Involvement Of MTOR Signaling In The Phosphorylation Of AMPA Receptor Subunit GluA1 Modulated By M1 Muscarinic Acetylcholine Receptor

M1 receptor(M1R),one of the five subtypes of muscarinic acetylcholine receptor,belongs to G-protein coupled receptors family members.MIR has the largest distribution in the brain and is expresses abundantly in the cortex and hippocampus.MIR plays a very important role in learning and memory.Activation of MIR can enhance long-time potentiation(LTP),and can induce the formation of the neuron dendritic spines and the head filiform pseudopodia so as to improve the synaptic plasticity.AMPA(a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid)receptor,the ionic glutamate receptors,mediates fast excitatory synaptic transmission.Dynamic expression of AMPA receptor in the postsynaptic membrane is closely related to the induction and maintenance of LTP and long-time depression(LTD)in central nervous system.Furthermore,AMPA receptor participates in regulating synaptic plasticity and learning and memory function.Our previous study revealed that the selective activation of M1R could induce the insertion of AMPA receptor subunits GluA1 to neuronal membrane and synapses and increase GluAl Ser845 of phosphorylation.Moreover,we found that GluAl Ser845 phosphorylation was crucial for M1R’s cognitive improvement.Besides,PI3K-Akt pathway was demonstrated to play an important role in the phosphorylation of GluAl Ser845 by MIR activation.Because mTOR is the downstream effector of and mTOR signaling influence synaptic plasticity by increasing the protein expression,what kind of role does mTOR play in the process of MIR phosphorylation of GluAl Ser845?Preliminary studies have shown that a short-term treatment of mTOR inhibitor rapamycin could inhibit carbachol phosphorylation of GluAl Ser845,therefore,mTORC1 could play an important role in the modulation of GluA1 by MIR.Thus,whether do ribosome protein subunit 6 kinase 1(S6K1)and the eIF4E-binding protein 1(4E-BP1),known as the downstream molecules of mTOR,participate in this process?In this study,using nonselective agonist carbachol,selective MIR agonist 77-LH-28-1 and mTOR ihibitor rapamycin with primary hippocampal neurons(18-21 days)and mice hippocampal tissue homogenate and slices,we studied the mechanism of mTOR signaling pathway in MIR modulation of GluAl through westerm blot,surface protein biotinylation labeling,live cell real-time imaging and immunofluorescencet staining.Our study demonstrated that:1.MIR activition increased the phosphorylation of mTOR Ser2448.2.Acute rapamycin treatment inhibited the MIR-induced increase of GluA1 Ser845 phosphorylation and membrane insertion of GluA1.3.In primary hippocampal neurons and hippocampus of mice,MIR activation enhanced phosphorylation of p85S6Kl,one of mTORCl downstream targets,whereas,MIR activation had no effect on phosphorylation of p70S6Kl and 4E-BP1.Furthermore,short-term incubation of rapamycin blocked MIR-increased phosphorylation of p85S6K1.4.ShRNA interference to reduce the p85S6K1 protein expression reversed MIR-increased phosphorylation of GluAl Ser845 and promoted GluA1 membrane insertion.The results of the study revealed that p85S6K1,one of downstream targets of mTORC1 complex,played a pivatal role in MIR modulated AMPA receptor GluA1 subunit.The findings clarified the molecular mechanism of M1R’s regulation on AMPA receptor GluA1 subunits and revealed the new p85S6K1 function.Thus,this study provides new insights into the molecular mechanism of learning and memory,and may provide the potential therapeutic target for cognitive function disorders such as Alzheimer’s disease.