The Role Of SAPPα-BACE1 Peptide In The Pathogenesis Of Alzheimer’s Disease

Background and AimsAlzheimer’s disease(AD)is the most common cause of dementia.Amyloid-beta(Aβ)deposition drives the pathogenesis of AD.Reducing the pro duction and aggregation of Aβ is an important approach for the treatment of AD.Aβ is mainly produced by APP β-amyloid precursor protein cleavage enzyme 1(BACE1)which is the key rate-limiting enzyme of AΒproduction.Inhibition of APP splicing by BACE1 is the main method to reduce Aβproduction.Soluble amylase precursor protein-alpha(sAPP-alpha)is produced by the cleavage of APP by α-secretase.It was found that sAPPα could bind to BACE1 and block the cleavage site of APP by BACE1 to inhibit the cleavage of APP by BACE1,thus reducing the production of Aβ.Based on this,we designed an extrinsic peptide(called sAPPα-BACE1peptide)that specifically binds to BACE1 according to EISEVKMDAEFR,an amino acid sequence on sAPPα that binds to BACE1,in an attempt to reduce the production of Aβ by inhibiting the splicing of APP by BACE1.The aim of this study was to investigate the effects of the peptide on APP metabolism,Aβ production,neurotoxicity and cognitive function in AD in vitro and in vivo.To clarify the therapeutic value of the peptide for AD.Materials and Methods1.The effects of the sAPPα-BACE1 peptide on Aβ level in SH-SY5YcellsThe working solution of the sAPPα-BACE1 peptide,saline and SH-SY5 Y cell line transfected with APP695 plasmid were used to co-culture.The levels of Aβ40 and Aβ42 in cell culture medium were detected by ELISA,and the levels of APP and BACE1 in cell supernatant were determined by western blotting.TUNEL staining and CCK8 assay were used to determine the cell activity.2.The effects of the sAPPα-BACE1 peptide on cognitive function of APP/PS1 miceEight-months mice were randomly divided into experimental group and control group.The experimental group was injected with the sAPPα-BACE1 peptide(100 nmol/kg)intraperitoneally,normal saline was injected into the control group once a day for 30 days.After one month,open field test,Y-maze,Morris water maze,and elevated cross test were performed to detect the cognitive function of mice.3.The effect of the sAPPα-BACE1 peptide on Aβ deposition in APP/PS1 mice brainImmunohistochemical staining with 6E10 antibody and Congo red staining were used to detect the proportion and number of Aβ positive areas in the brains of mice in experimental and control groups.4.The effect of the sAPPα-BACE1 peptide on Tau hyperphosphorylation in APP/PS1 mice brainWestern blot was used to measure the levels of Tau231 and Tau5 in the brains of APP/PS1 mice treated with sAPPα-BACE1 peptide and control mice.5.The effect of the sAPPα-BACE1 peptide on neuronal loss and synapse-associated proteins in APP/PS1 mice brainNeuN+Casepase-3 immunofluorescence staining was used to detect the apoptosis of neurons in the brains of APP/PS1 mice treated with the peptide.NeuN+MAP-2immunofluorescence staining was used to detect the survival of neurons in each group.Western blot was used to measure the expression of PSD95 and VAMP1 in mouse brain.5.The effect of the sAPPα-BACE1 peptide on glial activation in APP/PS1 miceThe levels of CD68 and GFAP antibody labeled microglia and astrocyte were used to detect and compare the activation of glial cells in each group.Results1.The sAPPα-BACE1 peptide reduced Aβ production in SH-SY5 Y cells.ELISA results showed that the sAPPα-BACE1 peptide significantly reduced the levels of Aβ40 and Aβ42 in the cells.Western blot results showed that there was no difference in APP and BACE1 expression between the two groups.CCK8 assay and TUNEL staining showed that the peptide had no effect on cell viability.This indicated that the sAPPα-BACE1 peptide could also reduce Aβ release in vitro without affecting cell viability.2.The sAPPα-BACE1 peptide could improve the cognitive function of APP/PS1miceThe open-field experiment showed that the distance travelled of mice in the experimental group was prolonged,which suggested that the peptide could improve the motor ability of AD mice.Y-maze experiment suggested that the number of spontaneous alteration increased in the experimental group,the results showed that the peptide increased the ability of spatial learning and memory in AD mice.The time in the target quadrant and annulus crossing in Morris water maze test was increased in the experimental group.Open arm entry and open arm time in Elevated Cross experiments was increased in the experimental group.3.The sAPPα-BACE1 peptide alleviated Aβ deposition in the brain of APP/PS1 mice.6E10 immunohistochemical staining showed that the proportion of the total area and the number of Aβ plaques in the hippocampus and neocorcortex of the experimental group were lower than those of the control group.Congo red staining showed a significant reduction in the number of dense plaques in the neocorcortex and hippocampus of the experimental group mice.This suggests that this mimic peptide reduces Aβ aggregation in APP/PS1 mice.4.The sAPPα-BACE1 peptide reduced the levels of phosphorylated Tau in the brain of APP/PS1 mice.Immunoblotting experiments suggested that the expression of Tau231 in the experimental experimental group was decreased,the content of Tau5 in the experimental group was lower than that in the control group.This suggests that the peptide reduces the hyperphosphorylation of Tau.5.The sAPPα-BACE1 peptide decreased the neuronal loss and enhanced the synaptic plasticity in APP/PS1 mice.The immunofluorescent staining showed that the NeuN positive staining intensity in the hippocampal CA1 region of AD mice in the experimental group was higher than that in the control group.The intensity of Caspase-3 positive staining in the hippocampal CA3 region was decreased in the experimental group.It is suggested that the peptide can reduce the neuronal damage.Immunofluorescence staining showed that the intensity of MAP-2 positive staining in the hippocampal CA1 region of AD mice in the experimental group increased.The immunoblotting indicated that the immunoreactivity of synapse-related proteins such as PSD95 and VAMP1 was enhanced in the experimental group.It is suggested that the peptide improves Synaptic plasticity function and attenuates neurodegeneration in mice.6.The sAPPα-BACE1 peptide inhibited the activation of glial cells in APP/PS1 mice.Compared with the control group,the positive staining of CD68 and GFAP in the experimental group decreased significantly.This suggests that the sAPPα-BACE1 peptide can inhibit the activation of microglia and astrocyte,and may reduce the neuroinflammatory response.ConclusionOur results showed that the sAPPα-BACE1 peptide could inhibit Aβ production without affecting cell activity in vitro.The peptide could improve the cognitive function of APP/PS1 mice,decrease Aβ plaque burden,decrease tau phosphorylation and inhibit neurodegeneration.Construction of fragment-based derived peptides may be an effective therapeutic approach.The peptide may be a potential therapeutic target for AD,and it may be a safe and effective new BACE1 inhibitor,which has some value in exploring new methods for the prevention and treatment of AD.