations, its Ala30Pro and Ala53Thr pathogenic mutation within code region of SNCA gene,were further constructed by the same methods.Results The objective band of 481 bp was obtained by PCR using recombinant pEGFP-C3-SNCA vectors as template,the objective band of 460 bp was obtained by th EcoRⅠand BamHⅠdouble digestion of recombinant pEGFP-C3-SNCA vectors and ultimately the E.coli DH5αtransformed with corresponding recombinant pEGFP-C3-SNCA vectors had been comfirmed successfully by gene sequencing.Conclusion The recombinant pEGFP-C3-SNCA vectors containing wild type SNCA synonymous mutation or its Ala30Pro and Ala53Thr pathogenic mutations had been constructed successfully PartⅡThe well-differentiated PC12 cells were stably transfected by the recombinant eukaryotic expressing vector pEGFP-C3.Objective To stably transfect well-differentiated PC12 cells by the recombinant eukaryotic expressing vectors pEGFP-C3-SNCA containing wild type SNCA synonymous mutation or its Ala30Pro and Ala53Thr pathogenic mutations,respectively.Methods PC12 cells were tranfected with the recombinant vectors pEGFP-C3-SNCA by means of liposome,and transfected PC12 cells were further screened with G418. The transfected monoclonal PC12 cells were obtained by means of limiting dilution assay.It was confirmed that SNCA gene isoforms were stably expressed in transfected PC12 cells by means of RT-PCR, Western blot and fluorescence microscope.Results The objective band of 481 bp was obtained from transfected PC12 cells by means of RT-PCR . The objective GFP-SNCA fusion protein band of 40KDa was obtained by means of Western blot.Green fluorescence was observed through fluorescence microscope.Conclusion The transfected monoclonal PC12 cells were obtained in which three SNCA gene isoforms had stable expression.PartⅢThe toxic effects of MPP+ on PC12 cells overexpressing human wild type,A30P and A53Tα-synuclein isoformsObjective To study the toxic effects of MPP+ on PC12 cells overexpressing human wild type,A30P and A53Tα-synuclein isoforms.Methods PC12 cells stably transfected with pEGFP-C3 empty vector were used as control group.Cell viability was measured by means of MTT methods.Apoptosis was observed through fluorescence microscope after Annexin V-FITC-PI dyeing. The common morphological changes of cell lines were observed through light microscope.The ultramicro-structural changes of cell lines were observed through transmission electron microscope.ROS levels in cell lines measured by means of DCF-DA probe.Results (1)After treatment of MPP+,cell viability of WT group was greater than control group,while those of both A30P and A53T groups were lower than control group. (2) After treatment of MPP+,cell apoptotic index in WT group was lower than control group,while those in both A30P and A53T groups were greater than control group.(3)Observing live cells of various PC12 cell groups through light microscope,we found cell morphous change after treatment with MPP+ in WT group was quiet,while in both A30P and A53T groups was significant. (4)Observing cell ultramicrostructure with electron microscope:Before treatment with MPP+,cells of both A30P and A53T groups had more lysosome and autophagosomes compared with both control and WT groups;After treatment with 1000μM MPP+ ,cells of WT group had no apoptosis except for autophagosomes and advanced stage lysosome,while cells in other three groups witnessed various apoptosis signs. (5)After treatment of MPP+,cell ROS levels in WT group went up with the lowest amplitude while those in both A30P and A53T groups did with the higher amplitude compared with control group.Conclusion Overexpressing human wild typeα-synuclein protect PC12 cells against the toxic influence of MPP+,while overexpressing human A30P or A53Tα-synuclein exacerbated the toxic influence of MPP+.PartⅣThe effect of MPP+ on DAT membrane-expression in different PC12 cell lines overexpressing human wild type,A30P and A53Tα-synuclein,respectivelyObjective To study the effect of MPP+ on DAT membrane-expression in different PC12 cell lines overexpressing human wild type,A30P and A53Tα-synuclein,respectively.Methods (1)Western blot detectedα-synuclein protein level.(2) Four PC12 cell lines were incubated with 131I-FP-β-CIT and intensity of radioactivity of membrane DAT-binding was detected with liquid scintillation.(3)Cell surface protein was biotinylated with sulfo-NHS-SS-biotin and DAT membrane-expression was detected.(4)Cellularα-synuclein mRNA and DAT mRNA were detected with real-time PCR.(5)Withα-synuclein and DAT marked by dipl–immunofluorescence, con- allo cation ofα-synuclein and DAT was detected through laser confocal microscopy.Results (1)After treatment with MPP+,the protein expression ofα-synuclein elevated in four groups.(2) After treatment with MPP+,131I-FP-β-CIT binding were attenuated in four groups. Under the same treatment conditions, the binding radioactivity in WT group were all lowerer than in control group; the binding radioactivity in A30P group were lowerer than in control group without MPP+ treatment,while higher in treatment with 1000μM MPP+; the binding radioactivity in A53T group was no statistical difference in no treatment,while were higher in treatment with 500,1000μM MPP+(.3)Cell surface protein was biotinylated with sulfo-NHS-SS-biotin and DAT membrane-expression was detected in four groups with or without MPP+ treatment.We found that DAT membrane-expression had a decline tendency dependent on MPP+ concentration in all four PC12 cell lines.Under the same treatment level, compared to control group for threeα-synuclein isoforms transfected PC12 cell lines,the DAT membrane-expression level in WT group was lower;the one in A30P group was lower in no treatment while higher in treatment with 1000μM MPP+.The DAT membrane-expression level in A53T group was higher only in treatment with 1000μM MPP+.(4)Following treatment with MPP+,α-synuclein mRNA levels in all four groups were elevated dependent on MPP+ concentration through real time PCR,while DAT mRNA levels in all groups had no statistical difference. (5) Withα-synuclein and DAT marked by dipl-immunofluorescence,con-allocation ofα-synuclein and DAT was detected through laser confocal microscopy in four groups with or without MPP+ treatment.Co-allocation of bothα-synuclein and DAT were ameliorated in all groups except for A53T group following treating with 1000μM MPP+.Conclusionα-synuclein isoforms had influence on the toxic cellular effect of MPP+ through altering cell membrane DAT internalization.Wild typeα-synuclein relieved the toxic cellular effect of MPP+ through facilitating cell membrane DAT internalization,while both A30P and A53Tα-synuclein aggravated the toxic cellular effect of MPP+ through slowing cell membrane DAT internalization.