The Role Of MTORC2 Signaling In Regulating Cocaine-induced Structural Remodeling In Striatum

BackgroundLong-term abuse of cocaine results in drug addiction,which is generally known being relevant to the structural plasticity of median spine neurons on the NAc(Nucleus Accumbens)of the striatum.mTOR is a serine/threonine kinase that originally defined as mammalian target of rapamycin,which is widely distributed in vivo and act in a variety of biological processes,such as cell growth,metabolism and protein synthesis.mTOR function in vivo as two distinct protein complexes,mTORC1 and mTORC2.One of the core components of mTORC2 is rictor(mAVO3)The AGC kinases family member Akt is one of the key down-stream substrates of mTORC2 complex.The phosphorylation on the 473 serine site of Akt depends on the activity of mTORC2 complex,which can be identified as a symbol of the activation of mTORC2 pathway.Evidence showed that Rapamycin could restore the cocaine-induced behavioral changes by suppress the activity of mTORC1 pathway specifically.However,the effect of mTORC2 in cocaine addiction was barely understood.We intend to figure out whether the mTORC2 complex is involved in the regulation of cocaine induced neuroplasticity.Method1.The activities of mTORC2/mTORC1 pathways were analyzed:The induction of rictor/raptor and the phosphorylation of Akt Ser 473/p70S6k1 were detecting by western blotting,respectively.2.Constructed the Akt S473 constitutively active AAV vectors and its control vector:Vectors were infused into the NAc brain region of the C57/BL6 mice by stereotaxic injection.3.Analyzed the basal behavioral phenotype of the Akt S473 constitutively activate mice:By openfield test which measures the basic motor capacity and exploring interest,in addition,by the elevated plus maze test and anxiety respectively.4.Analyzed the neuron structrual plasticity:Mice received repeated saline or cocaine i.p.administration for 7 days and sacrificed after 24 hours to perfuse and fix the brain tissue.The neuron branching morphology and the spine density of the neurons on the NAc were measured by immunofluorescence technology.5.Analyzed the addictive behaviors of mice:By adapting the locomotor activity test and cocaine conditioned-position preference test.6.Analyzed the relevance of the mTOR signaling and the Racl signaling by western blotting,GST-Pull down,coIP and immunofluorescence co-localization.In addition several cocaine induced neuroplasticity relevant gene the were detected.Result1.The activities of Akt S473 and the expression of rictor in the mTORC2 pathways were up-regulated while in the mTORC1 pathways was down-regulated.2.The Akt S473 constitutively activate AAV vector were constructed,which was applied smoothly in producing the NAc Akt S473 constitutively activate mice3.Akt S473 constitutive activation in the NAc reduced the exploring interest to the new environment.4.Akt S473 constitutive activation in the NAc reversed the cocaine induced increase of the dendrite branching numbers and the dendrite spine density of the MSNs onto the NAc.5.Akt S473 constitutive activation in the NAc attenuated the addiction behavior of mice,for instance the locomotor activity after cocaine treatment and the cocaine conditioned-position preference6.Akt S473 constitutive activation in the NAc up-regulated the Rac1-GTPasel activities,the induction of tiaml and the phosphorylation of cofilin,and attenuated the interaction effect between the tiaml and rictor in the NAc.It also decrease the phosphorylation of ERK and the expression of cfos and bdnf,while the activity of mTORC1 signal were up-regulated.ConclusionOur research indicated that mTORC2 signaling plays an essential role in the regulation of the cocaine induced structural plasticity of the MSNs and the addiction relevant behaviors by phosphorylating of Akt on the Ser 473 site.The interaction with tiaml-Rac1-PAK1-cofilin pathway is involved in this regulation.