The Study On The Antagonistic Effect Of Lixisenatide Against Amyloid β Protein-Induced Cognitive Dysfunction In Rats

Alzheimer’s disease (AD) is a chronic and primary degenerative disease in the central nervous system. Progressive cognitive dysfunction and learning and memory loss are the main clinical characteristics, with a severe dementia in the later phase of AD. One of the most typical pathological characteristics in AD is the presence of high density of senile plaques (SPs), mainly composed of amyloid β protein (Aβ). Aβ is the hydrolysis product of amyloid precursor protein (APP) by β secretase and γ secretase. The pathological regions in AD brain are mainly involved in those cognition-related area, especially the hippocampus and the temporal cortex. At present, the neurotoxicity of Aβ has been widely reported in vivo and in vitro, including the damage to the spatial learning and memory behaviors of animals, the impairment of synaptic plasticity and the cell death in cultured neurons. Therefore, the "Ap theory" in AD pathological mechanism has been widely recognized. However, the cellular and molecular mechanisms on the Aβ neurotoxicity are more complicated, and there is still no breakthrough in the drug research against the neurotoxicity of A β up to now. So, it is very important and urgent to search for effective drugs against the neurotoxicity of Aβ in the treatment of AD.Interestingly, a large number of epidemiological surveys show that another age-related degenerative disease type2diabetes mellitus (T2DM) has a close correlation with AD. There are many similar characteristics in the processes of AD and T2DM, including pathological changes and the clinical manifestations. So, a new strategy for AD treatment is using the method controlling T2DM to reduce the production and accumulation of Aβ, enhance the degradation of Aβ and inhibite the neurotoxicity of Aβ. Glucagon-like peptide-1(GLP-1) is a kind of incretin. Because it promotes the secretion of insulin in the glucose dependent way, it can not change the normal blood sugar. Because of this advantage, GLP-1has become the latest drug for the treatment of T2DM. Researches show that both GLP-1and GLP-1receptor (GLP-1R) are expressed in the brain, especially in the hippocampus. GLP-1can reduce the level of Aβ in the brain, resist the cell apoptosis and play the role of neurotrophic factor. However, natural GLP-1can be rapidly degraded by the enzyme dipeptidyl peptidase IV (DPPIV) and its half life is only1-2min in the blood plasma. So, the application of the natural GLP-1in AD is greatly restricted. Meantime, it is still almost unclear whether GLP-1can protect the cognitive function of rats against Aβ-induced behavioral disorder and hippocampal synaptic plasticity deficit. Morover, the neuroprotective mechanisms of GLP-1are still an open question.Lixisenatide is a novel GLP-1analogue, which can greatly resist DPPIV-indced degredation, with a longer half-life and four times affinity to GLP-1R than that of the natural GLP-1. Therefore, the present study, by using Morris water maze, in vivo electrophysiological recording and Western Blotting techniques, investigated the neuroprotective effects and the possible mechanism of GLP-1analogue lixisenatide. The experiments mainly include the following three parts:(1) Examing whether bilateral hippocampal injection of lixisenatide can effectively antagonize Aβ25-35-induced spatial learning and memory disorder by using Morris water maze test.(2) Observing whether bilateral hippocampal injection of lixisenatide can effectively reverse Aβ25-35-induced suppression of hippocampal long term potentiation (LTP)by recording in vivo hippocampal field potential in the CA1region.(3) By using Western Blotting, testing the effect of lixisenatide on the activitity of glycogen synthase kinase3β (GSK3P) in the PI3K-AKT signal transduction pathway and clarifying the possible molecular neuroprotective mechanism of lixisenatide.Part I Lixisenatide Protects Against Aβ25-35-Induced Impairments of Spatial Learning and Memory in RatsTo examine the neuroprotective role of lixisenatide in the brain, we investigated the effect of bilateral hippocampal injection of lixisenatide on the Aβ25-35-induced impairments of spatial learning and memory of rats by using Morris water maze (MWM) test. The normal male Sprague Dawley (SD) rats were used. After anesthesia, animals were placed in a brain stereotaxic apparatus. Drugs were slowly injected into the bilateral hippocampus by a micropump. Two weeks later, MWM test was performed, including three parts:hidden platform test, probe trial and the visual platform test. The escape latency (s) and distance (cm) in the hidden platform test, and the percentages of the total time and distance in the target quadrant in the probe trials were recorded. The swimming speed (cm/s) and the latency (s) to find the visible platform were also recorded in the visual platform experiment.The results showed that:(1) Bilateral hippocampal injection of Aβ25-35impaired the spatial learning and memory of rats. In the hidden platform test, the latencies and distances of rats for searching for the hidden platform in Aβ25-35alone group were significantly increased, compared with the control group during the2nd day to the5th day (P<0.05). For example, the escape latencies and distances were23.02±1.95s and452.90±26.31cm at the4th day, significantly larger than the values of16.43±1.67s and214.05±39.61cm in the control group (P<0.05). In the probe trial, the percentages of time and distances in the target quadrant in the5nmol Aβ25-35alone group were only25.30±3.85%and25.97±3.63%, significantly lower than46.24±4.59%and45.71±4.25%in the control group (P<0.05).(2) Lixisenatide alone did not affect the normal cognitive behavior.5nmol lixisenatide alone had no effects on the escape latencies and distances (P>0.05) in the hidden platform test, as well as the percentages of time and distances in the target quadrant in the probe trial (P>0.05).(3) Treatment with different concentrations of lixisenatide effectively protected against Aβ25-35-induced impairments of the spatial learning and memory in a dose-dependent manner.0.05nmol lixisenatide did not affect Aβ25-35-induced impairment in the spatial learning with the similar escape latencies and distances in the hidden platform test, as compared to the Aβ25-35alone group (P>0.05). However, treatment with0.5nmol and5nmol lixisenatide significantly reversed the spatial learning impairment induced by5nmol Aβ25-35from the2nd day to the5th day (P<0.05). For example, on the4th day of the hidden platform test, the escape latencies and distances in0.5nmol and5nmol lixisenatide plus5nmol Aβ25-35groups were18.09±1.17s and14.78±1.13s;326.45±25.95cm and226.41±25.50cm, respectively, showing a dose-dependent decrease (p<0.05). In the probe trial,0.05nmol lixisenatide did not affect5nmol A(325-35-induced impairment in the spatial memory with the similar percentages of time and distances in the target quadrant, as compared to the5nmol Aβ25-35alone group (P>0.05). The percentages of time and distances in the target quadrant were34.38±3.11%and42.22±3.33%;35.14±3.99%and44.42±3.41%, for0.5nmol and5nmol lixisenatide plus5nmol Aβ25-35groups, respectively, significantly higher than the5nmol Aβ25-35alone group (p<0.05), showing a significant dose-dependent increase (p<0.05).(4) All drugs did not affect the swimming speeds of rats and the latencies of rats to find the visiual platform (P>0.05). Part Ⅱ Lixisenatide Protects Against Aβ25-35-Induced Impairment of Long Term Potentiation in Rat Hippocampal CA1Region in vivoAfter the behavioral experiment, rats were anesthetized and placed in the brain stereotaxic apparatus for the in vivo field excitatory postsynaptic potential (fEPSP) recording. The binding electrode (bipolar stimulation electrode and unipolar recording electrode) was precisely inserted into the stimulation and recording regions of the hippocampus. By delivering the single stimulation, the paired pulses and of the high frequency stimulation (HFS) into the Schaffer collateral/commissural pathway of the hippocampus, basic fEPSP, paired pulse facilitation (PPF) and the long term potentiation (LTP) were recorded in the radiation layer of the hippocampal CA1region.The results showed that:(1) Aβ25-35did not affect the baseline fEPSP, but significantly suppressed LTP induced by HFS. After application of5nmol Aβ25-35, the fEPSP amplitudes after HFS were136.66±3.41%and119.01±3.82%at30min and60min, respectively, significantly lower than the values of168.13±3.16%and155.65±3.48%in the control group at the same time points (P<0.01).(2) There was no significant change in the fEPSP amplitudes after HFS at30min and60min in the5nmol lixisenatide alone group, as compared to the control group (P>0.05).(3) Treatment with different concentrations of lixisenatide protected against Aβ25-35-induced impairment of LTP. The fEPSP amplitudes in0.5nmol and5nmol, but not0.05nmol lixisenatide, plus Aβ25-35groups, were135.70±3.80%and145.71±3.81%at1h post-HFS, respectively, significantly larger than the values in the5nmol Aβ25-35alone group (P<0.01), showing a significant dose-dependent increase (p<0.05).(4) All drugs did not affect PPF (P>0.05), indicating that the influence of all drugs on LTP was not by presynaptic mechanism.Part Ⅲ Lixisenatide Suppresses Aβ25-35-Induced Activation of GSK3β in the HippocampusThe neuroprotection of GLP-1might be involved in the PI3K-AKT-GSK3β pathway. To investigate the possible neuroprotective mechanism of GLP-1on Ap-induced impairments of cognition and LTP, we observed the effects of lixisenatide and AP25-35on the activity of glycogen synthase kinase3β (GSK3β) in the PI3K-AKT signal transduction pathway. After the electrophysiological experiment, the hippocampi of rats were prepared for samples. By using Western Blotting method, we examined the levels of GSK3β and its two phosphorylation forms:pGSK3β(S9) and pGSK3β (Y216). The ratios of GSK3β/β-actin, pGSK3β (S9)/GSK3β and pGSK3β (Y216)/GSK3β were calculated.The results showed that:(1) The ratio of GSK3β/β-actin were1.41±0.07,1.40±0.05,1.40±0.05and1.41±0.06in control,5nmol Aβ25-35alone,5nmol lixisenatide and5nmol lixisenatide plus5nmol Aβ25-35group, respectively. There was no significant difference in all groups (P>0.05).(2) The ratio of pGSK3β (S9)/GSK3p in the5nmol Aβ25-35alone group was0.38±0.06, less than the control group (0.53±0.06, P<0.05). The ratio of pGSK3β (S9)/GSK3p of the5nmol lixisenatide plus5nmol Aβ25-35group was0.52±0.05, which was more than the value in Aβ25-35alone group (P<0.05). At the same time, there was no significant difference in the control group and the5nmol lixisenatide alone group (P>0.05).(3) The ratio of pGSK3β (Y216)/GSK3β in Aβ25-35alone group was0.51±0.05, more than the value in control group (0.35±0.07, P<0.05). The ratio of pGSK3β (Y216)/GSK3β in5nmol lixisenatide plus5nmol Aβ25-35group was0.36±0.06, which was less than the5nmol Aβ25-35alone group (P<0.05). There was no significant difference in the control group and the5nmol lixisenatide alone group (P>0.05).These results above indicated that:(1) Bilateral hippocampal administration of Aβ25-35seriously impaired the spatial learning and memory of rats, while treatment with lixisenatide effectively resisted Aβ25-35-induced impairments in a dose dependent manner;(2) Bilateral hippocampal administration of Aβ25-35seriously impaired LTP, while treatment with lixisenatide effectively reversed Aβ25-35-induced suppression of LTP in a dose-dependent manner;(3) Lixisenatide could reverse the Aβ25-35-induced activation of GSK3β. So, our electrophysiological result was well consistent with the behavioral result, which might partly explain the cellular mechanism of GLP-1and GLP-1analogue-lixisenatide in improving the spatial learning and memory function of rats. These findings strongly suggest that the activation of PI3K-AKT pathway and the inhibition of GSK3β activity might be the important mechanism for the neuroprotective function of GLP-1and GLP-1analogue-lixisenatide and the up-regulation of GLP-1or application of GLP-1analogue lixisenatide might be a promising strategy for the treatment of neurodegenerative diseases such as AD.In conclusion, the present study, by using MWM test, LTP recording and Western Blotting technique, observed the effects of lixisenatide on the spatial learning and memory, the hippocampal LTP in vivo and the activity of GSK3β in the PI3K-AKT signal transduction pathway and investigated the neuroprotective mechanism of lixisenatide against the neurotoxicity induced by Aβ25-35. These results indicated that lixisenatide could effectively protect against Aβ25-35-induced impairments of the spatial learning and memory and the hippocampal LTP in vivo; the neuroprotective mechanism of lixisenatide might be closely related to the modulation of the synaptic plasticity and the activity of GSK3β. Therefore, the present study provides behavioral and electrophysiological evidence for the neuroprotective effect of GLP-1and its analogue lixisenatide, and reveals the possible cellular and molecular mechanisms. We think that our these experimental results could be helpful not only to understand the mechanisms of Aβ, but also to promote the development of GLP-1analogue for the clinical treatment of AD in the future.