Immunoglobulin G Alterations In Brain Of APOE-TR Mice And Herpes Simplex Virus Type Ⅰ Induced Aβ Expression In SH-SY5Y Cells

Immunotherapy has been regarded as a potential disease-modifying treatment for Alzheimer’s disease in recent years.The clinical trials suggested that the APOEε4 allele was related to the effect of immunotherapy and the occurrence of adverse reactions.Some studies have suggested that in people carrying the APOEε4 allele,therefore,predisposed to develop AD,HSV-1 infection markedly increases the risk of AD.However,the mechanism is unclear.This topic mainly includes the following two parts:In the first part,we determined the alterations of IgG,B cell specific marker CD19,and Aβ in various brain regions of uninfected male and female APOE3-and APOE4-TR mice at the age of 3 or 10 months,by using immunochemical staining,western blotting,ELISA and flow cytometry to elucidate impacts of AD risk factors on alterations of brain IgG.Immunohistochemical staining showed that the positive staining for IgG was distributed across the brain,including neocortex,hippocampus,thalamus and cerebellum.The levels of IgG in the female group was higher than that in the male group.In the female group,compared with10-month-old APOE3-TR control mice,10-month-old APOE4-TR mice had lower IgG levels in AD susceptible brain regions such as neocortex,entorhinal cortex and hippocampus,but no significant changes in thalamus and cerebellum,two regions nearly intact in AD.Compared with 3-month-old APOE3-TR mice,the 10-month-old APOE3-TR mice significantly increased in the entorhinal cortex,neocortex,hippocampus and cerebellum,but APOE4-TR mice did not have this changes.In the male group,there was no statistically significant difference in IgG levels between age and genotype.Double-immunofluorescent staining revealed IgG positive staining in brain was mainly located on microglia,but not astrocytes.Some IgG positive neurons were also observed in mediodorsal thalamic nucleus,and none in other brain areas.In addition,the expression of CD19,a specific marker expressed on mature and activated B cells,was significantly reduced in the hippocampus of 10-month-old female APOE4-TR mice.Although there were no obvious differences between plasma IgG levels of APOE4-and age matched female APOE3-TR mice,flow cytometry showed significantly decreased B cell amount in blood of 10-month-old female APOE4-TR mice,compared with APOE3-TR control mice.Moreover,more obvious positive staining for Aβ,one major pathological mark of AD,was observed in the cortex of10-month-old female APOE4-TR mice than other groups.These results,for the first time,demonstrated that AD risk factors were associated with IgG alterations in various brain regions,which might result from the defects of humoral immunity and lead to the impairment of IgG-mediated clearance of Aβ by microglia,therefore facilitated AD progression。In the second part,we explored the effect of HSV-1 infection onβ-amyloid(Aβ)production and degradation in SH-SY5 Y cells.SH-SY5 Y cells were infected with 10 MOI for 12 h or 24 h.Immunofluorescence assay was performed to measure the expression of Aβ and APOE in the cells.The expression of APP,HSV-1 g C protein,APP,MME,APOE,GSK3β and p-GSK3β in cell lysates were analyzed using Western-blotting.We revealed morphological changes in SH-SY5 Y cells during infection with HSV-1.The cells displayed clear neurite retraction and few neurites remain by 12 hours infection.All cells appeared to be infected and rounded up at 24 hours.Meanwhile,the expression of Aβwas significantly increased while the expression of APOE protein was not changed.At 24 hours post infection,the expression of Aβ and APOE protein were increased.Western-blotting analysis showed the expression of the APP in the cell was decreased,Ser9 phosphorylation increased in infected cells,and the expression of MME was increased at 12 hours post infection.While at 24 hours post infection,the expression of MME decreased and the APOE protein was increased.The results suggest that early stage of HSV-1 infection is linked to modulation of GSK-3βphosphorylation to increase APP expression induced Aβ;while at late stage,HSV-1 inhibited the degradation of Aβ,and increased APOE protein expression,affecting the expression of Aβ.These results contribute to further elucidating the interaction between HSV-1 and the presence of Aβ in neurons.