The Effects Of (D-Ser2)oxm On The Learning And Memory In APP/PS1 Transgenic Mice And Its Electrophysiological And Molecular Mechanisms

Objective:Alzheimer’s disease(AD) is a neurodegenerative disease which seriously affects the health and life span in the elderly. Recently, more and more attention has been paied to the correlation of AD and type 2 diabetes mellitus. One of promising strategies in developing novel anti-AD is to use the antidiabetic drugs. In the present study, we investigated the effects of intraperitoneal injection of(D-Ser2)Oxm on the impairments in spatial learning and memory of APP/PS1 mice by using Morris water maze test; and examined electrophysiological mechanisms by recording in vivo hippocampal field excitatory postsynaptic potentials(f EPSPs) and its long term potentiation(LTP) in the CA1 region of APP/PS1 mice. In order to investigate the molecular mechanisms of(D-Ser2)Oxm, we used western blot technique to test the expression levels of p-PI3 K, p-Akt(S-473) and p-GSK3β(Y216) and also observed the cell viability, neuronal apoptosis, mitochondrial membrane potential and intracellular calcium concentration with cultured primary hippocampal neurons treated with Aβ1-42 and(D-Ser2)Oxm.Methods:Our experiments include two parts: in vivo transgenic mice experiment, and in vitro culture cell experiment.First, APP/PS1 transgenic mice(nine months age) and littermate control C57 were used in behavioral experiments. The animals were divided into four groups:control(C57+saline), AD model(APP/PS1+saline),(D-Ser2)Oxm alone(C57+Oxm)and(D-Ser2)Oxm treatment(Oxm+Aβ1-42). All animals were injected intraperitoneally(i.p.) with(D-Ser2)Oxm or saline once daily for 2 weeks. After injection, Morris water maze was used to test spatial learning and memory of mice.The learning ability of mice was examined using hidden platform test, in which mice was allowed to swim 4 times per training day for 5 days to record the escape latencies.The spatial reference memory of mice was examined using probe trial, in which mice was allowed to swim 2 times for recording the swimming time and distance elapsed in the target quadrant. After probe trial, the visual and motor ability of rats was examined using visual platform test. Escape latency in hidden platform test and swimming traces in all experiments were recorded for off-line analysis with animal behavior software.Next, we investigated the possible electrophysiological mechanism of(D-Ser2)Oxm by using the same AD animals and recording the field excitatory postsynaptic potentials(f EPSPs) of CA1 region in hippocampus and its plasticity induced by HFS. After electrophysiological recordings, we examined the expression levels of p-PI3 K, p-Akt(S-473) and p-GSK3β(Y216) using western blot.In cell culture experiment, cultured hippocampal cells were divided four groups:control(Vehicle+Saline), Aβ alone(Vehicle+Aβ1-42),(D-Ser2)Oxm alone((D-Ser2)Oxm+Saline) and co-application((D-Ser2)Oxm +Aβ1-42). Mature cells were exposed to Aβ1-42 and(D-Ser2)Oxm for 24 hours. Primary cultures were prepared from 1-3 day postnatal SD rats. Cultures were maintained in 5 % CO2 at 37℃ in incubator for 7 days. The cells cultured in 96-well plates were used in cell viability assay, in which another GLP-1 inhibitor group((D-Ser2)Oxm+Aβ1-42+Exendin(9-39)) was added. Annexin V/PI double-staining was used to test apoptosis, and JC-1 dye was use to test the changes in the mitochondrial membrane potential when measured by flow cytometry. Finally, intracellular free Ca2+ was measured with fluorescent dye Fluo-4/AM by utilizing confocal imaging system.Results:The results of animal behavioral and electrophysiological experiments showed that: 1) in hidden platform tests, a significant deficit in spatial learning was found in APP/PS1 transgenic mice, with longer escape latencies in searching for the under-water platform(P<0.01). There were significant main effects on escape latency in AD model group and(D-Ser2)Oxm treatment group(P<0.01). 2) In probe trials, a significant difference in memory retrieve was found between AD model group and control group, with a reduced swimming time in target quadrant in APP/PS1 transgenic mice(P<0.01). But,(D-Ser2)Oxm treatment effectively reversed the detrimental effects in AD animals(P<0.01). 3) In visible platform test, there is no significant difference in escape latency and swimming speed was found between all groups(P>0.05). 4) The hippocampal field potential recording experiments showed that the LTP value in APP/PS1 transgenic mice was significantly suppressed(P<0.001), which was moderately reversed after administration of(D-Ser2)Oxm(P<0.001). 5) There was no significant difference in PPF between groups(P>0.05). 6)The western blot assay showed that(D-Ser2)Oxm could increase the expression levels of p-PI3 K and p-Akt(S-473)(P<0.01), and decrease the expression level of p-GSK3β(Y216)(P<0.01) in AD animals.In addition, the results from cell culture experiments showed that: 1)pretreatment with(D-Ser2)Oxm partly and effectively prevented Aβ1-42-induced decline in cell viability(P<0.001), and this protective roles could be inhibited by the pretreatment with exendin(9-39), a GLP-1 receptor blocker. 2)(D-Ser2)Oxm treatment decreased Aβ1-42-induced early neuronal apoptosis(P < 0.001). 3)(D-Ser2)Oxm inhibited Aβ1-42-induced mitochondrial membrane potential depolarization(P<0.001). 4)(D-Ser2)Oxm reversed Aβ1-42-induced [Ca2+]i elevation(P<0.001).Conclusion:(D-Ser2)Oxm improved spatial learning and memory and the hippocampal synaptic plasticity in APP/PS1 transgenic mice, and also protected hippocampal neurons against Aβ1-42-induced cytotoxicity. The mechanisms of(D-Ser2)Oxm may be involved in the activation of GLP-1 receptors, the modulation of PI3K/Akt/GSK3βsignal pathway, and stabilization of intracellular free Ca2+ and mitochondrial membrane potential. All of these results provide a novel insight into the neuroprotective mechanisms of(D-Ser2)Oxm, and suggest that(D-Ser2)Oxm could be benifitial to the improvement of cognitive function in AD.