The Impact Of Early Immunization Of Aβ3-10 Vaccine On β-amyloid Protein In Alzheimer’s Disease

Objecive: Alzheimer’s disease is the most common type of dementia.It is a kind of progressive neurodegenerative disease with memory dysfunction,cognitive dysfunction,executive dysfunction as major manifestations.The main pathological changes of Alzheimer’s disease are β-amyloid deposits,microtubule-associated tau protein phosphorylation,neurofibrillary tangles,impaired synaptic function,neuronal loss and neuroinflammation.At present the cause of AD is not clear yet,of which "amyloid cascade hypothesis" is currently widely acceptted.This kind of hypothesis regards amyloid as the center of AD,the clearance of amyloid protein and protein production lost the balance that leading the amyloid accumulation and induces subsequent synaptic damage,tau protein phosphorylation,neuroinflammation and neuron death and other AD pathological changes.At first,people always focused their research on the removal of amyloid plaques.However,as the research goes deeper,many experimental results confirm that the clearance of amyloid plaques does not improve the disease pathology changes and symptoms.Therefore,Aβ oligomer has gradually become a new research hot issue.Based on the "amyloid cascade hypothesis",an "amyloid oligomer cascade hypothesis" has been proposed.It is gradually realized that the removal of the already formed amyloid plaque is not the crutial purpose of treating this disease but prevention of amyloid deposition is more important.The current clinical treatment of Alzheimer’s disease is still dominated by the control of disease progression,Aβ as the target of active and passive immunotherapy is the major direction of the investigation.Some Aβ-related vaccine trials in animals have come out some results.After immunization,Aβ deposition in experimental animals decreased and cognitive and learning function was also somewhat improved.Scientists have put some vaccines into clinical trials,but most have failed in the early stage.The successful establishment of a transgenic animal model plays a key role in the research on AD.AD pathological changes are very complex,so to establish fully expression of all pathological changes in AD animal model has great difficulty.Initially,most scientists used the APP single transgenic and APP / PS1 double transgenic models.Until 2003,Oddo and his team successfully established a triple transgenic mice of APP/PS1/tau,making it possible to study the interaction between Aβ and tau protein.This triple transgenic mouse is by far the most similar animal model to AD.Mice display agerelated pathological changes.First,Aβ production was detected in brain tissue.Over time,synaptic damage,inflammatory cells,and tau phosphorylation were gradually detected.Both synaptic plasticity and neuroinflammatory can cause cognitive impairment,and these two pathologies are also caused by upstream soluble Aβ oligomers.Therefore,through the early active immunotherapy,effective prevention of Aβ production and clearance of Aβ become the focus of this study.At the same time,by the removal of Aβ we detect the synaptic damage,tau protein phosphorylation and neurological inflammation changes.Methods: The synthetic peptide vaccine Aβ3-10 was conjugated to hemocyanin KLH and the triple transgenic AD mice model were immunized subcutaneously.The mice were injected at the age of 1 month until their 7 month old after seven immunizations.Another group of mice were injected the same amount of PBS in the same manner as a negative control.Before the first immunization and after each immunization,a certain amount of blood was collected from the internal canthal vein of mice and the supernatant was centrifuged to detect the concentration of Aβ antibody by ELISA.After the mice grew to 7 months of age,the brains were removed by perfusion with saline and half of them were used to prepare brain homogenate to detect the content of soluble and insoluble Aβ1-x protein and tau protein,and the brain was also detected by Western blot to find out Aβ oligomer and changes of synaptophysin,PSD-95,NR2 B and Dynamin 1.The other half of the brain tissue was fixed with paraformaldehyde for immunohistochemical staining to detect intracellular and extracellular Aβ protein,inflammatory response-related microglial cells and astrocytes activation,and compared with the same age of the wild-type background mice.Results: After seven immunizations with Aβ3-10-KLH,mice developed higher concentrations of anti-Aβ antibodies in vivo and there was some difference in antibody levels between the mice.Although non-immunized mice also had antibody production,but it maintained at a very low level.After immunization,the soluble and insoluble Aβ in brain tissue of mice decreased and the content of tau protein also decreased.The Aβ oligomer also showed a decreasing trend in Western blot.Western blot detection of pre- and post-synaptic membrane-associated marker protein in mice without immunotherapy came out that four protein content were significantly decreased compared with wild-type mice,while the immunized mice have four protein content elevated.The result of immunohistochemistry showed that the content of Aβ in the brain of mice after immunization was lower than that in the negative control group.The microglia and astrocytes in the brain of mice injected with PBS were significantly higher than those of wild type mice,while the number of microglia and astrocyte in the immunized mice decreased.Conclution:1,The Aβ3-10-KLH vaccine was administered to mice at the time when there was no AD-like pathological changes can be detected,and the vaccine induced a high level of antibody concentration after the last immunization.2,The antibody produced by the vaccine not only reduced soluble amyloid that contains toxic Aβ oligomers,but also reduced plaque-forming insoluble amyloid and tau protein in brain tissue.3,The vaccine reduced APP / PS1 / tau transgenic mouse intracellular and extracellular Aβ in the brain tissues.4,The vaccine produced an effective protective effect on the synapses of transgenic mice.5,The vaccine reduced the activation of microglia and astrocytes,thus reducing the extent of the inflammatory response.6,The vaccine against Aβ not only reduced the pathological changes of Aβ,but also decreased the tau protein,the pathological changes of synapse and neuroinflammation which are closely related to AD,which provided the basis for the establishment of the "amyloid cascade hypothesis".