| Background:Nowadays,along with the acceleration of the world's aging population,Alzheimer's disease(AD)has become a major global public health problem.The data has shown that the aging process in China has accelerated in recent years,meanwhile the prevalence of Alzheimer's disease has increased significantly.It is estimated that by 2050,the number of patients with Alzheimer's disease in China will reach 27 million.How to improve the life quality of the elderly in the later years by controlling Alzheimer's disease and reduce the social burden has become the focus of scientific research.Therefore,it is important to explore the cellular and molecular mechanisms,find effective early diagnosis,prognostic markers and therapeutic targets for the diagnosis and prognosis of Alzheimer's disease.Myristoylated alanine-rich C kinase substrate(MARCKS)is a specific substrate for protein kinase C(PKC)and is expressed at high levels in the nervous system.It plays an important role in cell morphology,cell movement,secretion,transmembrane transport,cell cycle regulation and neural development.MARCKS is present with the form of phosphorylated and non-phosphorylated,playing an important role in the G protein coupled receptor signaling pathway.Phosphorylated myristoylated alanine-rich C kinase substrate(P-MARCKS)translocates into the cytoplasm and is involved in membrane trafficking,cell movement and neurotransmitter release.P-MARCKS is localized to characteristic pathological changes-senile plaques(SP)in AD and possibly involved in its neurological dysfunction.Studies have reported that MARCKS phosphorylation can lead to changes in the morphology,quantity,density of dendritic spines,which is the initial event that triggers the pathological state of the synapse,far earlier than the clinical symptoms.P-MARCKS was found to be closely related to synaptic plasticity,which led us to conclude that P-MARCKS may also be associated with Alzheimer's disease,but the molecular mechanism of P-MARCKS on Alzheimer's disease remains unclear.All of the above evidences indicate that MARCKS phosphorylation plays a very important role in the pathogenesis of Alzheimer's disease.Therefore,this study plans to establish an AD cell model by interfering with A?1-42 oligomers in neonatal rat hippocampal,cortical neuron to study the neurotoxic effects of A?1-42 oligomers and further explore the expression and related signaling pathways of P-MARCKS in AD cell model.This research is dedicated to discover the influence of P-MARCKS on synaptic plasticity and its potential mechanism in Alzheimer's disease,providing new ideas for further elucidation of the pathogenesis of Alzheimer's disease,trying new possible directions for clinical treatment.Methods:1.In this experiment,healthy neonatal rats within 24 h of birth were used to extract primary hippocampal,cortical neuron.The A?1-42 oligomers intervent the primary neurons to make AD cell models.2.CCK-8 and HOECHST33258 were used to detect the effect of A?1-42 oligomers on cell viability and apoptosis,and NMDAR antagonist memantine pretreatment was used to verify whether this neurotoxicity is mediated by NMDAR.3.Western Blot was used to detect the effect of A?1-42 oligomers on P-MARCKS protein expression,and NMDAR antagonist memantine pretreatment was used to verify whether the protein expression change was mediated by NMDAR.4.Western Blot was used to detect the effect of A?1-42 oligomers intervention onSynaptic plasticity key protein PIP2 and SYP expression,and NMDAR antagonist pretreatment was used to verify whether the above protein expression changes were mediated by NMDAR.Results:1.CCK8 results showed that exposure of A?1-42 oligomers(500nM)to cultured neurons decreased OD values,with a reduction in cell viability observed at 24 h.Compared with the control group,the cell viability of the A? group was lower than that of the control group(P<0.05,Fig.1).However,memantine pretreatment reversed the cell viability decline.The OD value of A? + Me group was higher than that of A?1-42 group(P<0.05,Fig.1).2.The toxic potency of A?1-42 oligomers on cultured neurons was substantiated by a time-dependent increase in the number of Hoechst 33258-positive apoptotic neurons.After 3 h of intervention with A?1-42 oligomers alone,a small number of cell nuclei showed apoptosis changes compared with the control group(P<0.05,Fig.2A).After memantine pretreatment for 2 h,the number of apoptotic neurons was decreased compared withA?1-42 roup(P<0.05,Fig.2C).Moreover,when the exposure time of neurons to A?1-42 oligomers alone up to 24 h,a large number of cell nuclei showed typical apoptosis changes compared with the control group(P<0.05,Fig.2B).However,memantine pretreatment reduced the number of apoptotic neurons compared with the A?1-42 group(P<0.05,Fig.2C).3.To determine whether the phosphorylation of MARCKS was involved in A?-induced neurotoxicity,neurons were exposed to 500nM A?1-42 oligomers for various times(3 and 24 h)and then expression level of P-MARCKS was assessed by Western blotting.After 3 or 24 h of exposure to A?1-42 oligomers,the expression of P-MARCKS protein increased(3 h:P<0.05;24h:P<0.05,Fig.3).However,after memantine pretreation,the expression of P-MARCKS protein was decreased compared with the A?1-42 group(3 h:P<0.05;24h:P<0.05,Fig.3).4.After treatment with A?1-42 oligomers for 24 h,the expression of PIP2 protein in neurons was increased in comparison with the control group(P<0.05,Fig.4A).It illustrated the synaptic plasticity was damaged by A?1-42 oligomers.The level of synaptic maker protein SYP was decreased(P<0.05,Fig.4B).However,the expression of PIP2 and SYP were alleviated after pretreatment of memantine,which was different from that of A?1-42 group(PIP2:P<0.05;SYP:P<0.05,Fig.4).Conclusions:1.A?1-42 oligomers can cause the decline of primary neuron viability,while NMDAR antagonist memantine pretreatment can reverse the decline of cell activity,which proves that the neurotoxicity of A?1-42 oligomers is partly mediated by NMDAR.2.A?1-42 oligomers can cause apoptosis in primary neurons,while NMDAR antagonist memantine pretreatment can reduce the apoptosis rate,which proves that the neurotoxicity of A?1-42 oligomers is partly mediated by NMDAR.3.A?1-42 oligomers can increase the expression of P-MARCKS in primary neurons,while NMDAR antagonist memantine pretreatment can alleviate the expression changes of this protein,which proves that P-MARCKS protein expression level and related functions are partly mediated by NMDAR.4.A?1-42 oligomers can increase the expression of PIP2,while decrease the expression of SYP in primary neurons.NMDAR antagonist memantine pretreatment can alleviate above protein expression changes,demonstrating the changes in synaptic plasticity induced by A?1-42 oligomers are partly mediated by the NMDAR.SignificanceThese results suggest that P-MARCKS plays a key role in the pathogenesis of Alzheimer's disease.NMDAR antagonist memantine alleviates A?1-42 oligomer-mediated neurotoxicity and attenuates the decrease of synaptic plasticity caused by A?1-42 oligomers by inhibiting P-MARCKS expression and altering its downstream PIP2 and SYP expression. |
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