The Protective Effect And Its Underlying Mechanism Of Compound OAB-14 On Learning And Memory Impairment In APP/PS1 Mice

AimThe pathogenesis of Alzheimer’s disease(AD)is complex and there are multiple hypothesis,the most recognized one is the β-amyloid(Aβ)cascade hypothesis.According to the hypothesis,with the development of AD,a large amount of Aβ deposit in the brain of the patients,resulting in cognitive decline.It is reported that bexarotene can quickly clear the Aβdeposits in the brain of AD mice and improve learning and memory impairment in AD mice.However,its large number of adverse reactions may limit its use in long-term clinical treatment of AD.Our research group synthesized a series of compounds with bexarotene as the lead compound,and screened out a novel small molecular compound OAB-14.In this study,we selected APP/PS1 transgenic mice as AD model animals to investigate the effect of OAB-14 on learning and memory impairment and its effects on neurons and synaptic plasticity.We also investigated the effects of OAB-14 on Aβ from two aspects of Aβproduction and Aβ clearance,and explored the related mechanisms.The purpose of this study is to further clarify the related mechanisms of OAB-14 improving learning and memory disorders,and to provide the scientific basis for the development of anti-AD drugs.MethodsIn the first batch of experiment,8-months-old APP/PS1 double transgenic mice(age-matched C57BL/6 mice were used as a control group)were given OAB-14(50,100 and 200mg/kg),bexarotene(100mg/kg)and peanut oil(model group and control group were given the same volume of peanut oil)for consecutive 15 days.On the sixth day of administration,novel object recognition,Y maze,Morris water maze were conducted successively to investigate the effects of OAB-14 on learning and memory impairments in APP/PS1 mice.Western blot was used to assay the expressions of APP and Aβgeneration-related proteins(ADAM 10,BACE-1,PS1),Aβ degradation enzyme IDE,synapse-related proteins(SYP,PSD95,GAP43,NogoA),BDNF pathway-related proteins(BDNF,TrkB,raf,ERK),M1 microglia marker iNOS,M2 microglia marker ARG I,inflammatory cytokines(IL-1β,IL-6,TNF-a),ApoE pathway-related proteins(ApoE,ABCA1,ABCG1),ApoE subtypes(ApoE3,ApoE4),Ac-H3,H3 and NeuN.Native gel electrophoresis was applied to measure the amount of lipidated ApoE.Immunohistochemical staining was used to detect Aβ deposition and NEP level.Fibrosis Aβ was detected by Thioflavin-S fluorescence staining.The levels of soluble and insoluble Ap1-40 and Aβ1-42 in brain of mice were determined by enzyme-linked immunosorbent assay(ELISA).NeuN was used to mark hippocampal neurons in CA1 region by immunofluorescence.Double immunofluorescence staining was used to colocalizated Aβ and microglia,CD36 and microglia.The ultrastructure of neuronal nucleus,mitochondria and synapse in hippocampus CA1 region were observed by Transmission Electron microscopy(TEM).In the second batch of experiment,APP/PS1 mice(age-matched C57BL/6 mice were used as a control group)were given OAB-14(100,200 mg/kg),bexarotene(100 mg/kg),donepezil(1.3 mg/kg)and peanut oil(model group and control group were given the same volume of peanut oil)for consecutive 10 days,and then social interaction test and nest building test were conducted successively to investigate the effects of OAB-14 on social interaction ability and self-care ability in APP/PS1 mice.Results8-month-old APP/PS1 double transgenic mice showed significant impairments of image identify memory,working memory and spatial learning and memory in novel object recognition,Y maze and Morris water maze test,respectively.The social interaction test showed that the active contact time was shortened and the social interaction ability was reduced.The nesting test showed that the self-care ability was also declined significantly.Compared with the model group,OAB-14 200 mg/kg could significantly improve the Preferential index and distinguish index in novel object recognition test in APP/PS1 mice,increased the spontaneous alternation in Y maze test,shorten the escape latency and swimming distance in Morris water maze test,and improved the social interaction time in social interaction test and nesting ability in nest building test.The results suggest that OAB-14 can significantly improve image identify memory,working memory and spatial learning and memory disorder,and improve social interaction ability and self-care ability in APP/PS1 mice.After administration for 15 consecutive days,there were no significant effect on the expression of APP and Ap generation related protein ADAM 10,BACE-1 and PS1,suggesting that OAB-14 did not affect Aβ generation.OAB-14 significantly increased the expression of Aβ degrading enzyme NEP and IDE,promoted the expression of ApoE,ABCA1 and ABCG1,elevated the level of esterified ApoE,and promoted phagocytosis of Aβ by microglia,which suggest that OAB-14 may activate ApoE pathway,increase the expression of Aβ degrading enzyme and promote the clearance of soluble Aβ in brain.OAB-14 can also significantly improve the expression of CD36 on the surface of microglia,thereby promoting the clearance of fibrous Aβ.OAB-14 promoted the transformation of microglia from pro-inflammatory M1 to anti-inflammatory and phagocytic M2 phenotype,which increased the clearance of AP and reduced the expressions of inflammatory cytokines IL-1β,IL-6 and TNF-a.Results of TEM showed that OAB-14 could alleviate hippocampal neuron damage and improve the ultrastructure of synapses in APP/PS1 mice.Study on mechanisms found that OAB-14 could significantly increase the expression of ApoE3 and decrease the expression of ApoE4,elevate the acetylation level of H3 and increase the expression of BDNF,and activate the BDNF/TrkB pathway,then activate rafi/ERK pathway and enhance the expression of synapse-associated protein SYP,GAP43 and PSD95,and improve synaptic plasticity.OAB-14 also promoted the repair of damaged hippocampal neurons through the activation of BDNF,and increased the number of NeuN-labeled neurons and the expression of NeuN protein.ConclusionOAB-14 could significantly improved learning and memory impairment in APP/PS1 double-transgenic mice,decreased Aβ deposition in the brain,which might be related to the activation of ApoE pathway or the increase of expression of Aβ degradation enzymes,as well as the increase of expression of CD36 on the surface of microglia.Meanwhile,OAB-14 could also reduce the loss of hippocampal neurons and improve synaptic plasticity.The mechanism might be related to the reduction of the toxicity of Aβ to neurons and the activation of the BDNF/TrkB/raf/ERX signaling pathway.The results of this study provided the pharmacodynamic basis for the development of OAB-14 as an anti-AD drug.