Effects Of α-synuclein On The Endoplasmic Reticulum-Golgi Apparatus System And Apoptosis Of Astrocytes

Objective:Discuss the effect of overexpress a-synuclein of wide-type or mutations (A30P, A53T) on endoplasmic reticulum-golgi system and cell apoptosis of astrocytes (astrocyte, AST).Method:Isolated culture and purify1-3day SD rats of astrocytes, identity the purity of astrocytes (GFAP) with immunofluorescence method, identity the expression of endogenous a-synuclein with Western blot.Lentivirus package (wild-type, A30P, A53T) a-synuclein FUIGW-GFP-HA,GFP-FUIGW plasmid with293FT cells. Harvest virus supernatant,respectively transfect astrocytes, divided into Overexpression of wild-type group (WT), A30P group, A53T group and control group (FUIGW).5-7days after infection, use immunofluorescence and western blot to detect the expression of (wild type, A30P, A53T) a-synuclein-HA fuison protein in AST. Astrocytes treat with tunicamycin6hours as the positive control group, detect the changes of BIP, CHOPwith western blot; Immunofluorescence test GM130to observe changes in the form of golgi apparatus; flow cytometry to detect the changes of rates of cell apoptosis.Results:1) Astrocytes have the expression of endogenous a-synucleinwith Western blot.2)5-7days after lentivirus infection, we can detected the expression ofa-synuclein-HA fusion protein in overexpression of wild-type group (WT) and A30P group, A53T group with Immunofluorescence and Western blot, FUIGW group were negative.3) the BIP, CHOP index of overexpression of wild-type (WT) group and A30P group, A53T group have obvious changes compared with the control group (FUIGW)(P<0.05); The BIP, CHOP index of overexpression of wild-type (WT) group and A30P group, A53T group have no obvious changes(P>0.05).4) Golgi apparatus of the control group (FUIGW) distribute at the side of the nucleus, vesicles complete without fracture, but the overexpression of wild-type (WT) group and A30P group,A53T group disperse in the cytoplasm, vesicles rupture apparently. By chi-square test, the fragment percentage of golgi apparatus of overexpression of wild-type (WT) group and A30P group, A53T group are significantly higher than the control group (FUIGW)(P<0.01); The fragment percentage of golgi apparatus of overexpression of wild-type (WT) group and A30P group, A53T group have no obvious changes (.P>0.05).5)the AST apoptosis rates of the overexpression of wild-type (WT) group and A30P group, A53T group increased compared with the control group (group fuigw)(P<0.01); The AST apoptosis rates of overexpression of wild-type (WT) group and A30P group, A53T group have no significant difference (p>0.05).Conclusion:Overexpress a-synuclein of wide-type or mutation (A30P, A53T) may induced astrocytes apoptosis by endoplasmic reticulum and golgi apparatus system injury.