Study On The Effects Of Anti-A?31-35 Single Chain Antibody On The Pathology In APP/PS1 Transgenic Mice Of Alzheimer's Disease And Its Underlying Mechanisms

Objective:By observing the Amyloid-?(A?)clearing effect of the novel anti-A?31-35 single chain antibody(scFv17)in APP/PS1 transgenic mice brain,we studied its underlying neuroprotection mechanism from aspects of neuroinflammation and A?-related enzymes,to providing new immunotherapy strategy for Alzheimer's disease(AD).Methods:15-month-old APP/PS1 mice were assigned into three groups randomly: Saline(PBS injected for control),6E10(6E10 injected for a positive control antibody),and scFv17(scFv17 injected for treatment).For each group,frontal cortex and hippocampus of both hemisphere of mice brain were administrated with 2 ?l scFv17(or 6E10)with 1 mg/ml concentration,or 0.01 M PBS of same volume.7 days after one administration,brain of mice was collected for the following experiments.(1)Immunohistochemistry: after perfumed with formalin,brains were stored at-80?.Then immunohistochemistry experiment was performed.Brain slices were at a thickness of 25 ?m,goat serum was used for blocking,then primary antibodies were incubated overnight at 4?,secondary antibodies for 2 hours,after every incubation,PBS was used for washing redundant antibodies.At last,after DAB for chromogenic reaction,brain slices' images for A? plaques and activated microglia(CD45+ cells)in prefrontal cortex and hippocampus were collected for further analysis.(2)ELISA detection: fresh brains of mice in each group were collected and extracted for protein under high speed centrifuge,BCA measurement was performed for assessing total protein concentration of brain extraction,then anti-inflammatory cytokines(IL-10,TGF-?),pro-inflammatory cytokines(IL-1?,TNF-?),and IgG were measured according to instructions of ELISA kits.Based on the total protein concentration by BCA and target protein concentration by ELISA,we calculated target protein at a concentration of total protein in brain for analysis.(3)Western blot: fresh hippocampus of mice was collected for Western blot,protein was extracted by RIPA,and then BCA was performed for assessing protein concentration.Protein samples were added to the prepared SDS-PAGE gels for electrophoresis.80 V of electrophoresis were used first for samples on stacking gel and then 120 V were used for electrophoresis when samples reach the line between stacking gel and separating gel.Then proteins were transferred onto PVDF membranes from gels.PVDF membranes were blocked by 5% BSA,incubated with primary antibodies overnight at 4?,followed by secondary antibodies incubation for 2 hours.The optical density of the target strips(A?,sAPP?,BACE1,Neprilysin)were analyzed by Image-Pro Plus.Results:1.scFv17 reduced A? plaques and oligomers in APP/PS1 mice brainFirst,we used immunohistochemistry for A? to compare areas of A? plaques in prefrontal cortex and hippocampus of mice brain in each group,then western blot was used for assessing A? oligomers of hippocampus in mice brain.We found that:(1)By administration of scFv17 and 6E10,A? plaques area of prefrontal cortex and hippocampus in APP/PS1 brain were significantly reduced compared with saline group(P < 0.001).But there is no significant difference in A? plaques area of prefrontal cortex and hippocampus between sc Fv17 and 6E10(P > 0.05).(2)Compared to saline group,scFv17 had significantly reduced optical density of A? 12-oligomers(P < 0.001)and 6-oligomers(P < 0.001),but on significantly change in 3-oligomers(P > 0.05).While 6E10 had also reduced optical density of A? 12-oligomers(P < 0.001)and 6-oligomers(P < 0.001)compared to saline,but 3-oligomer had raised dramatically compared to saline(P < 0.001).Besides,optical density of A? 12-oligomers and 3-oligomers in scFv17 group significantly decreased compared to 6E10 group(P < 0.001),while no significantly change in 6-oligomers between sc Fv17 and 6E10 group(P > 0.05).2.scFv17 and 6E10 regulate inflammatory cytokines and activate microglia differentlyWe used immunohistochemistry and ELISA kit to detect activated microglia CD45+ cells in prefrontal cortex and hippocampus,and anti-inflammatory cytokines(IL-10,TGF-?),pro-inflammatory cytokines(IL-1?,TNF-?),and IgG concentration in prefrontal cortex of each group.We found that:(1)After scFv17 and 6E10 administration,CD45+ cells in prefrontal cortex did not have any significance compare to saline(P > 0.05),but CD45+ cells in scFv17 group significantly lower than 6E10 group(P < 0.05).For CD45+ cells in hippocampus,both scFv17 group(P < 0.01)and 6E10 group(P < 0.001)showed more CD45+ cells compared to saline group.(2)Pro-inflammatory cytokines(IL-1?,TNF-?),after scFv17 and 6E10 administration,didn't change significantly compared to saline group(P > 0.05).For anti-inflammatory cytokines(IL-10,TGF-?),after scFv17 administration,had significantly raised compared to both 6E10 and saline group(P < 0.05).(3)IgG in prefrontal cortex had decreased significantly(P < 0.05)compared to saline group after administrated with scFv17,while 6E10 group decreased the Ig G concentration in prefrontal but without any statistical significance(P > 0.05).3.scFv17 regulated A?-related enzymes in APP/PS1 mice brain.After extracted protein of hippocampus in APP/PS1 mice in each group,we detected A?-related enzymes(sAPP?,BACE1,neprilysin)by using western blot.We found that:(1)The optical density of sAPP? in hippocampus of scFv17 group had increased significantly compare to saline group(P < 0.001)and 6E10 group(P < 0.001).And optical density of sAPP? in hippocampus of 6E10 group did not have any significance compare to saline group(P > 0.05).(2)After scFv17 and 6E10 administration,optical density of neprilysin(NEP)decreased significantly(P < 0.001).Besides,scFv17 group had significantly decreased optical density in NEP compare to 6E10(P < 0.001).(3)In optical density of BACE1,scFv17 group did not show any significance(P > 0.05)compared to saline group,but optical density of BACE1 in 6E10 group decreased significantly compared with both scFv17 and saline group(P < 0.001).Conclusion:1.15-month-old APP/PS1 mice showed remarkably increased A? plaques and A? oligomers.Intracranial injection of scFv17 can reduce A? plaques and A? oligomers significantly.2.ScFv17 mildly activated microglia in hippocampus of APP/PS1 mice,and scFv17 also promoted secretion of anti-inflammatory cytokines(IL-10,TGF-?),meanwhile,sc Fv17 reduced IgG concentration in brain.These shows that scFv17 reduced A? pathology by regulates inflammatory cytokines.3.scFv17 administration dramatically increased sAPP?,and also reduced NEP remarkably,while 6E10 decreased BACE1,which shows that these two antibodies have different mechanism in regulates A?-related enzymes.