The Study On β₂ Adrenergic Receptor Mediated The Function Of Aβ Monomer In The Pathogenesis Of Alzheimer’s Disease

Molecular studies of Alzheimer’s disease（AD）began in the early 1980s.AD is an age-related neurodegenerative disorder,which is characterized by cholinergic neurodegeneration and progressive loss of memory.The two most prominent histopathological biomarkers of AD are extracellular plaques,mainly composed of Aβspecies,and intracellular neurofibrillary tangles（NFTs）made of hyperphosphorylated Tau protein.Imbalance of Aβleads to the loss of synapses,spines,and microtubules because of hyperphosphorylation of Tau proteins.Various forms of Aβproteins exist due to self-association of Aβ_1-42 including monomers（Aβ-M）,dimers（Aβ-D）,oligomers（Aβ-O）and polymers（Aβ-P）and fibrils in vivo.Aβ-O plays a major role in AD in early studies.Aβ-O mainly induces oxidative stress and promotes the production of reactive oxygen radicals which subsequently destroys the structure and activity of various proteins in neuronal cells.Aβinduces Tau phosphorylation at many sites in our early research.In our unpublished study,Aβ-M was found to induce Tau phosphorylation at Ser-214.In recent years,psychological stress has been identified to be a possible trigger for AD,and the roles of adrenergic receptors especiallyβ₂AR have attracted researchers’notice.Since the cortex and the hippocampus are major brain regions that are highly degenerated in AD and the cortex is the earliest to form Aβplaques.A study has shown that chronic treatment withβ₂AR agonist enhancesγ-secretase activity,and related Aβproduction and the formation of Aβplaques.In conclusion,β₂AR and Aβ-M may be a key factor in the pathogenesis of AD.Basing on our previous and other researchers’reports,in this study,we aim to investigate the biological function of Aβ-M through molecular biology,cellular biology,biochemistry and mass spectrometry,FPLC and other methods.And also to investigate the differential expression of genes in mice carrying both mutant presenilin 1 and amyloid precursor protein transgenes with or without knockout ofβ₂AR gene through microarray and transgenic technology.This study will benefit research communities to understand the mechanism of AD and to find targets for preventing and treating the disease.The results are as follows:1.pcDNA3.1-Aβ-Halo and pcDNA3.1-Halo vectors were constructed and transfected into SH-SY5Y cells.Proteins of the transfected cells were tested by western blot which showed that the Aβ-Halo and Halo fusion protein were expressed and Aβ-P was not detected.SH-SY5Y cells transfected with Aβ-Halo died in 48 h after the transfection while SH-SY5Y cells transfected with Halo had no significant effect,revealing that Aβ-M might induce apoptosis of SH-SY5Y cells.Apoptosis and necrosis kits identified that Aβ-M induced apoptosis.In the q PCR experiment,gene expression levels of proapoptotic factors Bax and Bid gene were up-regulated compared with the control group while the expressions of anti-apoptotic factors Bcl-2 and Bcl-x L were down-regulated,demonstrating that Aβ-M had its own cytotoxicity by inducing apoptosis.2.According to yeast two hybrid experiment results,Aβ-M directly interacted withβ₂AR.p ET41b-Aβ-His and p ET41b-Aβ_42-1-His vectors were constructed and transfected into Escherichia coli for expression and purification.The purified protein was used to incubate with the cell lysate of SH-SY5Y and the eluent of Pull-down assay were collected for western blot.Eluent of Aβ_42-1-His showed positive results againstβ₂AR verifying the interaction of Aβ-M andβ₂AR.3.Aβ-P,Aβ-O,Aβ-D and Aβ-M were successfully isolated from Aβsolution by FPLC method;the separated multiple forms of Aβwere used to stimulate SH-SY5Y cells and Tau phosphorylation at Ser-214,JNK at Thr-183/Tyr-185 and p70S6K at Thr-389 were tested by western blot.The results showed that only Aβ-M induced Tau phosphorylation at Ser-214,JNK at Thr-183/Tyr-185 and p70S6K at Thr-389.4.β₁AR inhibitor CGP 20712(10^-6M),β₂AR inhibitor ICI118551(10^-6M),PKA inhibitor Myristoylated PKI(10^-5M),MEK inhibitor PD0325901(2×10^-7M),Epac inhibitor ESI09(5×10^-6M),JNK inhibitor SP600125(2×10^-7M)and GRK2 inhibitorβARKct were pretreated for 10 min before Aβ-M stimulation in SY5Y cells.β₂AR-PKA-JNK pathway andβ₂AR-GRK2-Arrestin induced the phosphorylation of Tau at Ser-214,MEK inhibitor blocked the phosphorylation of p70S6K at Thr-389.Aβ₁AR inhibitor andβ₂AR inhibitor hardly affected the phosphorylation of p70S6K.5.Half of the brain of PS1/APP andβ₂AR-KO/PS1/APP mice were taken and used for RNA extraction through Rneasy lipid tissue kit.Microarray tests were applied to study the genomic expression effects ofβ₂AR knockout on AD mice.The results showed that:Genomic significance ofβ₂AR-KO was to influence protein-processing and presentation involving membrane structure and MHC class I and II protein complex,and protein degradation components,which are lysosome and hydrolase activity.In conclusion,Aβ-M induce the apoptosis of SH-SY5Y cells.Aβ-M interacts directly with the cell membrane receptorβ₂AR.Besides,it induces the phosphorylation of JNK,Tau and p70S6K while the phosphorylation of Tau protein Ser-214 is mainly affected by theβ₂AR-PKA-JNK andβ₂AR-GRK2 pathway.MEK is involved in Aβ-M-induced phosphorylation of p70S6K,however,it is not affected by eitherβ₂AR orβ₁AR.Genomic significance ofβ₂AR-KO was to influence protein-processing and presentation involving membrane structure and MHC class I and II protein complex,and protein degradation components,which are lysosome and hydrolase activities which might be critical for accumulation of Aβ.Aβ-M,the smallest unit of Aβmultiple forms together withβ₂AR,the membrane receptor which is related to AD need further research concerning their biological functions.The results may benefit research communities to understand the mechanism of AD and to find targets for preventing and treating the disease.