Effect Of LINGO-1 On Early Cognitive Function And Hippocampal Neurons Of APP/PS1 Transgenic AD Mice And Its Mechanism

Objective:AD is characterized by spatial learning and memory impairment,neuronal loss and Aβlesions,and the pathogenesis of AD is not clear.In recent years,the role of highly expressed LINGO-1 in neurodegenerative diseases such as AD has attracted much attention.Antagonizing highly expressed LINGO-1 not only has a positive effect on myelin injury in AD animal model,but also effectively prevent the injury of neurons in animal models of various neurodegenerative diseases.However,the effect of high expression of LINGO-1 on neuronal loss in AD lesions is not clear.In this study,APP/PS1 double transgenic mice in the early stage of behavioral change were used to simulate the early stage of AD.Firstly,the effect of LINGO-1 was blocked by intraperitoneal injection of antagonist to study the effects of LINGO-1 on the spatial learning and working memory and neurons in the hippocampus of APP/PS1 transgenic AD mice.Then,the transcriptome sequencing data of hippocampal tissue were used to analyze the related mechanisms and pathways of hippocampal neuron injury in APP/PS1 transgenic AD mice and its correlation with LINGO-1.Finally,after adeno-associated virus overexpressing or silencing LINGO-1 were injected into bilateral cerebral hippocampus,the effects of hippocampal LINGO-1 on the cognition function of normal or AD mice and the neuron related proteins and the proteins in the CB1R/BDNF/TrkB signaling pathway were investigated.At the same time,the mouse hippocampal neuron cell line(HT22 cells)transfected with APP/PS1 genes were used to simulate AD in vitro and explore the effect and mechanism of antagonizing LINGO-1 on AD hippocampal neurons in vitro in order to provide new scientific evidence for the study of the pathogenesis of AD.Methods:1.Effects of anti-LINGO-1 antibody on early cognitive function and hippocampal neurons of APP/PS1 transgenic AD mice were investigated as the followings.(1)Anti-LINGO-1 antibody was injected intraperitoneally to block the effect of LINGO-1 in APP/PS1 transgenic AD mice.Morris water maze and Y maze experiments were used to evaluate their spatial learning and memory ability and working memory ability respectively.LINGO-1 and Aβplaque in hippocampus were labeled with the immunofluorescence staining and thioflavin S staining respectively to analyze the content of LINGO-1 and the number of Aβplaque in hippocampus.The volume of hippocampus and the total number of neurons in hippocampus were analyzed with Nissl staining and stereological methods.(2)At the 5th week after anti-LINGO-1 antibody intervention,mice in each group were intraperitoneally injected with Brd U to label proliferating cells.DCX immunohistochemical staining was used to label immature neurons,and Neu N immunohistochemical staining was used to label mature neurons.GAD67 immunohistochemical staining was used to label GABAergic interneurons.The total number of immature neurons and GABAergic interneurons were evaluated with stereological three-dimensional quantitative method.Brd U,DCX and Neu N immunofluorescence stainings were used to label neurons,and the densities of newborn immature neurons(Brd U~+/DCX~+/Neu N~-cells),newborn mature neurons(Brd U~+/DCX~-/Neu N~+cells),the third type of newborn neurons between immature and mature neurons(Brd U~+/DCX~+/Neu N~+cells)and total newborn neurons(the sum of the first three)were analyzed.GABAergic interneurons were labeled with immunofluorescence stainings of GAD67,CCK8 and CB1R.The densities of GABAergic interneurons expressing CB1R(GAD67~+/CB1R~+cells)and CCK-GABAergic interneurons expressing CB1R(GAD67~+/CCK8~+/CB1R~+cells)were analyzed.GABAergic interneurons expressing CB1R(CB1R-GABA)were labeled with GAD67,LINGO-1 and CB1R immunofluorescence staining.The densities of CB1R-GABAergic interneurons that can express LINGO-1(GAD67~+/LINGO-1~+/CB1R~+cells)and CB1R-GABAergic interneurons that do not express LINGO-1(GAD67~+/LINGO-1~-/CB1R~+cells),and the density ratio between CB1R-GABAergic interneurons expressing LINGO-1 and the total CB1R-GABAergic interneurons(that is,the density ratio between GAD67~+/LINGO-1~+/CB1R~+cells and GAD67~+/CB1R~+cells)were analyzed.2.Study on the mechanism underlying the effects of LINGO-1 on hippocampal neurons in normal mice and APP/PS1 transgenic AD mice.(1)Transcriptome sequencing was used to analyze the expression of RNA in the hippocampus of normal WT mice and APP/PS1 transgenic AD mice.The differentially expressed genes related to neurons were analyzed by GO-P-Desc and GO-C-Desc,respectively.(2)The AAV overexpressing LINGO-1 was stereotactically injected into the bilateral cerebral hippocampus of normal mice,and then the spatial learning and memory ability were detected by Morris water maze test.LINGO-1 protein and the neuron related proteins(GAD67,DCX,Neu N),and CB1R and proteins in BDNF/TrkB/p-TrkB signal pathway in the hippocampus were detected by Western blot.(3)The AAV silencing LINGO-1 was stereotactically injected into bilateral cerebral hippocampus to antagonize LINGO-1 in the hippocampus of AD mice,and then the spatial learning and memory abilities were evaluated by Morris water maze test.The expressions of LINGO-1 protein,neuron related proteins(GAD67,DCX,Neu N),and CB1R and proteins in BDNF/TrkB/p-TrkB signal pathway in the hippocampus were detected by Western blot.(4)HT22 cells transfected with APP/PS1 in vitro were treated with anti-LINGO-1 antibody.Firstly,the proliferating cells were labeled with Edu.Then,the expression of LINGO-1 protein,neuron related proteins(GAD67,DCX,Neu N),CB1R and proteins in BDNF/TrkB/p-TrkB signal pathway were evaluated by Western blot.Finally,the expression of intracellular LINGO-1 and Aβ,the expression of newborn neurons(Edu~+/DCX~+/Neu N~-cells,Edu~+/DCX~-/Neu N~+cells,Edu~+/DCX~+/Neu N~+cells),the expression of CCK-GABAergic interneurons rich in CB1R,the ratio between CB1R-GABAergic interneurons expressing LINGO-1 and the total CB1R-GABAergic interneurons,and the expression of TrkB and p-TrkB on GABAergic interneurons,were evaluated with immunofluorescence staining.(5)HT22 cells transfected with APP/PS1 in vitro were treated with CB1R antagonist AM251 and activator ACEA.The expressions of intracellular LINGO-1,GAD67,DCX,Neu N,CB1R,BDNF,TrkB,p-TrkB proteins were evaluated by Western blot,and the effects of AM251 and ACEA on the expression ratio of intracellular LINGO-1 and TrkB were detected by immunofluorescence staining.Results:1.Effects of anti-LINGO-1 antibody on the early behaviors and neuropathology including hippocampal neurons of APP/PS1 transgenic AD mice.(1)After APP/PS1 transgenic AD mice were injected intraperitoneally with anti-LINGO-1 antibody to antagonize LINGO-1,their spatial learning and memory abilities and working memory ability were effectively improved,and the fluorescence intensity of hippocampal LINGO-1 protein was decreased.The density of hippocampal Aβplaque was reduced,and the atrophy of hippocampal volume was delayed.The loss of the immature neurons in hippocampal DG region and the loss of the total neurons and GABAergic interneurons in hippocampal DG and CA1 region were delayed.(2)After APP/PS1 transgenic AD mice were injected intraperitoneally with anti-LINGO-1 antibody to antagonize LINGO-1,the formations of three types of newborn neurons including Brd U~+/DCX~+/Neu N~-cells,Brd U~+/DCX~-/Neu N~+cells and Brd U~+/DCX~+/Neu N~+cells in hippocampal DG region were effectively promoted,and the number of CCK-GABAergic interneurons rich in CB1Rs in hippocampal subregions(DG,CA1 and CA2/3 region)were increased.The density ratio between CB1R-GABAergic interneurons expressing LINGO-1 and the total CB1R-GABAergic interneurons in hippocampal DG and CA1 region were significantly reduced.2.Mechanism underlying the effects of LINGO-1 on hippocampal neurons of normal mice and APP/PS1 transgenic AD mice.(1)Differential analyses showed that there were 3510 differentially expressed genes in the hippocampus of APP/PS1 transgenic AD mice and normal WT mice.GO-P-Desc analyses showed that 418 genes were related to neurons in the 3510 differentially expressed genes,including LINGO-1and DCX genes.Linear correlation analyses showed that there was a negative correlation between LINGO-1 and DCX gene expression.After GO-C-Desc and statistical analyses,it was found that the expression of GAD1 gene encoding GAD67 protein and NTRK2 gene(i.e.TrkB gene)in the hippocampus of APP/PS1 mice were decreased.(2)After overexpressing hippocampal LINGO-1,the level of LINGO-1protein in the hippocampus of normal mice was significantly increased.Although the spatial learning and memory abilities of mice were partially impaired and the level of CB1R protein was decreased,the levels of neuron related proteins(GAD67,DCX,Neu N)and proteins in BDNF/TrkB/p-TrkB signaling pathway in the hippocampus were not changed.(3)After silencing hippocampal LINGO-1,the level of LINGO-1 protein in the hippocampus of AD mice was significantly decreased,and the spatial learning and memory abilities of these AD mice were significantly improved.Among neuron related proteins in the hippocampus,only the level of DCX protein was increased,and the levels of GAD67 and Neu N protein were not changed.In addition,the levels of CB1R,BDNF and p-TrkB protein in the hippocampus were significantly increased,but the level of TrkB protein was not changed.(4)After anti-LINGO-1 antibody intervention,the level of LINGO-1protein in HT22 cells transfected with APP/PS1 was decreased,and the levels of Neu N,CB1R,TrkB,and p-TrkB protein were increased.The levels of GAD67,DCX,and BDNF protein were not changed.In addition,LINGO-1~+cells,Aβ~+cells and LINGO-1~+/Aβ~+cells were decreased.The numbers of Edu~+/DCX~+/Neu N~+cells and total newborn neurons(the sum of Edu~+/DCX~+/Neu N~-cells,Edu~+/DCX~-/Neu N~+cells and Edu~+/DCX~+/Neu N~+cells)were significantly increased.The number of CCK-GABAergic interneurons rich in CB1R was significantly increased.The ratio between GAD67~+/LINGO-1~+/CB1R~+cell density and GAD67~+/CB1R~+cell density was significantly decreased.The numbers of DAPI/GAD67~+/TrkB~+cells,DAPI/GAD67~+/p-TrkB~+cells and DAPI/GAD67~+/TrkB~+/p-TrkB~+cells were significantly increased.(5)For HT22 cells transfected with APP/PS1,after activating CB1R,the protein levels of CB1R,GAD67,DCX,Neu N,BDNF,TrkB and p-TrkB were significantly increased,and the protein level of LINGO-1 and the ratio between LINGO-1 and TrkB were significantly reduced.On the contrary,after antagonizing CB1R,the protein level of TrkB was not significantly changed,but the protein levels of CB1R,GAD67,DCX,Neu N,BDNF,and p-TrkB were reduced,and the protein level of LINGO-1and the ratio of LINGO-1 to TrkB were significantly increased.In addition,LINGO-1 antagonist had the same effect as CB1R activator.At the same time,antagonizing LINGO-1 could significantly reverse a series of changes caused by CB1R antagonist AM251.Conclusions:1.Either intraperitoneal injection of anti-LINGO-1antibody to antagonize LINGO-1 in hippocampus or brain stereotactic injection to specifically silence LINGO-1 in hippocampus can effectively improve the hippocampal dependent spatial learning and memory ability and protect hippocampal neurons in transgenic AD mice.2.Antagonizing LINGO-1 can significantly increase the formation and maturation of newborn neurons in the hippocampus of AD mice,reduce the expression of LINGO-1 on CB1R-GABAergic interneurons and increase the density of CB1R-GABAergic interneurons in the hippocampus,suggesting that CB1R-GABAergic interneurons may be the key target cells for LINGO-1 to regulate hippocampal neurogenesis in transgenic AD mice.3.Overexpression of LINGO-1 in the hippocampus of normal mice had no significant effect on the downstream CB1R/BDNF/TrkB signal pathway of hippocampal CB1R-GABAergic interneurons,while silencing LINGO-1in the hippocampus of AD mice could significantly enhance the downstream CB1R/BDNF/TrkB signal pathway of hippocampal CB1R-GABAergic interneurons.4.Animal and cell experiments showed that antagonizing LINGO-1could effectively improve the hippocampus-related behavioral abilities of AD mice through activating CB1R/BDNF/TrkB signal pathway and improve the expression of neuron-related proteins in the hippocampus of AD mice and HT22 cells transfected by APP/PS1.The current results might provide scientific bases for further studies that explore the mechanism for the effects of LINGO-1 on AD.