Influence Of Akt Deletion On Spatial Cognition In Mice And Its Molecular Mechanism

Research backgroundAkt/protein kinase B(PKB)proteins are members of a family of mammalian serine/threonine-specific protein kinases that includes Akt1(also called PKB?),Akt2(PKB?),and Akt3(PKB?).Genetic manipulation studies have found the isoform-specific effects of the three isoforms.For example,Akt1-null mice are retarded growth;Akt3-null mice have impaired brain development.However,the mechanism of Akt3 deficiency in spatial cognition is still unclear.Frequency-dependent long-term potentiation(LTP)induction in hippocampal CA1 region is considered to be the cellular model of learning and memory function.LTP can be divided into early-LTP(intracellular kinase activity mechanism)and late-LTP(intraspinous protein synthesis mechanism)according to the time when the slope of excitatory postsynaptic membrane potential(EPSP)increases after high-frequency stimulation.The former may be related to the increased phosphorylation of AMPAr subunits Glu A1 and Glu A2,while the latter may be related to the increased synthesis of AMPA receptors and other membrane proteins.It has been found that rapamycin,an inhibitor of m TOR,can prevent the production of late-LTP,but does not affect the induction of early-LTP.The deletion of Akt3 can reduce the phosphorylation level of m TOR,and the deletion of Akt1 does not affect the phosphorylation level of m TOR,therefore this study proposed the scientific hypothesis that "Akt3 deletion affects the production of protein synthesis-dependent LTP by inhibiting the m TOR-p70S6 k signaling pathway,resulting in cognitive impairment".Objective1.To explore the influence of Akt1 and Akt3 deletion in spatial cognition and synaptic plasticity.2.To clearify the molecular mechanisms underlying Akt1 and Akt3 deletion-altered synaptic plasticity.Materials and Methods1.The Akt knockout mice were presented by Professor Chen Guiquan at the Nanjing University model animal center.Akt1 and Akt3 knockout mice,hereinafter referred to as "Akt1-KO mice and Akt3-KO mice".The PCR methods were used to identify Akt1-KO mice and Akt3-KO mice.The wild type(WT)mice of Akt1-KO mice are called Akt1-WT mice.Twelve-week-old Akt3-KO mice and Akt3-WT littermates,Akt1-KO mice and Akt1-WT littermates were employed at the beginning of the experiment.All mice were randomly assigned to experimental groups.Each experiment was performed by two experimenters who were blinded to the experimental groups.2.Behavioral assessments: Three different behavioral tests were carried out under following sequence: open-field test ? Y-maze ? Morris water maze.The spontaneous activity of mice was detected by opening-field test.The spatial maze and memory function of mice was examined by Y-maze and Morris water maze test.3.Toluidine blue staining was used to observe the histological results and pyramidal cells in the hippocampal CA1 area.4.Field potential recording was used to study the basic properties of the Schaffer collateral-CA1 region synaptic transmission in the hippocampal CA region.LTP was induced by high frequency stimulation of 100Hz(HFS).We have adopted the following two strategies: One-train high-frequency(100 Hz,1 sec)stimulation(HFS×1)and a stronger four-train(HFS×4)with a 5 min intertrain interval were used to detect the changes of synaptic LTP formation.After 60 minutes of high frequency stimulation,the record was seen as early-LTP,and 180 minutes record was as late-LTP.5.Western blot analysis was used to examine Ca MKII,ERK1/2,m TOR,p70S6 K,4EBP2 and e IF4 E phosphorylation and theirs protein levels,and also detect the protein level of AMPAr and Glu R1 and Glu R2 subunits.Results: Part ?1.Open-field tests(OFT)showed that there was no significant difference in total distance traveled in the open-field test between Akt3-WT mice and Akt3-KO mice or Akt1-WT mice and Akt1-KO mice.2.The results of Y-maze test indicated that a tendency for Akt3-KO mice to have a lower alternation rate compared to Akt3-WT mice,but this trend failed to reach significance.Meanwhile,alternations in Akt1-KO mice did not differ significantly from Akt1-WT mice(P>0.05,n=10).Compared with Akt3-WT mice or Akt1-WT mice,there was no significant difference in the number of incoming arms between Akt3-KO mice and Akt1-KO mice in Y-maze test.3.Morris water maze task results showed that on day 1-2 of training,there was no difference in latency for reaching the visible platform between Akt3-WT mice and Akt3-KO mice or Akt1-WT mice and Akt1-KO mice.On days 3-7 of training,the latency to reach the hidden platform for mice with an Akt3 deficiency was significantly longer than for WT mice,but not Akt1-deficient.In the eighth day trajectory test,compared with Akt3-WT mice,Akt3-KO mice significantly reduced in swimming time and shuttle or stay time in the platform quadrant.However,there was no difference in swimming time between Akt1-WT mice and Akt1-KO mice in platform quadrant test.No significant difference was found in swim speed between Akt3-WT mice and Akt3-KO mice or Akt1-WT mice and Akt1-KO mice.4.Brains size of either Akt3-KO mice and Akt1-KO mice was smaller than WT brains,but there was no significant difference in hippocampal formation and no significant difference in the density of pyramidal cells in hippocampal CA1 area.5.Compared to Akt3-WT mice or Akt1-WT mice,the slope of excitatory postsynaptic potential(EPSP)in CA1 region recorded by stimulating Schiffer's lateral branches did not change significantly in Akt3-KO mice or Akt1-KO mice.Similarly,there was no significant difference in double pulse facilitating(PPF)ratio induced by double pulse stimulation in Akt3-KO mice and Akt1-KO mice.6.In Akt3-KO mice and Akt1-KO mice,HFS×1 conditioned stimulus could induce early-LTP.The amplitude of early-LTP in Akt1-KO mice was slightly lower than that in Akt1-WT mice.The LTP induction of Akt3-WT mice or Akt3-KO mice was blocked by the NMDAr antagonist MK801.7.In Akt1-KO mice,HFS×4 stimulus could induce late-LTP,but the amplitude of late-LTP was lower than that of Akt1-WT mice.In Akt3-KO mice,HFS×4 stimulus did not induce late-LTP.In Akt3-WT mice,MK801 could not block the induction of late-LTP,while rapamycin,a m TOR inhibitor,could block the induction of late-LTP.Results: Part ?1.The basal levels of Ca MKII protein and phospho-Ca MKII or ERK2 protein and phospho-ERK2 in the hippocampal brain region of Akt3-KO mice or Akt1-KO mice did not differ significantly from Akt3-WT mice or Akt1-WT mice.At 5 min post-HFS×1,the levels of phospho-Ca MKII and phospho-ERK2 were elevated in all Akt-KO mice or Akt-WT mice,but the amplitudes of Akt1-KO mice were significantly lower than those of Akt1-WT mice.Besides,HFS×1-induced increases in phospho-Ca MKII and phospho-ERK2 levels in mice were blocked by MK801.At 5min post-HFS×4,the increase of Ca MKII and ERK2 phosphorylation in Akt3-KO and Akt1-KO mice was no statistical difference from that in Akt3-WT mice and Akt1-WT mice.2.Compared to Akt3-WT mice,the basal levels of phospho-mTOR or after 5 minutes of HFS x 1 stimulate were notably lower in Akt3-KO mice,while the level of m TOR protein was no significant change.HFS×4 stimulate increased the level of m TOR phosphorylation in Akt3-WT mice,but did not increase Akt3-KO mice.The HFS×4-induced increase in phospho-m TOR levels in mice were abrogated in the presence of PI3 K inhibitor LY294002.Compared with Akt1-WT mice,the basic m TOR phosphorylation,the m TOR phosphorylation level after HFSx1 and HFSx4,and the level of m TOR protein in Akt1-KO mice were not significantly changed.3.Compared with Akt3-WT mice and Akt1-WT mice,the basic p70s6 k phosphorylation lever and the level of p70s6 k phosphorylation after 5 minutes of HFSx1 were not significantly changed in Akt3-KO mice and Akt1-KO mice.HFS x4 stimulate increased the level of p70s6 k phosphorylation in Akt3-WT mice,but did not increase Akt3-KO mice.LY294002 could block the increase of P70S6 K phosphorylation induced by HFSx4.4.Compared with Akt3-WT mice and Akt1-WT mice,the basis of 4EBP2 and e IF4 E phosphorylation and the level of 4EBP2 and e IF4 E phosphorylation in Akt3-KO mice and Akt1-KO mice was not significantly changed after 5 minutes of HFSx1 stimulate.HFS×4 stimulate could increase the phosphorylation of 4EBP2 and e IF4 E in Akt3-WT mice,but did not affect 4EBP2 and e IF4 E phosphorylation in Akt3-KO mice.LY294002 and rapamycin could block the increase of 4EBP2 and e IF4 E phosphorylation induced by HFS×4.5.After 180 minutes of HFS×4 stimulation,the protein levels of AMPA receptor subunit Glu R1 and Glu R2 increased significantly in Akt3-WT mice,Akt1-WT mice and Akt1-KO mice,but no increase in Akt3-KO mice.ConclusionThese results indicate that a deficiency in Akt3 via inactivation of m TOR impairs production of protein synthesis-dependent long-LTP and,consequently,long-term spatial cognitive disorder phenotypes.