The Effects And Mechanisms Of Cannabinoid Type Ⅱ Receptor Deficiency On Learning And Memory Impairment In A Mouse Model Of Alzheimer’s Disease

Objective:To investigate the impact of the CB2 receptor system on the progression and pathology in Alzheimer’s Disease.A colony of mice with a deleted CB2 receptor gene（cannabinoid receptor 2 knockout,CB2R KO）and a transgenic APP/PSEN1 mice（a transgenic human mutant APP background mice）were bred and produced APP/PS1/CB2^-/-mice.Based on this animal model,the effects of CB2R deletion on learning and memory impairment in AD model mice and possible pathological changes of AD and related molecular mechanisms involved in learning and memory impairment were investigated.Methods:The experiments were carried out on pre-symptomatic（4-month-old）,early symptomatic（6-months-old）,and the advanced symptomatic（10-month-old）mice in this study.The non-spatial and spatial learning and memory abilities of the model mice were evaluated by novel object recognition experiment and Morris water maze test.The level of mRNA expression of proinflammatory mediators in Interleukin-6（IL-6）,tumor necrosis factor-α（TNF-α）and inducible nitric oxide synthase（iNOS）were detected in the cortex and hippocampus in mice brain by RT-qPCR.The contents of soluble amyloid beta protein 1-42(Aβ_1-42)in the cortex and hippocampus of mice were detected by ELISA assay.In order to better understand the mechanisms underlying the severe progression of pre-symptomatic APP/PS1/CB2^-/-mice,high-resolution label-free mass spectrometry was performed on cortex tissues and differential abundant proteins were analyzed by Ingenuity IPA database.The molecular mechanisms of CB2R involved in the regulation of learning and memory impairment in AD disease and the changes of the main signaling pathways in APP/PS1/CB2^-/-mice affecting learning and memory impairment were identified by Western Blot.Results:1.The results of novel object recognition experiment showed that in 4-month-old mice,the recognition index（RI）of APP/PS1/CB2R^-/-group decreased by 31.7%（P<0.001）compared with APP/PS1 group.There was no significant difference between the Ctrl group and the APP/PS1 group（P>0.05）.In 6-month-old mice,RI of the APP/PS1/CB2R^-/-group decreased by 24.5%compared with the APP/PS1 group（P<0.05）.In 10-month-old mice,there was no significant difference in RI between the APP/PS1/CB2R^-/-group compared with the APP/PS1 group（P>0.05）,but the APP/PS1 group was significantly lower than the Ctrl group（P<0.001）.It is suggested that in the model mice of each age,the non-spatial learning and memory ability of the APP/PS1 group showed a progressive decline.Knockout of CB2R had no effect on the Ctrl group,but it would lead to the early appearance of non-spatial cognitive impairment in APP/PS1 group.2.Morris water maze test results showed that there was no significant difference in latency and number of cross the platform between all groups at4-month-old（P>0.05）.In 6-month-old,the latency of APP/PS1/CB2R^-/-group on day 5 was longer than that of APP/PS1 group（P<0.05）.Suggesting that knockout of CB2R has little effect on spatial cognitive function in APP/PS1mice.3.The results of RT-qPCR showed that,in 4-month-old mice,the expression of IL-6 in the cortex of APP/PS1/CB2R^-/-mice was significantly higher than that of CB2R^-/-mice（P<0.05）,while the expression of TNF-αand iNOS was not significantly changed（P>0.05）,and the inflammatory factors in the hippocampus were not significantly changed（P>0.05）.In 6-month-old mice,the mRNA expression level of IL-6 in APP/PS1/CB2R^-/-group was significantly higher than APP/PS1 group（P<0.05）.Compared with APP/PS1group,the mRNA expression level of TNF-αin APP/PS1/CB2R^-/-group was significantly increased（P<0.05）.There was no significant change in the mRNA expression level of iNOS（P>0.05）.Compared with APP/PS1 group,the mRNA expression level of IL-6 in hippocampus of APP/PS1/CB2R-/-group was significantly increased（P<0.05）.There was no significant change in the mRNA expression level of TNF-αand iNOS（P>0.05）.Suggested that the expression level of proinflammatory cytokines in APP/PS1 mice increased with age,knockout of CB2R leads to an increase in cortical proinflammatory factors in APP/PS1 mice and neuroinflammation may be involved in early non-spatial learning and memory impairment in APP/PS1 mice.4.The results of ELISA showed that the content of soluble Aβ_1-42 in APP/PS1/CB2R^-/-group was 1.67 times higher than that in APP/PS1 group（P<0.05）,and there was no statistical difference in hippocampus.There was no significant difference in the content of soluble Aβ_1-40 in cortex and hippocampus,suggesting that knockout of CB2R lead to an early increase in the content of soluble Aβ_1-42 in the cortex of APP/PS1 mice.5.The Ingenuity IPA database was used to analyze the metabolic pathway network of the whole proteome,and to explore the molecular mechanism and regulation pattern of pre-symptomatic（4-month-old）APP/PS1/CB2^-/-mice in learning and memory impairment.In this process,1737 proteins were identified.Among the differentially expressed proteins,there were 26differential proteins between the Ctrl group and the APP/PS1 group,out of which 12 were up-regulated and 14 were down-regulated.After CB2R deletion,there were 54 differential proteins between the CB2R^-/-group and the Ctrl group,of which 40 were up-regulated and 14 were down-regulated.There were 114 differential proteins between the APP/PS1/CB2^-/-group and the APP/PS1 group,of which 85 were up-regulated and 29 were down-regulated.Bioinformatics analysis of differential proteins showed that the differential proteins between the APP/PS1 group and APP/PS1/CB2^-/-group were involved in multiple signaling pathways such as mTOR signaling pathway,p38 MAPK signaling pathway,GABA signaling pathway,NF-κB signaling pathway,and RAN signaling pathway.These signaling pathways are associated with inflammatory regulation,synaptic remodeling,mitochondrial dysfunction,and NMDA receptor regulation.According to bioinformatics analysis,the abnormal expression of Syngr3,Hint1,Dnm1 and Ipo5 were identified in pre-symptomatic APP/PS1/CB2R^-/-mice.6.Western Blot results showed that CB2R deficiency activated the PI-3K/AKT/mTOR signaling pathway and Erk1/2 MAPK signaling pathway in APP/PS1/CB2R^-/-mice compared with APP/PS1 mice,while the p38 MAPK signaling pathway was not affected by CB2R deficiency.It is suggested that early cognitive dysfunction and related pathological changes in APP/PS1/CB2R^-/-mice are associated with abnormal PI-3K/AKT/mTOR signaling pathway and Erk1/2 signaling pathway.Conclusion:In this study,CB2R deletion led to earlier onset of non-spatial learning and memory impairment in the pre-symptomatic（4-month-old）and severe cognitive impairment in early symptomatic（6-month-old）APP/PS1 mice,respectively.Cognitive dysfunction in early symptomatic（6-month-old）APP/PS1 mice was closely related to microglial-mediated neurogenic inflammation in the brain.Furthermore,CB2R deletion also led to pre-symptomatic（4-month-old）AD transgenic mice with premature cognitive dysfunction associated with of Aβ_1-42 oligopeptides with neurotoxicity in the cortical brain region,neuroinflammation mediated by microglia overactivation in the cortex,and abnormal activation of PI-3K/AKT/mTOR signaling pathway and Erk1/2 signaling pathway.In addition,differential protein analysis of cortex tissue through proteomics from pre-symptomatic（4-month-old）mice suggested that abnormal expression and dysfunction of synaptic proteins may be important regulatory molecules in APP/PS1 mice earlier onset of cognitive dysfunction caused by CB2R knockout.