| Background:Alzheimer's disease(AD)is a progressive neurodegenerative disease and the most important age-related dementia.The incidence of AD is increasing year by year with the aging of the global population.The most typical clinical manifestations of AD are progressive cognitive impairment and mental-behavioral disorders.The major pathological features are the deposition of?-amyloid(A?)forming senile plaques(SPs),neurofibrillary tangles(NTF)produced by abnormal phosphorylation of tau protein,and the atrophy of brain parenchyma caused by neuronal degeneration and loss.As an important pathological change of Alzheimer's disease(AD),the deposition and metabolism of?-amyloid(A?)are often positively correlated with the severity of the impairment in cognition and memory.In recent years,many studies have found that soluble A?oligomers,especially A?1-42oligomers,are considered as the most neurotoxic A?production,which are found causing the loss of neurons and synapses,synaptic plasticity damage and chronic inflammation of the central nervous system.The receptors of A?can be divided into“good”receptors involved in A?metabolism and clearance from the brain,as well as“bad”receptors involved in the binding and cytotoxic action of A?.And one receptor at different pathological stage of AD,or combining with different A?fragments,may have a different function.The paired immunoglobulin-like receptor B(PirB)in the mouse brain and its homologous in humans,leukocyte immunoglobulin-like receptor B(Lilr B),are demonstrated as a high-affinity receptor of A?1-42 oligomers,which is considered to be involved in synaptic plasticity damage by A?1-42 oligomers.However,the influence on the pathological progress of AD and related mechanisms remains to be elucidated.In addition,previous studies have confirmed that the synthesized extracellular segment of PirB protein competitively inhibited the activity of PirB as receptor,and rescued the damage of cognitive and neurological function caused by ischemia and hypoxia.However,as the A?receptor,the role of PirB in the accumulation and degradation of A?,and its underlying mechanisms are still unclear.What's more,in the middle and late stages of AD,energy metabolism disorders occur,and the inhibition of AMPK/mTOR signaling pathway,induced by the disturbing of energy metabolism,impairs the function of autophagy.Activation of the AMPK/mTOR pathway can improve the cognitive impairment and pathological progress in AD transgenic model and caused by various incentives(high glucose,ischemia,hypoxia,etc.)Objects:1.The correlation between PirB and A?deposition in brain.2.The effects of PirB level on A?pathology and cognitive function and the underlying mechanism.Methods:1.Detect the levels of PirB in mice brains and its relationship with the level of A?:Take the hippocampus tissues of 9-month-old APP/PS1 transgenic mice,the same-month-old wild-type mice(6 mice per group),and different-ages wild-type C57BL/6 mice(3-month-old,9-month-old,and 12-month-old)(6 mice per group),and different brain tissues of 9-month-old C57BL/6 mice And detect the expression (Western blot,real-time fluorescent quantitative reverse transcription PCR),distribution (immunohistochemistry)of PirB,and the positional relationship between PirB and A? in the APP/PS1 mice brain.2.Detect whether overexpression of PirB affects the uptake of A?1-42 oligomers,and inhibition of autophagy function and AMPK/mTOR signaling pathway induced by A?:1)Transfect the plasmid of expressed PirB or vector on stable cultured HEK293 cells (DMEMbasic+10%FBS),exogenously give the synthesized A?1-42 oligomers carrying FTIC(1mM),treated for 2 hours,observe and the effect of overexpression of PirB on uptake of A?1-42 oligomers,under a fluorescence microscope.Then,transfect the plasmid of expressed PirB or vector on stable cultured HEK293 cells,exogenously give the synthesized A?1-42 oligomers or the solvent(containing 0.01%DMSO)for 2 hours,Divide the cells into 4 groups(Vector-DMSO,Vector-A?1-42,PirB-DMSO and PirB-A?1-42),and use CCK-8 reagent to detect the four groups of cells Inhibition index.2)Transfect the plasmid of expressed PirB or vector on stable cultured HEK293 cells, exogenously give the synthesized A?1-42 oligomers(1mM)for 2 hours,and detect the levels of P62 and LC3 protein in the four groups of cells(Western blot)reflecting the function of autophagy.Co-transform the GFP-LC3-RFP plasmid which can display autophagy flow with the plasmid overexpressing PirB or vector on the stably cultured HEK293 cells,and give artificially synthesized A?1-42oligomers(1mM),observe the autophagy flux under a fluorescence microscope,and count the ratio of autophagolysosomes to total autophagosomes.3)Overexpress PirB or vector by plasmid on stable cultured HEK293 cells,and treat them with A?1-42 oligomers for 2 hours,and detect the activity of AMPK/mTOR signaling pathway(Using Western bolt detecting AMPK,p T172-AMPK,mTOR,p S2448-mTOR).3.Detect the effect of overexpression of PirB on the cognitive function and A?pathology of APP/PS1 transgenic mice:1)18-week-old APP/PS1 transgenic mice(16 mice)were randomly divided into two groups,8 mice in each group,and inject stereotactically the adeno-associated virus (AAV)expressing PirB and vector into the hippocampal CA3 region(1?l),APP/PS1-PirB,APP/PS1-Vec,and use C57BL/6 mice identified as wild-type(WT) in the same cage and the same age as a control(16 mice),also divided into two groups,8 animals in each group,and inject with AAV(1?l)in the hippocampal CA3area to overexpress PirB and vectors(WT-PirB,WT-Vec).2)One month after expression,test the function of cognitive,learning and memory (new object recognition test,Morris water maze test),and collect materials.Detect the level of APP and A?in the hippocampus of the four groups mice(Western blot, ELISA).Observe the formation of A?plaque and the positional relationship between A?plaque and PirB(immunofluorescence).3)Detect the level of P62 and LC3 in the hippocampus of four groups of mice (Western blot)reflecting the autophagy function,and detect the AMPK/mTOR signaling pathway(Western blot detects AMPK,PT172-AMPK,mTOR,PS2448-mTOR)4.Detect whether knockdown PirB affects the uptake of A?1-42 oligomers,and inhibition of autophagy and AMPK/mTOR signaling pathway induced by A?:1)Transfect PirB-si-RNA plasmid or vector plasmid on stable cultured N2a cells(10% FBS+45%DMEM basic+45%Opti-MEM),and then give synthetic A?1-42oligomers(2mM)exogenously treated for 2 hours as the uptake group(Vec+A?1-42-uptake,siPirB+A?1-42-uptake),and replace the medium for another 6 hours as the degradation group(Vec+A?1-42-degrade,siPirB+A?1-42-degrade),and the solvent group with A?1-42 as the blank control(Vec-blank,siPirB-blank).And test the level of A?1-42 in the 6 groups of N2a cell lysate(ELISA)reflecting the uptake and degradation of A?1-42 oligomers.2)Transfect the PirB-si-RNA plasmid or vector plasmid on stable cultured N2a/APP cells(stably expressing human APPswe)(N2a/APP-Vec,N2a/APP-siPirB),and detect the levels of A?1-42 in the two groups of cells(ELISA)and the levels of APP and sAPP(immunoblotting)and The levels of P62 and LC3(Western blot)reflect autophagy and AMPK/mTOR signaling pathway(Western blot detects AMPK,PT172-AMPK,mTOR,PS2448-mTOR).5.Detect whether knockdown PirB improves the cognitive and memory function of APP/PS1 mice:2-month-old APP/PS1 mice(when A?deposition just appeared)were randomly divided into two groups,5 mice in each group,and stereotactic injection of adeno-associated virus(AAV)expressing si-RNA-PirB and vector in hippocampal CA3 area respectively (1?l),APP/PS1-siPirB,APP/PS1-Vec,to detect changes in cognitive function in the middle of the disease course(7-month-old)(new object recognition test,Morris water maze test).Results:1.The expression of PirB is positively correlated with the level of A?.1)The expression of PirB in the brain of wild-type C57BL/6 mice increases with age, and the expression of PirB in the hippocampus is higher than that of the cortex and slightly lower than that of the cerebellum.2)Compared with wild-type mice at the same month age in the same cage,the expression of PirB increased significantly in the hippocampus of 9-month-old APP/PS1 transgenic mice,and the increase was mainly distributed in the hippocampal CA3 and DG areas.3)Immunofluorescence staining showed that PirB protein co-localized with A?,and compared with wild-type mice in the same cage and the same month,the level of PirB increased in the pyramidal neurons in the hippocampal CA3 region of APP/PS1 transgenic mice at the age of 9 months.2.The overexpression of PirB promotes the uptake of A?oligomers(A?o)in cells,and aggravates the inhibition of autophagy function and AMPK/mTOR signaling pathway induced by A?o.1)The overexpression of PirB increased the uptake of A?o in HEK293 cells.2)The overexpression of PirB with exogenous addition of A?o increased the levels of P62 and LC3-? in the lysate of HEK293 cells,and decreased the ratio of LC3-?/LC3-?.3)The GFP-LC3-RFP plasmid was co-transformed with the co-transformed PirB plasmid with exogenous addition of A?o.It was observed under a fluorescence microscope that the ratio of autophagy lysosomes(RFP-LC3)to the total autophagy body(RFP-LC3+GFP-LC3)of the overexpression PirB group is reduced.4)The overexpression of PirB with exogenous addition of A?o reduced the levels of AMPK and p T172-AMPK in the HEK293 cell lysate,increased the level of p S2448-mTOR,but did not change the level of mTOR.3.The overexpression of PirB aggravates A?deposition and cognitive memory impairment in APP/PS1 mice.1)The overexpression of PirB caused the obvious impairment of cognition and memory in 22-week-old APP/PS1 mice,but had no significant effect in wild-type mice.In the object recognition test(ORT),the APP/PS1 mice with overexpressed PirB had a lower preference index for new objects,and there was no effect on the total exploration time.Morris water maze test(MWM),in the place navigation test,the APP/PS1 mice with overexpressed PirB had the increased periods for reaching the escape platform.In the spatial probe test,the APP/PS1 mice with overexpressed PirB had the increased periods and the decreased times crossing the platform area.2)The expression of AAV is showed in the immunofluorescence of brain slice (GFP/PirB)and observed PirB increased expression in hippocampal CA3 area.The overexpression of PirB increased the levels of APP and soluble APP(sAPP)in the hippocampal homogenate of APP/PS1 mice,and the levels of A?1-40(11)A?1-42and the ratio of A?1-42/A?1-40 were increased.However,in the wild-type mice,overexpression of PirB even slightly reduced the soluble APP(sAPP)in the hippocampus,with the level of A?1-40 constant,and the level of A?1-42 was decreased.3)The overexpression of PirB increased the levels of P62 and LC3 in the hippocampal homogenate of APP/PS1 mice,decreased the levels of AMPK and PT172-AMPK, and increased the levels of PS2448-mTOR and mTOR.There was no obvious change on the levels of P62 and LC3 in wild-type mice with overexpressed PirB, whereas,the levels of AMPK and PT172-AMPK were increased,the level of PS2448-mTOR was decreased,and the level of mTOR did not change significantly.4)The overexpression of PirB caused abnormal changes on synaptic protein in the hippocampus of APP/PS1 mice.The level of presynaptic protein Synapsin1 decreased and the level of postsynaptic protein PSD95 increased.4.Knockdown PirB reduces the uptake of A?1-42 oligomers by nerve cells,and reverses the inhibition of autophagy function and AMPK/mTOR signaling pathway induced by A?.1)Knockdown PirB reduced the uptake of A?1-42 oligomers in N2a cells,and improved the degradation of A?1-42after 6 hours.2)Knockdown PirB in N2a/APP cells,did not significantly change the expression of APP and sAPP,but decreased the level of A?1-42 in the cell lysate.3)Knockdown PirB in N2a/APP cells induced the level of P62 decreased,the level of LC3-? increased,without the level of LC3-? significantly changed,and the ratio of LC3-?/LC3-? decreased.Knockdown PirB in N2a/APP cells increased the level of AMPK and PT172-AMPK,decreased the level of PS2448-mTOR,without the level of mTOR significant changed.5.Knockdown PirB improves the cognitive and memory function of APP/PS1 mice.Knockdown PirB reversed the impairment of cognition and memory in 7-month-old APP/PS1 mice.In the object recognition test(ORT),the AAV-siPirB APP/PS1 mice had a higher preference index for new objects,and there was a slight improvement on the total exploration time.Morris water maze test(MWM),in the place navigation test,the AAV-siPirB APP/PS1 mice had the decreased periods for reaching the escape platform. In the spatial probe test,the AAV-siPirB APP/PS1 mice had decreased periods and the increased times of crossing the platform area.Conclusions:1.The expression of PirB is positively correlated with the production of A?in brain,and co-localizes with A?in the cells.2.PirB promotes A?deposition and aggravates A?-induced cognitive dysfunction,by inhibiting AMPK/mTOR-mediated autophagy function3.Knockdown PirB ameliorates A?pathology and related cognitive dysfunction,therefore,PirB may be a potential target for AD treatment. |
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