Interventional Effect And Molecular Mechanism Of Novel Immune Checkpoint Molecule B7x Blockade On Neurodegeneration In Alzheimer’s Disease Model Mice

Alzheimer’s disease(AD)is one of the most common neurodegenerative diseases.The specific etiology of AD is still unknown,the mechanism of AD has not been elucidated thoroughly now,and there is no effective therapy in the clinic.The main clinical manifestations are memory and cognitive function decline,language,behavioral and neuropsychiatric disorders,and hard to taking care of themselves.The main pathological features are extracellular amyloid Aβ deposition,intracellular Tau hyperphosphorylation,and neurofibrillary tangles(NFT),accompanied by neuronal loss and synaptic dysfunction.Aging is the main risk factor for AD,with the increase of age,the immune function of human body decreases,so the common feature of AD patients is immune system dysfunction.Recent studies shown that the immune system plays a key role in the pathogenesis of AD,and enhancing the immune system function may have a role in resisting AD pathology.Therefore,some scholars suggest that systemic immune enhancement should be given for neurodegenerative diseases(such as AD)to drive the immune-dependent cascade of brain repair,so as to improve the pathological and neurological functions of AD,which provides new ideas and strategies for the treatment of AD.T cell activation requires dual signal stimulation and plays an important role in anti-tumor,anti-infection and autoimmune diseases.B7 ligand and its receptor CD28 family are the main immune checkpoint regulating the differentiation of T cells and other immune cells,and are important molecules in the transduction of costimulatory signals.With the deepening of research,new members of the family and excitatory and inhibitory costimulatory pathways have been discovered,which can enhance or stop the immune response.Interestingly,it has been reported in recent years that PD-1/PDL1 blockade can alleviate neuropathological damage,improve cognitive dysfunction and improve learning and memory ability in 5×FAD and DM-hTAU mice.It has also been reported that PD-1 blockade can activate the systemic immune system,but does not change the load of Aβ protein in the three aged APP model mice.This difference may be related to the selection of immune checkpoint molecules,the type of AD mice and the different selection of disease course in AD mice.So far,the U.S.FDA has approved neutralizing antibodies to the immune checkpoint molecule PD-1/PD-L1 as immunotherapy agents for some advanced tumors,and has achieved encouraging clinical efficacy in some cases,but the overall clinical response rate is not high,and some serious adverse reactions have occurred.B7x is a newly member of immunosuppressive signal molecules in the B7/CD28 family,widely expressed in a variety of tumors and immune cells(such as macrophages,B cells,antigen presenting cells,etc.),which can mediate a variety of immunosuppressive pathways and promote tumor immune escape,and is expected to become a more promising immune checkpoint molecule than PD-1/PD-L1.It has been found that B7x plays an important role in bacterial infection model and autoimmune encephalomyelitis in mice.Similarly,the analysis of infiltrating immune cells in the central nervous system showed that the loss of endogenous B7x led to the enhancement of Thl and Th17 immune responses,suggesting that immune checkpoint molecule B7x may play an important role in neurological diseases.While,the role of B7x in the occurrence and development of AD has not been reported.ObjectivesIn order to reveal the effect of novel immune checkpoint molecule B7x blockade on AD.We used APP/PS1 and 5×FAD mice models as research objects,and took B7x blockade to enhance the immune response of the body as the starting point,and did not target any single disease factor in AD brain,so as to restore the immune repair mechanism of the body.We look forward to the comprehensively improve the pathology of AD,restore the neurological function,and preliminarily explore the molecular mechanism of its action.MethodsPart Ⅰ:Effects of B7x blockade on behavioral function in AD mice1.Effects of Anti-B7x on learning,memory,anxiety-and depression-related behaviors in AD miceTwo AD mice model(5×FAD and APP/PS1)were used to detect the effects of i.p.anti-B7x on the neural function of AD mice.1)5×FAD mice were divided into 5×FAD+IgG group,5×FAD+anti-B7x group and Wide type(WT)group.At the age of 5 month,each group mice were i.p.injected 200μg anti-B7x(or IgG)on day 0 and 7,respectively.After 15 days,learning and memory functions of each group mice were detected by Morris water maze,Y maze and T maze tests.The anxiety-and depression-like behaviors of each group were detected by open field test,elevated plus maze and tail suspension test.2)APP/PS 1 mice were divided into APP/PS 1+IgG,APP/PS1+anti-B7x and WT groups.200μg anti-B7x(or IgG)were respectively i.p.injected on day 0 and day 7 at the age of 6 month.Morris water maze test was performed to test the learning and memory of each group mice after 15 days.Open field test,novelty suppressed feeding test,tail suspension test,forced swimming test and sucrose preference test were used to detect anxiety-and depression-like behaviors of each group mice.2.Effects of B7x-/-on learning,memory,anxiety-and depression-like behaviors in 5×FAD miceB7x gene knockout(B7x-/-)mice were divided into 5×FAD group,B7x-/-group,5×FAD/B7x-/-group and WT group.At the age of 6 months,Y maze and T maze were used to test the learning and memory function of each group mice.The anxiety-and depression-like behaviors of each group mice were detected by open field test,elevated plus maze test and tail suspension test.Part Ⅱ:Effects of Anti-B7x on neuropathology in AD miceWe tested the effects of i.p.injection of anti-B7x on the neural function of 5×FAD and APP/PS1 mice.After the behavioral experiment,each group mice were sacrificed and sampled on day 30.Aβ deposition and the expression of Ibal and GFAP were detected by immunofluorescence staining.The expressions of inflammatory factors and anti-inflammatory factors in serum were tested by Elisa.The expression of p-Tau protein and senescence related protein in the brain tissues of each group mice was detected by Western Blot.Nissl staining,Tunel staining and Western blot were used to test nerve apoptosis in the cortex and DG region of each group mice.Nerve regeneration in hippocampus was detected by immunofluorescence staining.Glucose and lactic acid contents and the expression of glucose metabolism-related proteins in brain tissues of each group were detected by glucose and lactic acid content detection kit and Western blot.The integrity of meningeal lymphatic vessels and blood-brain barrier(BBB)were detected by immunofluorescence and Western blot.Part Ⅲ:Molecular mechanism of anti-B7x on neurodegeneration in AD miceFour mice were collected from 5×FAD+IgG,5×FAD+anti-B7x and WT groups,and the number of immune cell subsets in brain and spleen tissues of each group mice was detected by flow cytometry.First,a standard dataset of hippocampal transcriptome of 140 AD patients and controls was established based on a public dataset to analyze the differentially expressed genes and KEGG pathway prediction.Subsequently,RNA-seq experiment was performed and 5 mice of each group were selected,and total RNA of hippocampus tissue was extracted.Illumina platform was used for on-machine sequencing and data mining.Relationship between sample heat map,PCA analysis and Venn analysis were used to analyze the correlation between biological duplicates.The differentially expressed genes in 5×FAD+anti-B7x and 5×FAD+IgG groups were statistically screened by volcano map and heat map.Functional annotation and functional enrichment analysis of differentially expressed genes were performed by GO database and KEGG signaling pathway database.Subsequently,Western Blot was used to the verified the expression of NF-κB signal-related protein in hippocampus.ResultsPart Ⅰ:Effects of B7x blockade on behavioral function in AD mice1.Effects of anti-B7x on learning,memory,anxiety-and depression-like behavior in AD mice1)Morris water maze,Y maze and T maze showed that compared with 5×FAD+IgG group,the escape latency of 5×FAD+anti-B7x group mice was significantly decreased,and the time in target quadrant,the number of crossing platforms,the percentage of alternation,and the time spent in novel arm were increased significantly(P<0.05).The results of open field test,elevated plus maze and tail suspension test showed that i.p.injection of anti-B7x antibody did not affect the time spent in center and the time spent in open arm of 5×FAD mice(P>0.05),but reduced the immobility time of each test(P<0.05).2)Morris water maze test showed that compared with APP/PS1+IgG group,the escape latency of APP/PS1+anti-B7X group was significantly decreased,and the time in target quadrant,the number of crossing platform of APP/PS1+anti-B7x group were significantly increased(P<0.05).The results of open field test,novelty suppressed feeding test,tail suspension test,forced swimming test and sucrose preference test showed that compared with APP/PS 1+IgG group mice,the total number of rearings of APP/PS1+anti-B7x group was significantly increased(P<0.05),the time to enter the center and immobility time were significantly decreased(P<0.05).However,there was no significant difference in the total distance traveled and sucrose preference index(P>0.05).2.Effects of B7x gene knockout on learning,memory,anxiety-and depression-like behaviors in AD miceCompared with 5×FAD mice,the percentage of alternation in 5×FAD/B7x-/-mice was increased significantly in Y maze and T maze(P<0.05).During open filed test,elevated plus maze and tail suspension test.The time spent in center and the time spent in open arm had no significant difference(P>0.05),but the immobility time of 5×FAD/B7x-/-mice was reduced significantly(P<0.05).Part Ⅱ:Effects of anti-B7x on neuropathology in AD miceCompared with 5×FAD(or APP/PS1)mice i.p.injected with IgG,5×FAD(or APP/PS1)mice i.p.injected anti-B7x significantly decreased the expression of Aβplaque,GFAP and Iba1 in hippocampus and cortex(P<0.05).The expression of antiinflammatory cytokines(IL-4,IL-10)were increased,and the expression of inflammatory cytokines(IL-1β,TNF-α)were decreased(P<0.05).The expression of p-Tau proteins(P-Tau(Ser235),P-Tau(Ser396))and senescence related proteins(p16,p21 and p53)were significantly decreased in brain tissues(P<0.05).The number of Nissl bodies and the expression of anti-apoptosis protein Bcl-2 were significantly increased,while the number of Tunel+cells and the expression of apoptosis protein Bax were significantly decreased in the brain(P<0.05).DCX+cells and NeuN+cells were increased significantly in the hippocampal DG region(P<0.05).Glucose content was significantly decreased,lactic acid content and the expression of glucose metabolismrelated GLUT-1 and LDHA were significantly increased(P<0.05).The expression of meningeal lymphatic marker LYVE-1 and BBB permeability related proteins(ZO-1,Occludin and Claudin-1)were increased significantly(P<0.05).Part Ⅲ:Molecular mechanism of anti-B7x on neurodegeneration in AD miceFlow cytometry results showed that i.p.injection of anti-B7x could increase the number of various immune cells(such as CD4+T cells,CD8+T cells,B cells,monocytes,etc.)in spleen and monocytes in brain tissues of 5×FAD mice.By searching KEGG database,we found that B7x(B7H4)is mainly involved in T cell receptor signaling pathway.Our transcriptome dataset,based on public dataset,showed that 31 of 109 genes related to T cell receptor signaling pathway were differentially expressed in AD patients and controls(P<0.05).and these differentially expressed genes were significantly enriched in the NF-κB pathway.The NF-κB signaling pathway was significantly activated in the hippocampus of AD patients compared with controls.Our RNA-seq results showed that the three groups of mice had good biological replication within the group and significant differences between the groups.Anti-b7x i.p.injection could significantly regulate the gene expression level of 5×FAD mice,among which 106 genes were significantly up-regulated and 201 genes were significantly down-regulated.GO enrichment showed that the differential genes were significantly enriched in T cell receptor complex,adaptive immune response,and T cell activation regulation.KEGG signaling pathway enrichment analysis showed that differential genes were significantly enriched in cell adhesion molecules,Thl and Th2 cell differentiation,Th17 cell differentiation,T cell receptor signaling pathway and other related signaling pathways.B7x indirectly interacts with NF-κB pathway-related.proteins through T cell receptor-related proteins.Compared with the 5×FAD+IgG group,the downstream NF-κB pathway of T cell receptor signaling pathway was inhibited in the 5×FAD+anti-B7x group.Conclusion1.B7x blockade(anti-B7x i.p.injection and B7x gene knockout)can improve the learning and memory function of 5×FAD mice,alleviate the depression-like behaviors,but have little effect on anxiety-like behaviors.For APP/PS1 mice,anti-B7x can improve the learning and memory function and alleviate the anxiety-and depressionlike behaviors.2.Anti-B7x can reduce Aβ plaque deposition and activation of astrocytes and microglia in the hippocampus and cortex of APP/PS1 and 5×FAD mice,and reduce inflammation.Decreased the expression levels of p-Tau protein and senescence related protein in brain tissue,and neural apoptosis was reduced.Promote nerve regeneration in hippocampus.Enhance the glucose metabolism level in brain tissue,improve brain energy supply.Improve the permeability of damaged meningeal lymphatic vessels and blood-brain barrier.3.Anti-B7x can activate the level of immune response in 5×FAD mice,possibly by inhibiting the NF-κB pathway,reduce inflammation and Aβ deposition,thus improving the brain neuropathology and cognitive function in 5×FAD mice.