The Effects Of ST2-104 Peptide On Neuronal Apoptosis In Alzheimer’s Disease And Its Mechanism

Alzheimer’s disease（AD）is an irreversible progressive neurodegenerative disorder.Alzheimer’s disease is currently affecting approximately 50 million people worldwide,and its incidence is increasing.There is no effective way to treat the disease clinically.Therefore,it is particularly urgent to explore its potential pathogenesis and develop ideal therapeutic drugs.The main pathological characteristics of AD are neurofibrillary tangles（NFTs）and the formation of a large number of extracellular senile plaque（SP）.Studies have found that apoptosis occurs in the brains of nerve cells,animal models,and AD patients.The abnormal aggregation ofβ-amyloid（Aβ）induces neuronal apoptosis,which may be the basis for its neurotoxicity up-regulation.As a key factor in regulating apoptosis,autophagy is also believed to be involved in the development of AD disease.Some studies have suggested that Aβmay regulate Ca²⁺channels on cell membranes to promote extracellular Ca²⁺influx and cause calcium overload.And then Ca²⁺/Calmodulin-dependent protein kinase kinaseβ（CaMKKβ）activates AMP-activated protein kinase（AMPK）.Thereby the negative regulator of autophagy rapamycin target protein（mTOR）is inhibited by AMPK to activate autophagy.Thus the sensitivity of neurons to excitotoxicity and apoptosis is increased.CaMKKβ,an AMPK upstream kinase,regulates important neural functions.It is activated by calcium overload in the AD cell model to activate the downstream AMPK/mTOR signaling pathway.Its effect can be inhibited by the CaMKKβinhibitor STO-609.ST2-104 peptide is a novel small molecule peptide designed by Ca²⁺channel binding domain（CBD3）in recent years.It has nine arginine transmembrane peptide fusions that target calcium channels on nerve cell membranes such as N-methyl-D-aspartate receptors（NMDARs）.Previous studies have shown that ST2-104 peptide can inhibit Ca²⁺channel receptor over-activation,reduce calcium overload,and downregulate neuronal damage.Therefore,on this basis,this study will further explore the role of ST2-104 peptide in inhibiting neuronal apoptosis and its possible mechanism,providing a solid theoretical basis for the treatment of Alzheimer’s disease.Aims:In this study,we plan to use Aβ_25-355-35 to induced human neuroblastoma SH-SY5Y cell to establish a damage model.The autophagy agonist rapamycin（RAPA）and CaMKKβinhibitor STO-609 were administered to investigate the effect of ST2-104polypeptide on autophagy and apoptosis in SH-SY5Y cells through the CaMKKβ/AMPK/mTOR signaling pathway.Methods:In this study,Aβ_25-35-induced SH-SY5Y cells were used to establish a neuronal injury model,and ST2-104 peptide was pre-administered to intervene.The experiment was mainly divided into Con group,Aβ_25-355-35 group,ST2-104 group and Aβ_25-35+ST2-104 group.MTT assay was used to detect cell survival rate.Hoechst33258 fluorescence staining was used to observe the apoptosis level.MDC fluorescence staining was used to observe cell autophagy level.Flow Cytometry（FCM）was used to detect the concentration of intracellular Ca²⁺.And Western blot was used to detect apoptosis-related proteins（Bax,Bcl-2,caspase-3）,autophagy-related proteins（LC3B,Beclin-1）and CaMKKβ/p-AMPK/p-mTOR expression levels.To further explore the relationship between autophagy and apoptosis,SH-SY5Y cells were pretreated with autophagy agonist rapamycin.Hoechst 33258 fluorescence staining was used to observe the apoptosis level.And Western blot was used to detect the expression of apoptosis-related proteins（Bax,Bcl-2,caspase-3）.To investigate the role of CaMKKβin ST2-104 peptide protection of Aβ_25-35-induced cell damage,the CaMKKβinhibitor STO-609 was used to treat SH-SY5Y cells.Hoechst 33258 fluorescence staining was used to observe the apoptosis level.MDC fluorescence staining was used to observe cell autophagy.Expression levels of apoptosis-related proteins（Bax,Bcl-2,caspase-3）,autophagy-related proteins（LC3B,Beclin-1）and CaMKKβ/p-AMPK/p-mTOR were detected by Western blot.Results:MTT results showed that,compared with the Con group,Aβ_25-355-35 group significantly reduced cell viability at 5μM for 24 h.Hoechst 33258 fluorescence staining showed that Aβ_25-355-35 increased the condensation and fragmentation of nuclear.MDC fluorescence staining showed that the fluorescence intensity of the cell was increased by Aβ_25-35.FCM detected a significant increase in the concentration of intracellular Ca²⁺.Western blot results showed a significant increase in Bax,C-caspase-3 protein expression,but a significant decrease in Bcl-2 protein expression.And the expression of autophagy related proteins Beclin-1,LC3-II increased significantly,CaMKKβ,p-AMPK protein expression increased clearly,and p-mTOR protein expression decreased.Compared with the Aβ_25-355-35 group,5μM ST2-104peptide dramatically reversed the above situation after pretreatment of SH-SY5Y cells.SH-SY5Y cells were pretreated with RAPA.RAPA can block the protective effect of ST2-104 polypeptide on Aβ_25-35-induced apoptosis.To investigate the role of CaMKKβin ST2-104 peptide protecting Aβ_25-35-induced cell damage,CaMKKβinhibitor STO-609 was used to pretreat cells.STO-609 significantly antagonized Aβ_25-355-35 induced apoptosis,autophagy and suppressed CaMKKβ/AMPK/mTOR signaling pathway.In addition STO-609 could enhance the effects of ST2-104peptide.Conclusion:ST2-104 peptide can inhibit the apoptosis of SH-SY5Y cells induced by Aβ_25-35.The mechanism may be that ST2-104 peptide inhibits intracellular calcium overload induced by Aβ_25-35,and then inhibits the CaMKKβ/AMPK/mTOR signaling pathway to weaken autophagy and further attenuate apoptosis.