Resveratrol Protects Spatial Learning And Memory Function Against The Reduction Of SIRT1Expression And CREB Phosphorylation Induced By Aβ_1-42 In Rats

Alzheimer’s disease （AD） is the most prevalent neurodegenerative disease and most commoncause of dementia without known cure. AD affects approximately10%of the population olderthan65years of age. Its main neuropathological hallmarks include extracellular senile plaques,which consist of a core of β-amyloid peptide （Aβ）, and intracellular neurofibrillary tangle （NFT）formation, as well as the selective loss of neurons and synaptic connections. However, the causesand pathogenesis of AD are complex and unknown at present. The accumulation of oligomericand fibrillar Aβ has been regarded as a key factor in a cascade of harmful events. Currentpharmacotherapies, including several cholinesterase inhibitors and memantine, an NMDAreceptor antagonist, have been approved for use, but neither cholinesterase inhibitors normemantine are effective in mild cognitive impairment. Many novel pharmacological approachesinvolving strategies to reduce amyloid deposition are in progress. Although results of pivotalstudies are awaited, results to date have been equivocal and no disease-modifying agents areeither licensed or can be currently recommended for clinical use. New strategies to slow and/orreverse the pathogenesis of patients with AD are greatly needed.Resveratrol （trans-3,4,5’-Trihydroxystilbene）, as a natural polyphenolic compound, ispresent in more than70different plants, including the skin of grapes and nuts, pomegranates, andis present at high levels in red wine. A broad range of beneficial effects of resveratrol has beendescribed in mammals, including cardioprotective, chemopreventive, anti-inflammatory, as wellas neuroprotective activities. The initial study showed that the red wine intake protected againstAD and attenuated AD type cognitive deterioration and amyloid neuropathology. Numerous invitro and in vivo experiments focusing on it and demonstrating the direct and indirect effects ofresveratrol against Aβ pathology progress. The potential direct mechanisms of resveratrol againstAβ pathology are summarized: inhibit synthesis of Aβ, inhibit fibril formation/aggregation of Aβ,remodels of Aβ and promoting degradation/clearance of Aβ. And the possible indirect effect ofresveratrol against Aβ neurotoxicity includes anti oxidative stress and anti neruoinflammationinduced by Aβ. Recently, more and more evidence suggest another potential protectivemechanism of resveratrol is independent of its antioxidant properties, but as an agonist of thesirtuins （also called SIRT or silent information regulator two proteins）, which belong to the histone deacetylase family. So current researches are focusing on and trying to understanding themechanisms involved in the ability of resveratrol to increase the activity of sirtuin1（SIRT1） andthe intracellular pathways activated or regulated by SIRT1. But the mechanism is not clear.Recently, there exist studies may give us some clues. They showed that SIRT1is essentialfor normal cognitive function and synaptic plasticity. Activation of SIRT1enhances, whereas itsloss-of-function impairs, synaptic plasticity and these effects were mediated viapost-transcriptional regulation of cAMP response binding protein （CREB） expression by abrain-specific microRNA, miR-134. It is well know that phosphorylation at Ser-133is critical forthe transcriptional activity of CREB, and disruption of CREB function specifically interfereswith activity-dependent synaptic plasticity ranging from long term potentiation （LTP） to longterm memory. Interestingly, there was report showed that a reduction of p-CREB levels has beenobserved in the postmortem AD brain. Furthermore, the following study confirmed that neuronalactivity induced CREB phosphorylation is suppressed by sublethal levels of Aβ_1-42. So the role ofAβ in neuron dysfunction in the initiation stage may be mediated through alterations in signaltransduction pathways involving CREB. Another recent research showed a significant reductionof SIRT1（messenger RNA [mRNA],-29%; protein,-45%） in the parietal cortex of AD patients,and that loss of SIRT1is closely associated with the accumulation of Aβ and tau in the cerebralcortex of persons with AD.Taken together, evidences suggest that the accumulation of Aβ may attenuate the SIRT1protein level as well as the phosphorylation of CREB in AD pathology. Whether resveratrol caninterfere the both changes of levels of SIRT1protein and phosphorylation of CREB induced byAβ? Based on these observations, we provide our hypothesis: resveratrol may prevent theimpairment of learning and memory function induced by Aβ_1-42against the reduction of proteinlevels of SIRT1and the phosphorylation of CREB. To tested this hypothesis, we use thehippocampal injection of Aβ_1-42to rats and investigate the changes of spatial memory, LTP, theprotein level of SIRT1and phosphorylated CREB by pretreated with resveratrol.1. Part I Effects of resveratrol on learning and memory impairment induced by Aβ_1-42inrats（1） OBJECTIVEβ-amyloid （Aβ） deposition, as a key factor in a cascade of harmful events, is considered to bepartly responsible for the impairment of learning and memory in Alzheimer’s disease （AD）.Reports have showed that resveratrol can attenuated the impairment of spatial memory inTg2576mice and p25transgenic mouse （two type of AD model）, and these result are alsoconfirmed in the cognitive impairment rat induced by Aβ25-35（i.c.v）. To investigate resveratrol effect on learning and memory function with the present of Aβ_1-42, we will use the bilateralhippocampus injection of Aβ_1-42or/and resveratrol in SD rats, and test the reference memorywith a Morris water maze task that is known to require hippocampal function.（2） MATERIALS AND METHODSAdult male Sprague-Dawley （SD） rats （weighting range220to240g） were assigned randomlyinto8groups （n=9） and treated differently:1. Vehicle control group;2. Aβ_1-42（0.22nM/1μl） group;3. Resveratrol （0.05nM/1μl,0.22nM/1μl,0.44nM/1μl） group;4. Resveratrol （0.05nM/1μl）+Aβ_1-42（0.22nM/1μl） group;5. Resveratrol （0.22nM/1μl）+Aβ_1-42（0.22nM/1μl） group;6. Resveratrol （0.44nM/1μl）+Aβ_1-42（0.22nM/1μl） group.Eight days after the hippocampus injection surgery, Morris water maze test were performed.（3） RESULTS1) The bilateral hippocampus injection of Aβ_1-42impaired the learning and memory behavior ofrats in Morris water maze testIn the acquisition training session, the Aβ_1-42-treated group exhibited longer escape latencies （thetime taken to find the platform） throughout training trial sessions than did the vehicle-treatedcontrols （p<0.05）. In the probe trail, vehicle-treated group rats spent more time in the targetquadrant （TQ）, where the platform had been located during training, than in the other quadrants.But the swimming times in the TQ for the Aβ_1-42-treated group were significantly lower thanthose of the vehicle-treated control group （p<0.05）.2) Resveratrol treatment only did not affect the learning and memory functionResveratrol （0.05nM,0.22nM or0.44nM） treated only, there were no significant affects inescape latencies during the acquisition test compared with the vehicle-treated controls （p>0.05）.And in the probe trail, no difference of the time in the TQ was detected among thevehicle-treated control group,0.05nM,0.22nM and0.44nM resveratrol-treated group （p>0.05）.3) Resveratrol pretreatment inhibited the impairment of learning and memory induced byAβ_1-42in ratsResveratrol （0.05nM and0.22nM） pretreated significantly attenuated the effects of Aβ_1-42onescape latency （p<0.05）, while0.44nM resveratrol pretreated didn’t show the effect （p>0.05）. Inthe probe trail, the pretreatment of resveratrol （0.05nM and0.22nM） improved the performanceof rat which search in the TQ more than the Aβ_1-42-treated group （p<0.05）. Resveratrol pretreated group rats remembered where the platform was the previous days and spent approximately thesame time as vehicle-treated rats （p>0.05）.4) Both resveratrol and Aβ_1-42did not affect the vision and the swimming speed of ratsA visible platform trial performed after the probe trials did not reveal any difference in the timeto the platform among all groups （p>0.05）. In addition, no alterations of the swimming speed inthe5days of successive learning acquisition among all groups （p>0.05）, indicating no motor orvisual deficits in these animals.（4） SUMMARY1) The bilateral hippocampus injection of Aβ_1-42impairs the learning and memory in rats;2) Resveratrol prevents learning and memory impairment induced by Aβ_1-42in rats;3) The inhibition effect of resveratrol on learning and memory impairment induced by Aβ_1-42by a concentration independent way.2. Part II Effects of resveratrol on hippocampal LTP impairment in vivo induced byAβ_1-42in rats（1） OBJECTIVEAcute exposure to synthetic or naturally secreted Aβ has been shown to impair hippocampallong-term potentiation （LTP） in slices, a widely accepted cellular model of learning and memory.We test weather the hippocampal injection of Aβ_1-42affects the basic synaptic transmission andHFS-induced LTP. Furthermore we will investigate whether the resveratrol can inhibit theattenuation of LTP induced by Aβ_1-42in rats.（2） MATERIALS AND METHODSTo test for the electrophysiology （in vivo LTP） effects of Aβ_1-42and resveratrol on animals, adultmale Sprague-Dawley （SD） rats （weighting range220to240g） were assigned randomly into4groups （n=6for each group） and treated differently:1. Vehicle control group;2. Aβ_1-42（0.22nM/1μl） group;3. Resveratrol （0.22nM/1μl） group;4. Resveratrol （0.22nM/1μl）+Aβ_1-42（0.22nM/1μl） group.（3） RESULTS1) Hippocampal injection of Aβ_1-42significantly suppressed HFS-induced LTP in CA1regionFirst we wanted to confirm that the hippocampal injection of Aβ_1-42inhibits HFS-induced LTP in vivo. We monitored the fEPSPs for30min after hippocampus injection of Aβ_1-42（0.22nM）, andthe synaptic responses were further recorded for1h after HFS delivery. We found thathippocampus injection of Aβ_1-42had no discernible effect on baseline synaptic transmission.However, Aβ_1-42significantly suppressed the induction of hippocampal LTP （Aβ:121.5±2.73%,vehicle:149.1±2.08%, p <0.05）.2) Resveratrol alone did not affect the baseline synaptic transmission and the LTPTo examine whether resveratrol pretreatment can protect against Aβ_1-42-induced LTP impairment,the effects of resveratrol alone on baseline synaptic transmission and HFS-induced LTP werefirst observed. Hippocampus administration of0.22nM resveratrol30-min prior to HFS had nosignificant effect on baseline synaptic transmission and HFS-induced LTP compared with invehicle-treated control group （resveratrol:141.1±2.44%, vehicle:149.1±2.08%, p>0.05）.3) Pretreatment with Resveratrol protected against Aβ_1-42induced impairment of LTPWe next examined the effects of coadministration of resveratrol with Aβ_1-42on the baselinesynaptic activity and HFS-induced LTP. The baseline synaptic transmission was not altered.However, with the pretreatment of0.22nM resveratrol, a significant inhibition of theimpairment of LTP induced by Aβ_1-42was found （Aβ:121.5±2.73%, resveratrol plus Aβ:137.3±3.42%, p <0.05）.4) PPF is not affected by Aβ_1-42and ResveratrolNormal PPF was recorded in vivo from all drugs-treated group rats, indicating that the vehicle,Aβ_1-42and the resveratrol injection to the hippocampus does not cause a significance alternatedeffect on basal synaptic transmission or presynaptic events at Schaffer collateral CA1synapses.（4） SUMMARY1) Hippocampus injection of Aβ_1-42significantly suppressed HFS-induced LTP in CA1region;2) Resveratrol pretreatment inhibits the attenuation of LTP induced by hippocampus injectionof Aβ_1-42.3. Part III Effects of resveratrol on the reduction of hippocampal levels of SIRT1proteinand phosphorylation of CREB induced by Aβ_1-42in rats（1） OBJECTIVESIRT1is essential for normal cognitive function and synaptic plasticity, and modulates synapticplasticity and memory formation via a microRNA-mediated mechanism. And effects weremediated via post-transcriptional regulation of CREB expression by a brain-specific microRNA, miR-134. Recently, report showed a significant reduction of SIRT1in the parietal cortex of ADpatients, and that loss of SIRT1is closely associated with the accumulation of Aβ in the cerebralcortex of persons with AD. In addition, another report also showed a⁴0%decrease in pCREBlevels at baseline in the hippocampi of3xTg-AD mice （a widely used animal model thatdevelops Aβ and tau accumulation associated with cognitive dysfunction） compared with theNonTg mice. In the following experiment, we investigated the protein levels of SIRT1and thephosphorylated CREB in hippocampus of rat. First we wanted to confirm that the hippocampalinjection of Aβ_1-42inhibits both the SIRT1expression and the phosphorylation of CREB. Then,we will investigated the effects of resveratrol on the reduction of hippocampus levels of SIRT1protein and phosphorylation of CREB induced byAβ_1-42in rats.（2） MATERIALS AND METHODSThe rats, after the behavioral test （Part I）, from each group were decapitated under anesthesia,and their brains were rapidly removed on ice and washed with ice-cold normal saline.Hippocampus were dissected out, immediately snap-frozen in liquid nitrogen, and stored at-80°C for western blotting analyzing.（3） RESULTS1) Effects of resveratrol on the reduction of SIRT1induced by Aβ_1-42in hippocampal of ratsA significance decline in the hippocampal SIRT1protein levels were detected in Aβ_1-42-treatedrat compared with the vehicle-treated group, while the decline were reversed by pre-treatedresveratrol. However, no significance alternations of the hippocampus SIRT1protein levels wereobserved in the resveratrol-treated only group.2) Effects of resveratrol on the reduction of CREB phosphorylation induced by Aβ_1-42inhippocampal of ratsA significance decline in the hippocampal CREB phosphorylation were detected in Aβ_1-42-treatedrat compared with the vehicle-treated group, while the decline were reversed by pre-treatedresveratrol. However, no significance alternations of the hippocampus CREB phosphorylationwere observed in the resveratrol-treated only group. And there were no dose-dependent effects ofresveratrol inhibiting the reduction of hippocampal CREB phosphorylation induced by Aβ_1-42inrats.（4） SUMMARY1) Resveratrol prevents the reduction of SIRT1induced by Aβ_1-42in rats;2) Resveratrol prevents the reduction of CREB phosphorylation induced by Aβ_1-42in rats. 4. Conclusions（1） The hippocampal injection of Aβ_1-42can change the behavior in Morris maze test andattenuate the hippocampal LTP, impairing the learning and memory function in rats;（2） Resveratrol can inhibit the changes of behavior in Morris maze test and the attenuation ofhippocampal LTP induced by Aβ_1-42, reversing the impairment of learning and memoryfunction in rats;（3） Resveratrol can reverse the reduction of SIRT1expression and CREB phosphorylationinduced by Aβ_1-42, which maybe involve in the neuroprotection effect of resveratrol againstAβ_1-42.