Multiple Mild Stimulations Attenuate Cognitive Deficits Caused By A Transient Ischemic Attack Regulated By Membrane Distribution Of Microglial ANXA1 And CX3CR1 In Mice

【Purpose】Transient ischemic attack(TIA)is a transient neurological disorder caused by ischemia in the central nervous system,most TIA patients are led to highly recurrent neurological deficits due to the lack of timely treatment.Therefore,it is particularly important to explore new interventions to improve the neurological deficits caused by TIA.This study aims to improve the neurological deficits caused by TIA via multiple mild stimulations(MMS).Meanwhile,this study explores the mechanisms by which MMS improves learning and memory deficits after TIA at the cellular level of microglia-neurons interaction and the molecular level of Annexin A1(ANXA1)binding with C-X3-C motif chemokine receptor 1(CX3CR1).【Methods】(1)A TIA model in mice was constructed by a bilateral common carotid artery occlusion(BCCAO).Three days later,the mice were subjected to MMS for 5 weeks.(2)The Adenoassociated virus(AAV)was injected into the hippocampus of Cx3cr1-cre mice to construct a mouse model that ANXA1 overexpressed specifically in hippocampal microglia.(3)Morris water maze(MWM),novel object recognition(NOR)tests were used to evaluate learning and memory.Elevated plus maze(EPM),open field test(OFT),forced swimming test(FST),tail suspension test(TST)and sucrose preference test(SPT)were conducted to detect the emotional state of mice.(4)MED64 was performed to detect hippocampal LTP.Golgi-Cox staining was used to detect neuronal processes and dendritic spines.(5)Primary cultured microglia were infected with adenovirus overexpressed ANXA1.Hippocampal neurons were primary cultured and infected with adenovirus expressed GFP.After coculture of microglia and neurons,the dendritic spines and microglial phagocytosis were detected by the confocal microscope.(6)TUNEL staining was to detect apoptosis.(7)IF was conducted for the proteins expression,interactions and subcellular distribution.(8)WB was performed to detect the total and the membrane/cytoplasmic protein expression.Co-IP was conducted to evaluate the protein binding.【Results】(1)MMS enhanced the spatial reference and hippocampal-dependent learning and memory after TIA.The hippocampal LTP was inhibited,the dendritic spine density and neuronal branches were reduced after TIA,while MMS improved the synaptic structure and function impaired by TIA.MMS reversed the hyperactivation of microglia,decreased the over-interactions between microglia and neurons and inhibited the over-phagocytosis of microglia after TIA.(2)After overexpression of ANXA1 in primary cultured microglia,the binding of ANXA1 and CX3CR1 was increased,the CX3CR1 protein level on the membrane was improved,and the binding of CX3CR1 and co-cultured neuronal CX3CL1 was enhanced.The ANXA1 overexpression in microglia promoted the phagocytosis of microglia to FITCdextran and PSD95 of co-cultured neurons,and reduced the density of spine synapses,which further demonstrated that overexpression of ANXA1 in microglia promoted dendritic spine pruning by microglia.(3)The hippocampal ANXA1 protein level was upregulated in TIA-treated mice,especially on the membrane,which promoted the membrane distribution of CX3CR1 and the interaction of CX3CR1 and CX3CL1;whereas MMS reversed the elevation of ANXA1 on membrane caused by TIA.MMS abolished the increasement of ANXA1 and CX3CR1 interactions after TIA,reduced the CX3CR1 protein level on the membrane,and finally reversed the increasement of CX3CR1-CX3CL1 binding after TIA.(4)After TIA and MMS treatments,ANXA1 overexpression specifically in hippocampal microglia promoted the binding of CX3CR1 and CX3CL1,reduced the PSD95 protein level,and decreased the synaptic spine density,which demonstrated that the dendritic spines were over-pruned.Meanwhile,the learning and memory were impaired after ANXA1 overexpression in microglia,which means that the improvement of MMS in learning and memory after TIA was inhibited.【Conclusions】MMS rescues the cognitive function and hippocampal synapses impaired by TIA in mice.ANXA1 promotes the membrane distribution of CX3CR1 in microglia,upregulates the subsequent interactions between microglial CX3CR1 and neuronal CX3CL1,and decreases the dendritic spine density in neurons.TIA promotes the interactions between hippocampal ANXA1 and CX3CR1 via increasing ANXA1 protein level,increases the distribution of CX3CR1 in the membrane,and thus enhances the binding of CX3CR1 and CX3CL1;whereas MMS reduces the ANXA1 protein level,attenuates the CX3CR1-CX3CL1 binding promoted by TIA and provides neuroprotective effects in TIA-treated mice.