Effect And Mechanism Of Microglial Atg5 Conditional Knockout On Social Memory In Mice

Objectives:This group unexpectedly found that microglia/monocyte-macrophage Atg5 knockout（Atg5 cKOLyz,3-5 Mon）mice had social memory deficits,but no movement disorders and repetitive behaviors.This thesis was aimed to explore the effect of microglial autophagy related gene 5（Atg5）conditional knockout on social memory and other behaviors in mice,and the underlying cellular and molecular mechanisms involved.Methods:Microglia Atg5 conditional knockout mice were constructed by Atg5fl/fl and CX3CR1cre mice crossbreeding.For behavior tests,3-5-month-old,male microglia Atg5 conditional knockout mice(Atg5fl/fl;CX3CR1cre/+,referred to as Atg5 cKO^Cx3cr1 microglia/monocyte-macrophage Atg5 knockout)and their littermates Atg5fl/fl（WT）were mainly used in this study.For three-chamber sociability test,female mice were also applied.For in vitro study,primary microglia cultures were prepared from neonatal（P0-P3）A tg5 cKO^Cx3cr1 and WT mice after genotyping.For genotyping,genomic DNA was extracted from mouse tail and amplified by polymerase chain reaction（PCR）.Microglial Atg5 knockout efficiency was verified by western blot analysis using both the brain homogenates and primary microglia cultures from Atg5 cKO^Cx3cr1 and WT mice.In terms of behavior tests,mouse social memory was evaluated by three-chamber sociability test.Short-term working memory was evaluated by T maze test.Repetitive behavior was evaluated by self-grooming and marble burying test.Olfactory function was evaluated by olfactory preference（peanut）test.Mouse motor function was evaluated by rotarod test,open field and gait analysis.The neuronal structure（dendrites and spines）in the hippocampus of 3-month-old Atg5 cKO^Cx3cr1 and WT mice were estimated using Golgi staining.Western blot and immunofluorescent staining were applied to study the expressions and difference of synaptic-associated proteins PSD95（postsynaptic marker）,VGLUT2（excitatory presynaptic marker）and VGAT（inhibitory presynaptic marker）,as well as complement C3 in the hippocampus of Atg5 cKO^Cx3cr1 and WT mice.Immunofluorescent co-staining was applied to evaluate the synaptic density in the hippocampus of Atg5 cKO^Cx3cr1 and WT mice,and to study the uptake of synapse-related proteins and the expression of complement receptor CR3 and lysosomal marker CD68 in microglia.Meanwhile,the CR3 protein and mRNA levels in wildtype and Atg5 deficient microglia was detected by western blot and Q-PCR.Flow cytometry was used to analyze the phagocytosis of fluorescent beads in BV2 cells,which were pretreated with autophagy inhibitor chloroquine（CQ）or pbs buffer（controls）.Finally,to explore whether the transcription factor C/EBPa regulates CR3 transcription,the expressions of CR3 and C/EBPa in BV2 cells with C/EBPa transiently knockdown or overexpression were determined by Q-PCR and western blot.Results:（1）Atg5 cKO^Cx3cr1 mice had gender-independent deficits in social memoryThere was no difference of social interaction during in the first stage of three-chamber sociability test between Atg5 cKO^Cx3cr1 and WT mice,both of which recognized and communicated more with the cage containing mice.However,during the second stage of test there was no difference in the time of Atg5 cKO^Cx3cr1 mice spent to communicate between an unfamiliar mouse and its littermate.As expected,WT mice preferred to interact more with unfamiliar mice but not their littermates.This suggested that Atg5 cKO^Cx3cr1 mice have social memory defects but no difficulty in social recognition.3-month-old female Atg5 cKO^Cx3cr1 mice had similar phenotype,indicating that the social memory defects of Atg5 cKO^Cx3cr1 mice were gender independent.Compared with WT mice,there was no difference in short-term working memory,olfactory or locomotor activity in 3-month-old male Atg5 cKO^Cx3cr1 mice.（2）The decrease of dendritic branch and increase of dendritic spines were accompanied with decreased expressions of hippocampal complement molecule C3 and its receptor CR3 in microglia in 3-month-old Atg5 cKO^Cx3cr1 mice compared to their WT littermatesCompared with WT group,the number of neuronal dendritic branches and branch points in the hippocampus of 3-month-old Atg5 cKO^Cx3cr1 mice were significantly reduced,but the density of dendritic spines in the CA2 region was increased.The expression of synaptic proteins including PSD95 and VGLUT2 in the hippocampus,especially in CA2,of 3-month-old Atg5 cKO^Cx3cr1 mice was higher than that of WT group.Co-staining of PSD95 and Synaptophysin（presynaptic markers）showed that the number of co-localized puncta of PSD95+and Synaptophysin+in the CA2 region of Atg5 cKO^Cx3cr1 mice was higher than that in WT mice.Both PSD95+ and VGLUT2+puncta in Ibal+ microglia cells in the hippocampus of 3-month-old Atg5 cKO^Cx3cr1 mice were fewer than those in WT mice.In addition,CD68+and CR3+ fluorescent intensity in hippocampus microglia of Atg5 cKO^Cx3cr1 mice were lower than those in WT mice.The mRNA and protein expressions of CR3 in Atg5 deficient microglia were lower than that in normal primary microglia.Moreover,the expression of complement molecule C3 protein in the hippocampus of 3-month-old Atg5 cKO^Cx3cr1 mice was decreased compared with WT littermates.Flow cytometry showed that the bead fluorescence intensity in CQ（an autophagy inhibitor）-treated BV2 cells was lower than that in control group.（3）The transcription and expression of C/EBPa and CR3 were decreased in Atg5 deficient microgliaThe C/EBPa protein expression in the hippocampus CA2 zone of 3-month-old Atg5 cKO^Cx3cr1 mice was lower than that of WT group.This was consistent with the data showing that the protein and mRNA levels of C/EBPa in Atg5 deficient microglia were lower than those in normal microglia.In C/EBPa knockdown and overexpressing BV2 cells,CR3 mRNA and protein expression were found to be affected simultaneously,implying that transcription factor C/EBPα may regulated the transcription of CR3 in microglia.Conclusion:3-5-month-old microglial Atg5 knockout(Atg5 cKO^Cx3cr1)mice showed gender independent deficits in social memory.The number of neuronal dendritic branches and branch points were decreased;however,the density of dendritic spine and number of excitatory glutamate synapse was increased in the hippocampus CA2 region in 3-month-old Atg5 cKO^Cx3cr1 mice compared to their WT littermates.These changes were accompanied with decreased expressions of hippocampal complement molecule C3 and its receptor CR3 in microglia.Atg5 deficient microglia showed a decreased phagocytosis ability.In sum,the findings in the thesis suggest that the alterations in dendritic structure and synaptic number in the hippocampus of Atg5 cKO^Cx3cr1 mice may be related to decreased expressions of C/EBPa and CR3 caused by Atg5 depletion in microglia,which may lead to reductions in complement-mediated synaptic pruning.