Regulatory Molecules Screening And Mechanism Exploration Of Classical Hallucinogens Induced Head-twitch Response Based On 5-HT_2AR/mGluR2 Dimer

Background:Serotonin（5-HT）2A receptor(5-HT_2AR)agonists with hallucinogenic effects are also known as classical hallucinogens.Classical hallucinogens are very dangerous in drug addiction and abuse,so studying the key mechanism of hallucinogenic effects induced by classical hallucinogens is of great significance for studying high-efficiency and low-toxicity adversaries and further weakening drug addiction and abuse.The rodent head-twitch response（HTR）is a common model for evaluating the hallucinogen-induced effects of classical hallucinogens.The classical hallucinogens lysergic acid diethylamide（LSD）and 2,5-dimethoxy-4-iodoamphet-amine（DOI）are metabotropic glutamate receptor 2（m Glu R2）knockout mice significantly reduced the number of HTR induced compared with wild-type mice,but could be reversed by virus-mediated overexpression of m Glu R2,indicating that m Glu R2 is required for classical hallucinogens to induce HTR.At the same time,5-HT_2AR and m Glu R2 can form heterodimers,and the two affect each other’s conjugated signaling pathways and induced animal behaviors,which provides a new direction for the exploration of key regulatory molecules and mechanisms of classical hallucinogens such as LSD,DOI and 2,5-dimethoxy-4-methylamphetamine（DOM）.Objective:To investigate the generalization and signaling pathway mechanisms of m Glu R2agonists LY379268,LY354740,LY404039 and inverse agonist LY341495 in regulating HTR induced by various classical hallucinogens,and to perform quantitative phosphorylation proteomics based on 5-HT_2AR/m Glu R2 heterodimers,and then to explore the mechanisms associated with classical hallucinogens-induced HTR and new signaling molecules and pathways associated with classical hallucinogens-induced HTR.Methods:1.Effect of m Glu R2 agonists and inverse agonists on LSD/DOM-induced HTRC57BL/6J（C57）mice were given different doses of LY379268（LY37）,LY354740（LY35）,LY404039（LY40）or LY341495（LY34）by intraperitoneal injection,and the number of HTR was observed and recorded within 30 min via intraperitoneal injection of LSD or DOM.C57 mice were given different doses of LY37,LY35,LY40 and LY34 by intraperitoneal injection,and the number of HTR was recorded 1 h after administration.C57 mice were given different doses of LY37,LY35,LY40 and LY34 by intraperitoneal injection,and the total distance of spontaneous activity within 1 h and the segmented distance within every 10 min after the administration were recorded.2.Study of the signaling pathway related to DOM-induced HTR by m Glu R2agonists and inverse agonists affectingHEK-293T cells co-transformed with 5-HT_2AR and m Glu R2:after treated with LY35 or LY34 for 5 min,G_qand G_ssignal changes were detected by adding different concentrations of DOM for 15 min;after treated with 5-HT_2AR antagonist M100907 for 5min,the effect of LY34 on DOM-induced G_ssignal was detected.Co-transformed5-HT_2AR and m Glu R2 3A mutant HEK-293T cells:after treated with LY35 or LY34 for5 min,G_qand G_ssignal alterations were detected by adding different concentrations of DOM for 15 min of induction.3.LY34-enhanced LSD/DOM-induced differential phosphorylation for quantitative proteomics studyHEK-293T cells stably expressing 5-HT_2AR were transiently transfected with m Glu R2 and the interaction between 5-HT_2AR and m Glu R2 was verified using anti-FLAG^?M2 magnetic bead Co-Immunoprecipitation（Co-IP）assay.HEK-293T cells co-transfected with 5-HT_2AR and m Glu R2 were divided into 5 groups:Vehicle（0.9%saline）group,LSD group,DOM group,LY34+LSD group and LY34+DOM group.Cells were first treated with Vehicle or LY34 for 30 min,then LSD or DOM were added,respectively,and cell protein samples were collected after 30 min of incubation,and the collected samples were subjected to phosphorylated proteomics analysis using isotope-labeled relative and absolute quantitative techniques.Venn diagrams were drawn after two-by-two comparison of Vehicle,LSD,LY34+LSD groups and Vehicle,DOM,LY34+DOM groups,respectively,and literature research and analysis of differentially phosphorylated proteins in overlapping regions were performed.4.Correlation study of PDHA1 and RAPTOR with classical hallucinogens-induced HTRBoth m TORC1 regulatory associated protein of MTOR complex 1（RAPTOR）and m TOR are important constituents of m TORC1.HEK-293T cells stably expressing5-HT_2AR were transiently transfected with m Glu R2 and incubated with DOM for 30 min to detect changes in the phosphorylation levels of P70S6,RPS6,Akt and pyruvate dehydrogenase E1 alpha subunit（PDHA1）downstream of m TOR.C57 mice were given single doses of pyruvate dehydrogenase kinase（PDK）inhibitor DCA,m TOR classical inhibitor Rapamycin or m TORC1 selective inhibitor Torin 1,XL388,OSI-027,respectively,and 30 min later were injected intraperitoneally with LSD,DOI or DOM were observed to record the number of HTR in mice within 30 min.C57 mice were given different doses of DCA,Rapamycin,Torin 1,XL388 and OSI-027 in a single dose and the total distance of spontaneous activity within 1 h after administration and the segmented distance within every 10 min were recorded.Results:1.Effect of m Glu R2 agonists and inverse agonists on LSD/DOM-induced HTRLY37,LY35 and LY40 dose-dependently inhibited LSD or DOM-induced HTR in mice,while LY34 enhanced LSD or DOM-induced HTR in mice.Each dose of LY37,LY35,LY34 and 0.5 mg/kg LY40 had no significant effect on spontaneous activity in mice,while 2 and 5 mg/kg LY40 had inhibitory effects on spontaneous activity in mice.2.Study of the signaling pathway related to DOM-induced HTR by m Glu R2agonists and inverse agonists affectingIn normal dimeric cells,DOM-induced G_ssignaling pathway could be inhibited by LY35 and enhanced by LY34,while DOM-induced G_qsignaling pathway was not affected.5-HT_2AR antagonist M100907 attenuated the enhancement of DOM-induced G_ssignaling pathway by LY34.In mutant dimer cells,the inhibitory effect of LY35 on DOM-induced G_ssignaling pathway was completely lost;the enhancing effect of LY34on DOM-induced G_ssignaling pathway was significantly enhanced.3.LY34-enhanced LSD/DOM-induced differential phosphorylation for quantitative proteomics studyThe results of Co-IP assay showed that 5-HT_2AR interacted with m Glu R2.PDHA1,which was differentially phosphorylated in both LY34+DOM＿Vehicle and LY34+DOM＿DOM,and RAPTOR,which was differentially phosphorylated in both DOM＿Vehicle and LY34+DOM＿Vehicle,might be associated with hallucinogenic effects.4.Correlation study of PDHA1 and RAPTOR with classical hallucinogens-induced HTRDOM enhanced the phosphorylation of P70S6,RPS6,Akt and PDHA1.DCA,Rapamycin,Torin 1,XL388 or OSI-027 all significantly inhibited LSD,DOI or DOM-induced HTR in mice,and none had significant effects on spontaneous activity in mice.Conclusions:m Glu R2 agonists and inverse agonists can regulate classical hallucinogens-induced HTR through 5-HT_2AR-mediated G_ssignaling pathway,and 5-HT_2AR/m Glu R2heterodimer is crucial in it.On this basis,the involvement of PDHA1 and RAPTOR in classical hallucinogens-induced HTR was confirmed by phosphorylated proteomic screening and experiments,providing a new mechanism of action and research direction for classical hallucinogens-induced HTR.Innovation points:1.Innovative discovery that m Glu R2 agonists and inverse agonists regulate DOM-induced HTR in mice through the G_ssignaling pathway.2.The first discovery and confirmation of the involvement of PDHA1 and RAPTOR in classical hallucinogen-induced HTR in mice through quantitative phosphorylation proteomics studies and signaling pathway studies.