The Study Of Electrophysiological And Molecular Mechanisms Of GLP-1/GIP/Gcg Triagonist In Improving Learning And Memory Of 3xTg-AD Mice

Objective:Alzheimer's disease?AD?is a neurodegenerative disease that occurs more commonly in older age and is characterised by progressive decline in the function of learning and memory.However,there is no cure for AD at present.It is interesting that numerous epidemiological and clinical studies have found that type 2 diabetes mellitus?T2DM?is a risk factor for AD.Moreover,the close relationships between AD and T2 DM have been displayed in the neuropathology and clinical treatment.Therefore,anti-T2 DM drugs may play an important role in the prevention and treatment of AD.Glucagon-like peptide-1?GLP-1?and glucose-dependent insulinotropic polypeptide?GIP?are incretin hormones for the treatment of T2 DM,which have shown neuroprotective properties and improving learning and memory in AD animal models.Recent studies show that GLP-1/GIP/Gcg triagonist?Triagonst?,a novel monomeric triagonist equally activating the GLP-1,GIP and glucagon receptors,has a longer biological half-life.Moreover,Triagonist has been reported to have significantly benefical effects in treating T2 DM animal models.However,whether the Triagonist has functions in improving AD and its electrophysiological mechanisms remain unknown.In this study,we first observed the ameliorative effects of chronic intraperitoneal injection of Triagonist on 3xTg-AD mice working memory and long-term memory in behavioral experiments by using Y maze and Morris water maze.Then,in order to elucidate the electrophysiological and molecular mechanisms of Triagonist improving cognitive behavior in 3xTg-AD mice,hippocampal LTP in the CA1 region was recorded by using in vivo field potential recording technique and the expression of S133p-CREB,T286p-CAMKII and S9p-GSK3? protein in hippocampus were analyzed by Western blot.In addition,we also investigated the effects of amyloid-? protein on the transmembrane Ca²⁺ flux in the hippocampal slices of mice by using the latest non-invasive micro technique?NMT?.Methods:1.Behavioral,electrophysiological and molecular biological experiment3xTg-AD mice?7 months?and wild-type?WT?C57BL/6 mice were randomly divided into four groups: WT+Saline,3xTg+Saline,WT+Triagonist and 3xTg+Triagonist.GLP-1/GIP/Gcg triagonist?10 nmol/kg?or equivalent saline?0.9% NaCl?was intraperitoneally injected once-daily for 30 days,and then the following tests were performed:?1?Y maze spontaneous alternation testThe total number of arm enteries and the percentage of correct spontaneous alternations in each group were observed to estimate the working memory ability of mice.?2?Morris water maze testThe place navigation test and the probe test were performed respectively to evaluate the spatial learning and memory ability of mice by recording the escape latencies and the number of platform crossings in different groups.?3?In vivo hippocampal LTP test By redording the hippocampal field excitatory postsynaptic potential?fEPSP?in the CA1 region of mice,paired pulse stimulation-induced paired-pulse facilitation?PPF?and high frequency stimulation-induced long-term potentiation?LTP?were observed respectively to reflect the changes of synaptic plasticity in the brain.?4?Molecular biological experiment The expression of S133p-CREB,T286p-CAMKII and S9p-GSK3? in the hippocampus of mice were detected by using Western blot to explore the possible molecular mechanisms of neuroprotective effect of Triagonist.2.Non-invasive micro-test technique?NMT?experiments on acutely dissociated hippocampal brain slicesAcutely dissociated hippocampal slices were prepared from male C57BL/6 mice?8 months?.NMT system was used to detect the influx and efflux of Ca²⁺,as shown in below.?1?Ca²⁺ influx experiment Acute administration with different concentration of Glu and A?31-35 respectively to induce the influx of Ca²⁺;the effects of different concentrations of A?31-35 on the Glu-induced Ca²⁺ influxes were observed;the changes of Glu-induced Ca²⁺ influxes by pretreatment with A?31-35 combined with D-APV or CNQX were observed to investigate the mechanisms of A?31-35 on the Glu-induced cell excitability.?2?Ca²⁺ efflux experiment Low calcium artifical cerebrospinal fluid?aCSF?-induced transmembrane Ca²⁺ efflux in the presence of A?31-35,KB-R7943 and TFH was recorded to explore the effects and mechanisms of A?31-35 on Ca²⁺ efflux.Results:1.In Y maze spontaneous alternation test,there were no differences in the total number of arm entries among the four groups?P > 0.05?;but control 3xTg-AD mice showed a decrease in the percentage of correct spontaneous alternations compared with wild-type mice?P < 0.01?;whereas Triagonist-treated 3xTg-AD mice significantly increased the the percentage of correct spontaneous alternations compared with control 3xTg-AD mice?P < 0.01?.2.In Morris water maze place navigation test,compared with control wild-type group,3xTg-AD mice displayed significantly longer escape latency on day 4?P < 0.01 or P < 0.001?and day 5?P < 0.01 or P < 0.001?.Importantly,Triagonist-treated 3xTg-AD mice exhibited shorter escape latency than the saline-treated 3xTg-AD mice from the 3rd day?P < 0.01 or P < 0.001?.In the probe test,the number of platform crossings was significantly decreased in the 3xTg-AD mice compared to the wild-type mice?P < 0.01?;while the number of platform crossings in the Tragonist-treated 3xTg-AD mice had a significant increase compared to that in the saline-treated 3xTg-AD mice?P < 0.01?.In the reversal place navigation test,the average escape latency of mice to find the hidden platform gradually decreased with the increase of training day among four groups.During reversal training,3xTg-AD mice showed significantly longer escape latencies compared with wild-type mice on day 8?P < 0.05?and day 9?P < 0.01?,whereas in Triagonist-treated 3xTg-AD mice were significantly decreased?P < 0.05 or P < 0.01?.Then in the probe test,3xTg-AD mice showed the number of target platform crossing was significantly reduced compared with wild-type mice?P < 0.01?,and this reduction was significantly reversed by Triagonist treatment?P < 0.05?.3.In hippocampal field potential LTP experiment,there was no difference in the percentages of PPF among the four experimental groups?P > 0.05?.Compared with wild-type mice,a significant reduction of the percentage of fEPSP slope was found during the last 10 min post-HFS in 3xTg-AD mice?P < 0.001?,which was obviously rescued by Triagonist?P < 0.001?.4.In Western blot experiment,compared with wild-type mice,the expression level of S133p-CREB,T286p-CAMKII and S9p-GSK3? were decreased in the hippocampus of control 3xTg-AD mice?P < 0.05 or P < 0.01?,which were reversed by the treatment with the Triagonist?P < 0.05 or P < 0.01?.5.In NMT experiments:?1?acute administration of Glu in normal aCSF condition evoked an initially rapid,subsequently slowing but stable-keeping Ca²⁺ influx in the hippocampal slices?P < 0.05 or P < 0.001?;pretreatment with A?31-35 dose-dependently increased average Ca²⁺ influx induced by Glu?P < 0.001?and the average Ca²⁺ influxes were increased significantly during 5 min after administration?P < 0.01 or P < 0.001?,which could be effectively blocked by NMDA receptor antagonist D-APV?P < 0.001?.?2?low Ca²⁺ aCSF induced an obvious and continuous transmembrane Ca²⁺ efflux in the hippocampal slices?P < 0.001?,which could be mostly blocked by specific Na+/Ca²⁺ exchanger antagonist KB-R7943?P < 0.001?;A?31-35 pretreatment partially inhibited the Ca²⁺ efflux?P < 0.05?.Conclusion:1.Intraperitoneal injection of GLP-1/GIP/Gcg triagonist effectively improved the short-term and long-term spatial memory in 3xTg-AD mice;2.Intraperitoneal injection of GLP-1/GIP/Gcg triagonist alleviated the impairments of in vivo hippocampal synaptic plasiticity in 3xTg-AD mice;3.Intraperitoneal injection of GLP-1/GIP/Gcg triagonist prevented the down-regulation of the expression of S133p-CREB,T286p-CAMKII and S9p-GSK3? in the hippocampus of 3xTg-AD mice.4.A?-induced calcium overload was not only related to the increase of Ca²⁺ influx,but also associated with the inhibition of Ca²⁺ efflux.A? facilitated Glu-induced excitotoxicity is mainly mediated by enhancing NMDA receptor and damaging Na+/Ca²⁺ exchanger.In summary,GLP-1/GIP/Gcg triagonist improved learning and memory in cognitive behavior in 3x Tg-AD mice,which may be involved in the up-regulation of S133p-CREB,T286p-CAMKII and S9p-GSK3? in the hippocampus of 3xTg-AD mice,and the reduction of A?-induced Ca²⁺ overload and synaptic plasticity deficits.These results suggeste that GLP-1/GIP/Gcg triagonist might have positive significance on the treatment of neurodegenerative diseases such as AD.