Study On The Role Of Scaffold Protein Rapsyn In Microglia

Backgrounds: Microglia are immune phagocytes of the central nervous system.Rapsyn,a scaffold protein,is specifically highly expressed in microglia in the central nervous system.Although a genome-wide association study(GWAS)suggests that Rapsyn is associated with Alzheimer’s disease(AD),there is no other research on the function of Rapsyn in the central nervous system.Our previous study found that the expression of Rapsyn was related to the development of mice,and Rapsyn knockout affected microglia development.In this study,mice with conditional knockout of Rapsyn in microglia were utilized to identify the role of Rapsyn in the microglia.Objective and Methods:(1)To obtain mice with conditional knockout of Rapsyn in microglia,we first constructed Rapsyn flox mice and then crossed the mice with microglia-specific Cx3cr1-Cre mice to produce Cx3cr1-Cre;Rapsyn flox/flox(Cx3cr1-Rapsyn)mice.(2)The number,morphology of microglia in Cx3cr1-Rapsyn mice were detected by immunofluorescence.(3)In order to find the effect of Rapsyn on learning memory and cognitive function,several animal behavior tests were carried out on male adult mice,such as Open Field Test(OPT),Morris Water Maze(MWM),Y-maze,Novel Object Location Test(NOL)and Novel Object Recognition Test(NOR).(4)Amyloid beta(Aβ),one of the characteristic proteins of AD,was injected into the cerebral cortex of mice via stereotactic positioning in monomer format,and then the formation of Aβ plaques in the cerebral cortex was detected.Moreover,the distribution and morphology of microglia around Aβ plaques were checked.In addition,the Aβ was added into the culture medium,and the quantity change of Aβ consumed by Bv2 cells that overexpressed or knocked out Rapsyn was detected.(5)To explore the molecular mechanism of microglia phagocytosing Aβvia Rapsyn,a quantitative polymerase chain reaction(q PCR)was used to access the relative expression of AD-related genes.Then coimmunoprecipitation(Co IP)was engaged to examine whether RAPSYN interacted with AD-related genes at the protein level.Results:(1)Cx3Cr1-Rapsyn mice with conditional knockout of Rapsyn in microglia were successfully obtained.(2)In mice with Rapsyn conditioned knockout in microglia,the density of microglia decreased;and Rapsyn deficient in microglia did not affect the cell body size,branch length and branch number of the microglia.(3)Rapsyn deficient in microglia did not change the motor function and the learning memory of the adult mice.(4)There was the fewer formation of Aβ plaques in the mice with Rapsyn conditioned knockout in microglia than in the control mice.Rapsyn inhibits Bv2 cells from phagocytizing Aβ.(5).Except that Rapsyn knock affects the expression of some AD-related genes,Co IP results suggest that there may be interaction between RAPSYN and AD-related protein TREM2.Conclusions:(1)Rapsyn maintains the number distribution of microglia;(2)Rapsyn affects the transcription of phagocytosis-related genes and regulates the phagocytosis of microglia.