Effects Of Aβ3-10-KLH On AD Transgenic Mice And Its Mechanism

Objective: Alzheimer’s disease(AD)is the most common cause of dementia,mainly characterized by cognitive impairment and memory loss.The pathological changes of AD mainly consist of senile plaques formed by β-amyloid(Aβ)aggregation,neurofibrillary tangles formed by tau protein phosphorylation,neuron loss and synaptic dysfunction.Among several hypotheses of AD,the amyloid cascade hypothesis is the most famous theory.The amyloid cascade hypothesis indicates that the formation and accumulation of Aβ in early AD stage is the key section of the pathogenesis of Alzheimer’s disease.The continuous accumulation of Aβ in brain can lead to neurodegenerative cascade reactions,including synaptic damage,inflammatory reaction accompanied by astrocyte and microglia activation,destruction of neuronal ion homeostasis,oxidative stress,changes in kinase / phosphatase activity and formation of neurofibrillary tangles,which eventually lead to synaptic failure and neuronal death,causing cognitive impairment.In addition,mitochondrial dysfunction occurs in the early stage of AD,which is also an important section in the pathogenesis of AD.Mitochondrial dysfunction can lead to the formation of reactive oxygen species(ROS),the decrease of ATP production,synaptic dysfunction,apoptosis and other pathological changes.Meanwhile,the microbiota-gut-brain axis(MGB axis)theory provides a new perspective for the pathogenesis of AD.Bidirectional microbiota-gut-brain axis is an information communication system between gut and brain,which is composed of immune,vagus and neuroendocrine pathways.Gut microbiota can regulate brain function and behavior through MGB axis.It has been confirmed that the composition of intestinal microflora in AD patients is different from that in healthy people,and the decline of learning and memory ability in AD animal model is also related to the microflora dysbiosis.Therefore,improving the microflora dysbiosis of AD has gradually become one of the targets of AD treatment.At present,there is no effective treatment for AD.Clinical drugs such as acetylcholinesterase inhibitor(including donepezil,galanthamine and kabalatin)and N-methyl-D-aspartate(NMDA)receptor inhibitor(memantine)have been proved to only improve the cognitive impairment symptoms of AD,which can not prevent disease progression or reverse the potential neurodegenerative pathological changes.In recent years,Aβ immunotherapy for has become one of the most promising treatments for Alzheimer’s disease.Aβ immunotherapy mainly uses synthetic peptides and monoclonal antibodies to reduce the Aβ load in the brain,thus changing the process of the disease.However,since the first Aβ active immunization vaccine AN-1792 entered clinical trials in 2002,most Aβ immunotherapy related clinical trials have failed due to the occurrence of side effects such as encephalitis.Therefore,in order to avoid the occurrence of side effects and preserve the immunogenicity of the peptide,our research group constructed Aβ3-10-KLH synthetic peptide based on Aβ3-10 peptide,which was used for active immunotherapy of AD transgenic mice.Previous studies have shown that early immunization of AD transgenic mice with Aβ3-10-KLH can induce high concentration of anti-Aβ antibodies in transgenic mice and prevent the formation of Aβwithout obvious side effects.However,whether Aβ3-10-KLH can clear Aβ that has already be formed,or,whether Aβ3-10-KLH has a therapeutic effect on dementia,is still not completely clear.In addition,previous studies on the effect of Aβ3-10-KLH on other pathological mechanisms of AD have not been involved.In this study,Aβ3-10-KLH was applied to 6-month-old APP/PS1 mice to analyze whether Aβ3-10-KLH could(1)clear Aβthat has already be formed in the brain of APP/PS1 mice;(2)improve mitochondrial dysfunction;(3)change the composition of intestinal flora of APP/PS1 mice.Methods: 1.Aβ3-10-KLH peptide was synthesized by coupling Aβ3-10 peptide with hemocyanin(KLH),which was completed in Gen Script Co.,Ltd.,China.2.Six-monthold APP/PS1 transgenic mice were randomly divided into the Aβ3-10-KLH group and the APP/PS1 group.Mice in the experimental group were subcutaneously injected with Aβ3-10-KLH,and mice in the negative control group were injected with same amount of PBS buffer at the same time point.In addition,6-month-old C57BL/6 wild-type mice were raised as blank control group without any treatment.Since the age of 6 months,the mice were immunized biweekly for 6 times,and the related markers were detected at the age of8.5 months.3.The concentration of anti-Aβ antibodies in serum was detected by enzyme linked immunosorbent assay(ELISA).4.Morris water maze was used to test the cognitive function of mice.5.Using ELISA,immunohistochemistry and Thioflavin S staining,different aggregation forms of Aβ in brain tissue were detected.6.TUNEL staining was performed to detect neuronal apoptosis in the brain tissue.7.ELISA,Western blot and fluorescence staining were performed to detect the level of Prep in mitochondria,the level of Aβ in mitochondria,the markers of mitochondrial morphology,oxidative stress and the activities of key enzymes in mitochondrial respiratory chain.7.The structure and abundance of intestinal flora in mice were observed by 16 S r DNA sequencing.Results: 1.Aβ3-10-KLH induced high concentration of anti-Aβ antibodies in 6-monthold APP/PS1 mice.2.Aβ3-10-KLH peptide improved the spatial memory and learning ability of APP/PS1 mice.3.After immunization with Aβ3-10-KLH,Aβ1-40,Aβ1-42,Aβoligomer and senile plaques in the brain of APP/PS1 mice were eliminated.4.TUNEL staining showed that Aβ3-10-KLH reduced the apoptosis of neurons in the brain of APP/PS1 mice.5.Aβ3-10-KLH increased the level of mitochondrial Prep in APP/PS1 mice and decreased the level of mitochondrial Aβ.6.After immunization with Aβ3-10-KLH,the expression of ROS and MDA in brain tissue of APP/PS1 mice decreased,while the expression of Mn SOD increased.7.After immunization with Aβ3-10-KLH,the expression of MFN1,MFN2 and OPA1 in cortex and hippocampus increased,while the expression of DRP1 and FIS1 decreased.8.After immunization with Aβ3-10-KLH,the expression of CCO and SDH in the brain tissue of APP/PS1 mice increased.9.After immunization with Aβ3-10-KLH,the relative abundances of Actinobacteria,Firmicutes and Lactobacillus increased significantly,while the relative abundances of Bacteroidetes decreased significantly.Conclusion: 1.We immunized APP/PS1 double transgenic mice with Aβ3-10-KLH synthetic peptide at 6-month-old,at which point Aβ has already appeared.After the last immunization,a high level of anti-Aβ antibodies was generated.2.Aβ3-10-KLH peptide improved the cognitive function of APP/PS1 mice.3.Aβ3-10-KLH effectively eliminated different forms of Aβ in APP/PS1 mice.4.Aβ3-10-KLH can inhibit apoptosis and deletion of neurons in the brain of APP/PS1 mice.5.Aβ3-10-KLH attenuated oxidative stress in brain tissue.6.Aβ3-10-KLH improved mitochondrial function in the brain of APP/PS1 mice.7.The intestinal microbial structure of APP/PS1 mice changed after Aβ3-10-KLH immunization.