Investigation Of The Effects Of Hippocampal LINGO-1 On The Pathogenesis And Treatment Of AD Based On Oligodendrocytes

Objective:Alzheimer’s disease(AD)is characterized with memory and cognitive dysfunction.However,the pathogenesis of AD is still unclear,and there is still no effective treatment measures to cure AD.Recently,it has been found that Leucine-rich repeat and immunoglobulin-like domain-containing nogo receptor-interacting protein 1(LINGO-1),a nerve growth inhibitor,is able to affect axon growth,oligodendrocyte differentiation or myelination.LINGO-1 is highly expressed in many animal models of AD and even in AD patients.However,the role of LINGO-1 in the pathogenesis of AD remains obscure.In the current study,we explored the expression of LINGO-1 in the hippocampus of the APP/PS1 transgenic mouse model,and the effect of LINGO-1 on hippocampal-dependent spatial learning and memory capacity and hippocampal oligodendrocytes.Then,we investigated whether blocking LINGO-1 in the hippocampus by peripheral administration of anti-LINGO-1 antibody could restore hippocampal-dependent spatial learning and memory in APP/PS1 mice,and the abnormalities of oligodendrocytes in the hippocampus.Subsequently,the effect and mechanism of anti-LINGO-1 antibody on primary oligodendrocytes were further studied.Our study aimed to explore the effects of LINGO-1 in the pathogenesis and treatment of AD and provide a scientific basis and a potential target for the prevention and treatment of AD.Methods:1.To investigate the effects of hippocampal LINGO-1 on spatial learning and memory in APP/PS1 mice,we performed brain stereotaxic injection of the adeno-associated virus(AAV)to disturb the expression of LINGO-1 on wild-type(WT)and APP/PS1 mice,respectively.The expression of hippocampal LINGO-1 was determined by q RT-PCR,western blot,and ELISA,and the spatial learning and memory abilities were determined with Morris water maze(MWM).2.To investigate the effects of hippocampal LINGO-1 on the oligodendrocytes in APP/PS1 mice,we performed as the followings.(1)We used transcriptome sequencing to analyze the differentially expressed genes that are related to hippocampal oligodendrogenesis and myelination and the relationship between these genes and the LINGO-1 and its downstream signaling molecules factors between WT and APP/PS1 mice.(2)MWM was used to detect the spatial learning and memory abilities between WT and APP/PS1 mice,the hippocampal LINGO-1 expression was measured with western blot,and the number of hippocampal oligodendrocytes was measured with the three-dimensional quantitative methods,the stereological methods.Then,the correlations between behavior tests and the hippocampal LINGO-1 protein levels and the number of oligodendrocytes were analyzed.(3)After down-regulation of the hippocampal LINGO-1 level with brain stereotaxic injection of AAV,and the expression of LINGO-1 and its signaling pathway molecule Rho A,ROCK2 and GSK3β,as well as the molecular markers of oligodendrocyte differentiation and maturation in hippocampus Olig2,PDGFR-α,and CNPase were measured with western blot and q RT-PCR.3.To investigate the therapeutic effect of anti-LINGO-1 antibody intervention on APP/PS1 mice and the mechanism for the effects,we performed the followings.(1)After APP/PS1 mice were intraperitoneally injected with anti-LINGO-1 antibody,the Morris water maze was performed to evaluate the spatial learning and memory ability of the mice.The density of LINGO-1~+cells,the deposition of Aβ,the volume of each subfield,the number of oligodendrocyte differentiation and maturation markers,PDGFR-α,Olig2and CNPase positive cells,and the Binary Area of myelination marker MBP in the hippocampus were determined with immunohistochemical,immunofluorescence and stereological techniques.(2)In the primary culture of mouse brain oligodendrocytes in vitro,the cells were added with Aβto simulate the AD cells in vitro,and the cells were treated with anti-LINGO-1 antibody.The expressions of newborn PDGFR-α~+cells and Olig2~+/CNPase~+in the primary cell were analyzed with immunofluorescence.Wnt signal pathway was down-regulated and up-regulated with activators and inhibitors,respectively,and then the cells were treated with anti-LINGO-1 antibody,and the changes of Wnt/β-catenin signaling pathway and the proliferation of new-born PDGFR-α~+cells were analyzed with western blot and immunofluorescence.Results:1.The effects of hippocampal LINGO-1 on the spatial learning and memory abilities of APP/PS1 mice:(1)Up-regulation of the hippocampal LINGO-1 level impaired the WT mice’s spatial learning and memory abilities.(2)Down-regulation of the hippocampal LINGO-1 level restored the APP/PS1 mice’s spatial learning and memory abilities.2.The effects of hippocampal LINGO-1 on oligodendrocytes in the hippocampus of APP/PS1 transgenic AD mice:(1)RNA-seq analysis indicated that compared with WT mice,the APP/PS1 mice displayed significantly DEGs in the hippocampus that were related to LINGO-1 and its downstream signal molecules(Rho A,ROCK1 and ROCK2)as well as oligodendrocyte differentiation and myelination related genes.Significantly correlation between LINGO-1 and its downstream signal genes and oligodendrocyte differentiation and myelination-related genes were formed.(2)APP/PS1 transgenic AD mice displayed spatial learning and memory impairment,and meantime significantly high expression of hippocampal LINGO-1,which was significantly negatively correlated with water maze performance.APP/PS1 transgenic AD mice displayed decreased in hippocampal CNPase positive cells number,which was significantly positively correlated with water maze performance.(3)APP/PS1 mice displayed a high level of hippocampal Rho A,ROCK2 and GSK3β.The levels of hippocampal Rho A,ROCK2 and GSK3βwere significantly decreased in the APP/PS1 mice with the inhibition of LINGO-1.Besides,APP/PS1 mice displayed a high level of hippocampal Olig2,and PDGFR-α,and a low level of CNPase.The levels of hippocampal Olig2 and PDGFR-αwere significantly decreased and hippocampal CNPase was significantly increased in the APP/PS1 mice with the inhibition of LINGO-1.3.Therapeutic effects of anti-LINGO-1 antibody treatment on APP/PS1transgenic AD mice and the mechanisms for the effects include the followings.(1)Intraperitoneal injection of anti-LINGO-1 antibody significantly improved spatial learning and memory dysfunction in APP/PS1mice,decreased the density of LINGO 1 positive cells in the hippocampus,reduced the deposition of Aβplaques in the hippocampus,alleviated hippocampal atrophy,and reduced the loss of mature oligodendrocyte marker CNPase and myelin marker MBP positive cells.Besides,the abnormal changes of oligodendrocyte lines marker(Olig2)positive cells and oligodendrocyte progenitor cells maker(PDGFR-α)positive cells were reversed after anti-LINGO-1 antibody intervention.(2)Anti-LINGO-1antibody promoted the differentiation and maturation of primary oligodendrocyte progenitor cells and alleviated the injury of Aβto OPC.After anti-LINGO-1 antibody administration or up-regulation of Wnt pathway,the expression of GSK3βwas significantly decreased and the expression ofβ-catenin was significantly increased.Besides,the down-regulation of Wnt pathway,the expression of GSK3βwas significantly increased,and the expression ofβ-catenin was significantly decreased,and the proliferation of OPC was significantly inhibited.Moreover,anti-LINGO-1 antibody could reverse the proliferation inhibition of OPC with Wnt inhibitor.Conclusions:1.Overexpression of the hippocampal LINGO-1 resulted in hippocampal spatial learning and memory impairment,while inhibition of abnormal high expression of LINGO-1 reversed hippocampal spatial learning and memory impairment in AD mice,which indicated that LINGO-1 was involved in the impairment of hippocampal-depend spatial learning and memory ability.2.Both LINGO-1 and oligodendrocyte changes in the hippocampus played important roles in spatial learning and memory abilities in APP/PS1mice.GSK3βnot only negatively regulated the differentiation and maturation of oligodendrocyte in APP/PS1 mice through LINGO-1 and its downstream signaling pathway factors Rho A,and ROCK2 but also be involved in the regulation of genes that were related to the proliferation,differentiation and maturation of oligodendrocytes,which led to abnormal changes of oligodendrocytes in the hippocampus.3.Peripheral administration of anti-LINGO-1 antibody effectively restored spatial learning and memory impairment in APP/PS1 mice and promoted the proliferation,differentiation and maturation of oligodendrocytes in the hippocampus.The mechanisms underlying the effects might be that anti-LINGO-1 antibody inhibited LINGO-1 and further activated the WNT/GSK3βsignaling pathway,thus promoting the differentiation and maturation of oligodendrocytes.