Phosphodiesterase 4 Inhibitor Chlorbipram Improves Animal Memory Deficits And The Mechanisms Involved

OBJECTIVE The molecular pathogenesis of Alzheimer's disease?AD?involves aggregation of amyloid-? into amyloid plaques,hyperphosphorylation of tau protein into neurofibrillary tangles,and neuronal loss.The clinical manifestation of AD includes memory loss and progressive cognitive disorder.This research used microinjection of AP25-35 into rat hippocampus to establish Alzheimer's disease model and APP/PS1 transgenic mice model,simulating Alzheimer's disease pathology,to evaluate the memory improvement of a new phosphodiesterase 4?PDE4?inhibitor,chlorbipram,and study the mechanisms of underlying the learning and memory.Microinjection of A?25-35 35 into rat hippocampus can lead to learning and memory decline,thus becoming a widely used animal model of AD.APPswe/PS1dE1 transgenic mice overexpress mutative amyloid precursor protein?amyloid protein precursor,APP?and y-secretase,resulting in A? excessive accumulation and therefore can simulate AD main pathological changes,such as senile plaque deposition,nerve neurofibrillary tangles.In a word,it is the ideal model for studying the pathogenesis of AD and drug screening.PDE4 specifically catalyzes the hydrolysis of the second messenger cyclic adenosine monophosphate?cAMP?,and interacts with adenylate cyclase?AC?,which plays an opposite effect to catalytic synthesis of cAMP,to maintain the balance of intracellular cAMP concentration.cAMP is an ubiquitous second messenger which mediates biological responses to a variety of extracellular signals,including numerous neurotransmitters and therefore has important physiological and pathological significance.Numerous studies have shown that a modest increase of intracellular cAMP levels can improve the learning and memory function.Classic PDE4 inhibitors rolipram has been shown to significantly improve memory,but serious adverse reactions including nausea and vomiting have hindered its application in clinical practice.To overcome the traditional PDE4 inhibitor-induced nausea and vomiting adverse reactions,we designed and synthesized chlorbipram by studying the structure and the catalytic activity of the enzyme PDE4.Our early studies using a method of standard PDE assay to determine its IC50 for PDE enzyme showed that chlorbipram exhibited favorable selectivity for PDE4 and a slightly higher inhibitory activity to the catalytic domain than rolipram.Preliminary research found that chlorbipram had a very low potential to cause vomiting,compared with rolipram.This paper aims to explore the effects of chlorbipram,the new PDE4 inhibitor,on enhancement of learning and memory and its underlying mechanisms,utilizing the model of memory deficits in AD rats and APP/PS1 transgenic mice.METHODS 1.The rat model of learning and memory deficits with AD was used by bilateral microinjection of A?25₃5 into the CA1 region of the hippocampus.Then the rats were randomly divided into six groups:sham-operated group,A?25-35 microinjected?model group?,chlorbipram-treated?0.05 and 0.15 mg·kg-1?group,rolipram-treated?0.05mg·kg-1?and donepezil?1.0mg·kg-1?group.The effects of chlorbipram on memory behavioral performance were evaluated with Morris water maze and step-through passive avoidance test,and the open field test was performed to determine the animal locomotor activity.After the last behavioral performance test,the hippocampus was dissected for further molecular analysis.The protein level of BDNF and the phosphorylation of PKA and CREB were analyzed by western blotting;and the mRNA level of BDNF in the hippocampus was detected by real-time PCR.2.Mice whose genotype was identified using the method of agarose gel electrophoresis of PCR.And then were divided into two groups:wide type and transgenic according to their genotype.Total 48 mice,composed of 40 APP/PS1 transgenic mice and 8 wide type mice,were grouped.40 APP/PS1 transgenic mice were randomly divided into 5 groups:model group,chlorbipram-treated?0.10?0.20?0.40 mg·kg-1?group and rolipram-treated?0.05mg·kg-1?group.The remaining 8 wide type mice were acted as negative control.The effect of chlorbipram on memory behavioral performance was evaluated with Morris water maze,and the open field test was performed to determine the animal locomotor activity.After the last behavioral performance test,the hippocampus was dissected for further molecular analysis.The protein level of BDNF and the phosphorylation of PKA and CREB were analyzed by western blotting;the mRNA level of BDNF in the hippocampus was detected by real-time PCR.RESULTS 1.Microinfusion with A?25-35 produced impairment of spatial memory in behavioral tests,which was reversed by either PDE4 inhibitor or donepezil administration.Chlorbipram,rolipram and donepezil increased the number of crossing and percent of time in the target quadrant in Morris Water Maze probe trial.In step-through passive avoidance test,the 24h latency was significantly decreased in rats treated with either chlorbipram or positive control drugs,while no significant differences were observed in total locomotor activity among groups.Western blot analyses showed that Ap25-35-microinjection decreased the phosphorylation of PKA and CREB and inhibited the protein expression of BDNF in the hippocampus.Chlorbipram,rolipram and donepezil reversed the reduction of the phosphorylated PKA and CREB induced by A?25-35.Moreover,these drugs also enhanced both the mRNA and protein levels of BDNF.2.The 6-month-old APP/PS1 mice exhibited impairment of spatial memory in behavioral tests,which was reversed by either chlorbipram or rolipram administration.Chlorbipram and rolipram increased the number of crossing and percent of time in the target quadrant in the Morris Water Maze probe trial,while no significant differences were observed in total locomotor activity among the groups in the open field test.Western blot analyses showed that the 6-month-old APP/PS1 mice were characterized by a decrease of the phosphorylation of PKA,CREB and protein level of BDNF in the hippocampus.Chlorbipram and rolipram reversed the reduction of the phosphorylated PKA and CREB.Moreover,these drugs also enhanced both the mRNA and protein levels of BDNF.CONCLUSION Chlorbipram produces a significant improvement of learning and memory in AD rats and APP/PS1 transgenic mice,and this effect may be due to chlorbipram activates cAMP/PKA/CREB signal pathway by increasing intracellular cAMP levels,then promotes the transcription and translation of BDNF.