The Experimental Research Of Lamotrigine On Cognition Improvement In APPswe/PS1dE9transgenic Mouse Model Of AD

Alzheimer’s disease is characterized with amyloid plaques consisted of β-amyloid (AP) and neurofibrillar tangles formed by phosphorylated tau protein. Accumulation of β-amyloid and phosphorylated tau, cause neuroinflammation, synapses and neuronal loss and eventually cognitive deficits. Therefore, targeting accumulated Aβ and phosphorylated tau are the main strategies in Alzheimer’s disease therapy.Alzheimer’s disease patients have an increased incidence of epileptic seizures. Hyperexcitability has also been observed in various Alzheimer’s disease transgenic mice. Such aberrant increases in network excitability and compensatory inhibitory mechanisms in the hippocampus may contribute to the cognitive deficits in Alzheimer’s disease transgenic mice and, possibly, also in Alzheimer’s disease patients. Thus, inhibition of overexcitation may prevent the cognitive deficits in Alzheimer’s disease. A recent report suggests that levetiracetam (LEV), an antiepileptic drug (AED), rescues the cognitive deficits of Alzheimer’s disease in human amyloid precursor protein (APP) transgenic mice via modulating the abnormal network. These studies have brought a new strategy, especially the potential application of AEDs, in Alzheimer’s disease therapy.In contrast, the sodium channel blockers phenytoin and carbamazepine, as well as phenobarbitaland benzodiazepines, can worsen cognitive function in patients with AD. We examed whether lamotrigine might attenuate the cognitive deficits of Alzheimer’s disease in APP/PS1mice remains unknown. Lamotrigine (LTG) is a broad-spectrum AED, used in treatment for the generalized seizures of Lennox-Gastaut syndrome and primary generalized tonic-clonic seizures, partial seizures and absence epilepsies in adult and pediatric patients aged over2years. LTG is particularly beneficial in the treatment of women and elderly patients with epilepsy. In addition, LTG has been applied in treatment for bipolar disorders, schizophrenia and other neurological conditions such as migraines and neuropathic pain. Moreover, LTG has been reported to have slight beneficial effects on memory in healthy adults and epilepsy patients. Treatment with LTG in11patients with the diagnosis of probable Alzheimer’s disease improved world recognition, naming and depressed mood on Alzheimer Disease Assessment Scale. These results suggest that LTG may be effective in Alzheimer’s disease therapy. However, whether and how LTG functions in preventing Alzheimer’s disease pathology remains unknown. In this study, we have shown that the chronic treatment with LTG attenuates the deficits in synaptic plasticity, neuronal loss and accumulation of amyloid plaques, and therefore rescues the cognitive deficits in APP/PS1mice, which express human mutant APP and presenilinl (PS1). We have further found that the chronic LTG treatment suppresses abnormal spike activity, whereas it increases the levels of brain-derived growth factor (BDNF) and nerve growth factor (NGF) in the brains of APP/PS1mice. Chapter I Chronic LTG treatment rescues deficits in learning and memory of APPswe/PS1dE9miceObjective:To exam an antiepileptic drug (AED), lamotrigine, whether LTG might attenuate the cognitive deficits of Alzheimer’s disease in human amyloid precursor protein (APP) transgenic mice.Methods:APP/PS1transgenic mice and wild-type littermates were randomly assigned into four groups:Lamotrigine-treated (TG+LTG) and Control (TG) APP/PS1transgenic mice; Lamotrigine-treated (WT+LTG) and Control (WT) wild-types. Treated Mice were received a diet of standard laboratory chow supplemented with lamotrigine (30mg/kg/d)(sodium salt; Glaxosmithkline;), Control TG and WT mice received the same chow withiout lamotrigine. The mice were feeded with lamotrigine at3months old and were subjected behavioral analysis and other experiments at8months old.Results:The effect of LTG on spatial memory was examined by using the Morris water maze test and novel object recognition test. In morris water maze test, Control APP/PS1mice showed that learning in locating the submerged escape platform was impaired, as indicated by the increased escape latencies and swimming length in the consecutive trials compared to wild-type (WT) mice. In contrast, LTG-treated APP/PS1mice showed escape latencies and swimming length were reduced compared to control APP/PS1mice. To exclude the possibility that the reduced escape latencies in LTG-treated APP/PS1mice were due to the increased swimming speed, we normalized the escape latencies in the first trial day of each group of the mice to1.0. The relative escape latencies in the following trial days have been quantified to that of in the first trail day. In this way, we could compare the learning trend of each group of the mice despite their differences in swimming speed. Compared to WT mice, which showed a learning capability as indicated by the shortening escape latencies as days of training, control APP/PS1mice failed to show a learning trend. In contrast, LTG-treated APP/PS1mice showed a comparable learning trend to WT mice. The memory retention of each group of mice has been analyzed in the probe trial experiment. LTG-treated APP/PS1mice exhibited improved memory retention as indicated that they swam cross over the target site more times than control APP/PS1mice. No differences were observed between LTG-treated and control WT mice in escape latency, learning trend, swimming path length and the times crossed over the target site.To support this notion, we performed novel object recognition test, a behavioral test which recently has been used in evaluating the cognitive deficits in APP/PS1mice. No significant difference was observed in location preference during the training phase, indicating the location of the objects does not affect the exploratory behavior of mice. In the testing phase, as demonstrated previously, control APP/PS1mice displayed a shorter time period with recognition index (RI) than WT mice, indicating a deficit in their memory for the familiar object. In contrast, LTG treatment improved the time period with RI in APP/PS1mice.Conclusion:These results indicate that the chronic treatment with LTG rescues deficits in learning and memory in APP/PS1mice.Chapter II. The experimental research of the therapeutic role of lamotrigine in AD pathology and abnormal neural activity in APPswe/PS1dE9Objects:To test whether lamotrigine could modulate abnormal network activity and prevent Alzheimer’s disease pathology in APPswe/PS1dE9miceMethods:APP/PS1transgenic mice and wild-type littermates were randomly assigned into four groups:Lamotrigine-treated (TG+LTG) and Control (TG) APP/PS1transgenic mice; Lamotrigine-treated (WT+LTG) and Control (WT) wild-types. Treated Mice were received a diet of standard laboratory chow supplemented with lamotrigine (30mg/kg/d)(sodium salt;Glaxosmithkline;), Control TG and WT mice received the same chow withiout lamotrigine. The mice were feeded with lamotrigine at3months old and were implanted for chronic video-EEG monitoring at6months old. After behavior testing, animals were sacrificed and the brain was removed. The brain was fixed and stored in4%paraformaldehyde in phosphate-buffered saline (PBS) and later dehydrated, the coronal brain sections were cut on a microtome, treated for Immunohistochemistry and Immunofluorescence. Immunohistochemistry was used to detect the levels of Aβ plaque in brains of APP/PS1transgenic mice; Immunofluorescence used to detect the levels of NPY, neural cell bodies, astrocytes and microglia in brains of APP/PS1transgenic mice.Results:More epileptic spikes were detected in the cortex and hippocampus of APP/PS1transgenic mice. In LTG-treated APP/PS1mice, numbers of epileptic spikes were reduced in both the cortex and the hippocampus compared to control APP/PS1mice. In both LTG-treated WT mice and control WT mice, little spikes were observed in the cortex and hippocampus. Comparing to WT mice, APP/PS1mice showed an enhanced NPY immunoreactivity in both the cortex and hippocampus. In contrast, the levels of NPY immunoreactivity in LTG-treated APP/PS1mice decreased compared to that in control APP/PS1mice. Quantification analysis showed that the numbers and size of Aβ plaques were reduced in the cortex and hippocampus of LTG-treated APP/PS1compared to control APP/PS1mice. The numbers of NeuN+cells were quantified. Control APP/PS1mice displayed less NeuN+cells in the prefrontal cortex and parietal cortex compared to WT mice. Whereas the numbers of NeuN+cells were enhanced in LTG-treated APP/PS1mice compared to that in control APP/PS1mice. However, the numbers of NeuN+cells in the hippocampus showed no significant differences among the groups. increases in the volume of both astrocytes and microglia were observed in both the cortex and the hippocampus of APP/PS1mice compared to WT mice at8months old of age, indicating an activation of both astrocytes and microglia in APP/PS1mice. In contrast, LTG-treated APP/PS1mice exhibited decreased volume of both astrocytes and microglia compared to control APP/PS1mice. We failed to observe differences in the volume of either astrocytes or microglia in between the cortex and hippocampus of LTG-treated and that of control WT mice.Conclusion:Chronic LTG treatment reduces the abnormal spikes in the brains of APP/PS1Mice; LTG treatment inhibits accumulation of Aβ plaques in APP/PS1mice; Chronic LTG treatment prevents neuronal loss in APP/PS1mice; LTG suppresses activation of microglia and astrocytes in APP/PS1mice.Chapter Ⅲ. Evaluation of synaptic plasticity improvement of Chronic LTG treatment in APP/PS1miceObjective:To observe whether Chronic LTG treatment could alleviate deficits in synaptic plasticity in APP/PS1miceMethods:APP/PS1transgenic mice and wild-type littermates were randomly assigned into four groups:Lamotrigine-treated (TG+LTG) and Control (TG) APP/PS1transgenic mice; Lamotrigine-treated (WT+LTG) and Control (WT) wild-types. Electrophysiology was used to test LTP in the CA1region of8-month-old APP/PS1mice which had been treated with LTG for5months and control mice. Golgi staining was used to examine whether chronic LTG treatment might prevent loss of spines in the brains of APP/PS1mice, and immunofluoresense staining with an antibody against synaptophysin was used to investigate the effect of LTG treatment on synaptic loss in the hippocampus of four groups of mice.Results:Long term potentiation (LTP) was induced by high frequency stimulation (HFS; four100Hz and1s trains were delivered20s apart) in the CA1region. Control APP/PS1mice showed a lower LTP as indicated by a reduction in f-EPSP slope, f-EPSP peak amplitude and initial areas compared to WT mice. Compared to control APP/PS1mice, LTG-treated APP/PS1mice exhibited an enhanced LTP, even on a level comparable to WT mice. There were no differences in LTP in between LTG-treated WT and control WT mice. The density of spines exhibited a significant reduction in the cortex and hippocampus of APP/PS1mice compared to that in WT mice. LTG-treated APP/PS1mice, however, showed increased spine density in the cortex and hippocampus compared to control APP/PS1mice. In contrast, LTG-treated and control WT mice exhibit no significant differences in spine density in the cortex and hippocampus. The immunoreactive levels of synaptophysin in the hippocampal CA3subfield, an area enriched with synapses, were analyzed. Control APP/PS1mice exhibited less synaptophysin immunoreactivity in the CA3region than WT mice, consistent with a previous report. In contrast, LTG-treated APP/PS1mice exhibited enhanced synaptophysin immunoreactivity compared to control APP/PS1mice, even in a comparable level to WT mice. No significant differences in synaptophysin immunoreactivity were observed between LTG-treated and control WT mice.Conclusion:Chronic treatment with LTG could alleviate deficits in synaptic plasticity in APP/PS1mice. Chapter IV. The experimental research of the neuroprotective role of lamotrigine in APPswe/PS1dE9miceObjective:To determine the effects of lamotrigine on neuroprotection and the possible mechanism in APP/PS1mice.Methods:APP/PS1transgenic mice and wild-type littermates were randomly assigned into four groups:Lamotrigine-treated (TG+LTG) and Control (TG) APP/PS1transgenic mice; Lamotrigine-treated (WT+LTG) and Control (WT) wild-types. Treated Mice were received a diet of standard laboratory chow supplemented with lamotrigine (30mg/kg/d), Control TG and WT mice received the same chow withiout lamotrigine. The mice were feeded with lamotrigine at3months old and were subjected behavioral analysis and other experiments at8months old. The levels of BDNF, NGF, NT-3in brains of APP/PS1mices were detected by ELISA. Western blotting was used to detect the levels of p-CREB, CREB, p-GSK3β, BACE1, APP, APP-CTFs in the brains of APP/PS1transgenic mices.Results:In APP/PS1mice, levels of BDNF were decreased in the cortex compared to WT mice. Increased levels of BDNF were detected in the cortex of LTG-treated APP/PS1mice compared to that in control APP/PS1mice. In the hippocampus, APP/PS1mice showed a trend of decreased BDNF levels compared to WT mice. The lack of significant statistic difference in BDNF levels in the hippocampus of betweeen APP/PS1and WT mice may due to the fact that neuronal degeneration in the hippocampus occurs later than that in the cortex. LTG-treated APP/PS1mice showed a trend of enhanced levels of BDNF in the hippocampus compared control APP/PS1mice. Enhanced levels of NGF were detected in the hippocampus of LTG-treated APP/PS1mice compared to that in control APP/PS1. In contrast, neurotrophin-3(NT-3), which protects neurons from degeneration, remained unchanged in each group of mice. In APP/PS1mice, levels of p-CREB were decreased in the brain compared to WT mice. Increased levels of p-CREB were detected in the brains of LTG-treated APP/PS1mice compared to that in control APP/PS1mice. The levels of CREB showed no significant differences among the groups. In APP/PS1mice, levels of p-GSK3p, BACE1, APP-CTFs were increased in the brain compared to WT mice. LTG treatment significantly decreased the levels of GSK3P, BACE1and APP-CTFs in brains of APP/PS1transgenic mice.Conclusion:Chronic LTG treatment enhances the levels of BDNF and NGF in the brains of APP/PS1mice; LTG inhibited Aβ generation by suppressing BACE1activity; GSK3β/BAlCE signaling pathway may be involved in regulating APP processing.