Mechanism Of M1 Acetylcholine Receptors Regulating The GluA1 Subunit Of AMPA Receptor

Cholinergic neuronal degeneration and cholinergic dysfunction are important pathological features associated with Alzheimer’s disease(AD).M1 muscarinic acetylcholine receptor(M1R)is widely expressed in the central nervous system,especially hippocampus,and is closely related to advanced cognitive function.α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate(AMPA)receptor,an ionotropic glutamate receptor,is involved in the regulation of synaptic plasticity,learning and memory activities.Our previous studies have found that activation of M1 R enhances phosphorylation of AMPA receptor GluA1 subunit Ser845 via the PKA-PI3K-Aktm TOR signaling pathway,allowing AMPA receptors to move to the cell surface.M1 R is typically coupled to Gαq to activate protein kinase C(PKC)and Ras or ectopically to Gαs to activate protein kinase A(PKA).Previous experiments have also found that both PKC inhibitor and Ras inhibitor inhibit the increase in phosphorylation of GluA1S845 induced by M1 R.Here,we examined whether PKC and Ras participated in the M1R-mediated regulation of GluA1.In addition,it has been shown that activated M1 R increases the phosphorylation level of p85S6K1,a downstream target of m TORC1.Sh RNA interference to reduce p85S6K1 protein expression reverses the effects of cholinergic receptor agonist carbachol(CCh)to increase GluA1 Ser845 phosphorylation and to promote GluA1 translocation into the cell membrane.So we investigated whether p85S6K1 could participate in the regulation of cognition.In this study,primary hippocampal neurons,mouse hippocampi and APP/PS1 mice were used.Western blot was used to detect protein and its phosphorylation level,and biotin surface protein labeling was used to detect protein on the cell surface.Open field,Y maze,Morris water maze were used to explore the effect of overexpression of p85S6K1 on cognitive function in APP/PS1 transgenic mice.Our results showed that: 1.In the primary hippocampal neurons,the M1 R highly selective agonist 77-LH-28-1 promoted translocation of intracellular GluA1 to the cell membrane,while the PKC inhibitor G?6983 and Ras inhibitor Salirasib inhibited such an effect.2.Injection of PKC inhibitor G?6983 and Ras inhibitor Salirasib into hippocampi inhibited M1R-induced increase in GluA1 Ser845 phosphorylation,Akt Thr308 phosphorylation and m TOR Ser2448 phosphorylation.3.In primary hippocampal neuronal cells,reduced p85S6K1 protein expression by Sh RNA interference reversed the effect of M1 R to increase GluA1 Ser845 phosphorylation and inhibited GluA1 translocation to the cell membrane.4.Over expression of p85S6K1 in APP/PS1 double transgenic mice improved learning in Y maze and Morris water maze.Therefore,these data demonstrated that PKC and Ras were involved in M1 muscarinic receptor-mediated regulation of the AMA receptor GluA1 subunit and the m TOR downstream signaling molecule p85S6K1 could attenuate cognitive dysfunction in APP/PS1 mice.