Abstract:

Objective To discuss the differential effects of apolipoprotein E （APOE） gene polymorphism in the neurotoxicity-reactive astrocytes， and to provide the theoretical basis for the study of the pathogenesis of Alzheimer’s disease （AD）. Methods The primary cortical astrocytes from the APOE-knockout mice （APOE ^-/- ） were isolated and cultured in vitro，and the purity of the cells was identified by immunofluorescence staining. The human APOE3 and APOE4 recombinant over-expression plasmids were constructed and separately transfected into the primary APOE ^-/- astrocytes， and the APOE ^-/- primary cells were regarded as control. Western blotting method was used to detect the expression levels of APOE and glial fibrillary acidic protein （GFAP） proteins in the cells； enzyme-linked immunosorbent assay （ELISA） method was used to detect the APOE level in the cellular culture supernatant. The inflammatory models were prepared with the primary astrocytes transfected with APOE3 and APOE4 and co-stimulated with interleukin-1α（IL-1α）， tumor necrosis factor （TNF）， and complement C1q.The cells were divided into APOE3+PBS group， APOE4+PBS group， APOE3+IL-1α+TNF+C1q group， and APOE4+IL-1α+TNF+C1q group. Cell immunofluorescence staining method was used to observe the morphology of the cells in various groups； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of glypican 4 （Gpc4）， glypican 6 （Gpc6）， thrombospondin 1 （Thbs1）， thrombospondin 2 （Thbs2）， SPARC-like protein 1 （Sparcl1） and glial cell line derived neurotrophic factor （GDNF）， C3，and S100 calcium binding protein B （S100B） mRNA in the cells in various groups； microsphere phagocytosis assay was used to detect the phagocytic capacities of the cells in various groups； Western blotting was used to detect the protein expression levels of B-cell lymphoma 2 （Bcl-2），and cysteinyl aspartate specific protease-3 （Caspase-3） proteins in the cells in various groups. Results Compared with APOE ^-/- group， the expression levels of APOE and GFAP proteins in the cells and the APOE level in the cellular culture supernatant in transfected APOE3 and transfected APOE4 groups were increased （P<0.01）. The fluorescence microscope observation results showed that compared with APOE3+PBS and APOE4+PBS groups， the astrocytic processes in APOE3+IL-1α+TNF+Cq1 group and APOE4+IL-1α+TNF+Cq1 group became shorter and the cell bodies became larger； compared with APOE3+IL-1α+TNF+Cq1 group， the astrocytic processes in APOE4+IL-1α+TNF+Cq1 group were even shorter. Compared with APOE3+PBS and APOE4+PBS groups， the expression levels of Gpc4， Gpc6， Thbs1， Thbs2，and Sparcl1 mRNA in the cells in APOE3+IL-1α+TNF+Cq1 group and APOE4+IL-1α+TNF+Cq1 group were significantly decreased （P<0.01）； compared with APOE3+IL-1α+TNF+Cq1 group， the expression levels of Gpc4， Gpc6，Thbs1， Thbs2， and Sparcl1 mRNA in the cells in APOE4+IL-1α+TNF+Cq1 group were significantly decreased （P<0.05 or P<0.01）. Compared with APOE3+PBS and APOE4+PBS groups， the expression levels of GDNF mRNA in the cells in APOE3+IL-1α+TNF+Cq1 group and APOE4+IL-1α+TNF+Cq1 group were decreased（P<0.01），and the expression levels of C3 and S100B mRNA were increased（P<0.01）；compared with APOE3+IL-1α+TNF+Cq1 group， the expression level of GDNF mRNA in the cells in APOE4+IL-1α+TNF+Cq1 group was decreased（P<0.05），and the expression levels of C3 and S100B mRNA were increased（P<0.05）. Compared with APOE3+PBS group and APOE4+PBS group，the numbers of hagocytosis of microspheres in the cells in APOE3+IL-1α+TNF+Cq1 group and APOE4+IL-1α+TNF+Cq1 group were significantly decreased； compared with APOE3+IL-1α+TNF+Cq1 group，the number of hagocytosis of microspheres in the cells in APOE4+IL-1α+TNF+Cq1 group was significantly decreased. Compared with APOE3+PBS group and APOE4+PBS group，the expression levels of Bcl-2 protein in the cells in APOE3+IL-1α+TNF+Cq1 group and APOE4+IL-1α+TNF+Cq1 group were decreased （P<0.05 or P<0.01）and the expression levels of Caspase-3 protein were significantly increased（P<0.01）；compared with APOE3+IL-1α+TNF+Cq1 group， the expression level of Bcl-2 protein in the cells in APOE4+IL-1α+TNF+Cq1 group was decreased（P<0.01），and the expression level of Caspase-3 protein was increased（P<0.05）. Conclusion The APOE4 genotype has a stronger ability to induce the inflammatory factors compared with APOE3；it can lead to a neurotoxicity-reactive astrocyte phenotype，increase the neurotoxicity，affect the astrocyte apoptosis， and aggravate the neuron damage.

Key words: Alzheimer’s disease, Astrocyte, Apolipoprotein E, Gene polymorphism, Apoptosis, Neurotoxicity