Effects Of Simvastatin On Cognitive Function And Its Molecular Mechanism

Statins are inhibitors of 3-hydroxy-3-methyl-glytarylcoenzyme A(HMG-CoA)reductase,which are now widely used to treat atherosclerosis.Epidemiological evidence from various populations has showed that statin users have low incidence of Alzheimer’s disease(AD).Simvastatin(SV)has been reported to improve cognitive functions in aging normal mice and affect the long-term potentiation.However,the mechanisms underlying SV-regulated spatial memory remain largely unknown.Long-term potentiation(LTP)in hippocampal CA1 region is a cellular model of learning and memory and cognitive processes.LTP induction is dependent on the influx of calcium.NMD A receptor(NMDAR)and a7nAChR are the Ca2+ channel receptors in hippocampus neurons.Recent study has reported that the calcium influx of NMDAR and a7nAChR has a great role in regulating the LTP induction.In adition,a7nAChR-related Akt and ERK1/2 signaling pathways also can affect the LTP induction.Statins not only reduce de novo cholesterol biosynthesis but also prevent the conversion of HMG-CoA to mevalonate,resulting in a reduction of nonsterol intermediates,known as isoprenoids.Isoprenoids,such as farnesyl-pyrophosphate(FPP)and geranylgeranyl-pyrophosphate(GGPP),serve as lipid attachments for the small GTPase superfamilies to regulate the membrane localization of Ras,Rho,and Rab proteins.Recently,it has been reported that the activity of NMDAR,the trafficking and function of a7nAChR are increased in the hippocampus of H-Ras null mice.We examed if statins through reducing the isoprenoids affect the activity or expression of NMDAR and a7nAChR,then induce hippocampal LTP-augmentation or LTP-facilitation,which regulate the spatial cognitive potentiation.In this way,we focused on the effect of SV on the synaptic properties and plasticity,and regarded the NMDAR and a7nAChR as molecular target to explain the molecular mechanism underlying SV-enhanced spatial memory.Objective1.Make sure the influence of SV on spatial memory and synaptic plasticity;2.Clarify the molecular mechanism underlying SV-regulated NMDAR activity;3.Clarify the molecular mechanism underlying SV-regulated a7nAChR activity.Materials and Methods1.Administration of SV solution:SV(Enzo Life Sciences International)was activated by alkaline lysis.In this study,SV at dose of 20 mg/kg(200 μ1)was intragastrically administered daily to adult ICR male mice for 30 days(30d-SV-mice)(Part Ⅰ).SV at dose of 20 mg/kg was intraperitoneal inject(i.p.)to postnatal 25-30 days ICR male mice for 5 days(5d-SV-mice)(Part Ⅱ).For in vitro experiment,the SV stock solution was neutralized with 1 N HC1 to pH of7.4 and diluted in artificial cerebral spinal fluid(ACSF),then incubated the slices of mice at the concentration of 10μM for hours(SV-slices).2.Morris water maze task(hidden platform task and probe trial)and Y maze were performed to test the hippocampus-dependent ability of spatial memory.3.Field potential recording were used to exam the basal property(Input/output(I/O)curve,Paired-pulse facilitation(PPF))and LTP of hippocampal Schaffer collateral-CA1 synapses.4.Whole cell patch-clamp recording were performed to record the current of NMD A,AMPAR anda7nAChR.5.Western blot were used to exam the protein levels of GluN2A.GluN2B,and phosphorylation protein levels of a7nAChR、GluN2A、GluN2B、Src、PKC、PKA、CaMKII、Akt、Erk.6.Real-time RT-PCR was performed to analysis the expression of genes of GluN2A、GluN2B.7.Slices biotinylation and western blot to test the cell surface trafficking of a7nAChR.8.Immunoprecipitation to exam the phosphorylation of a7nAChR in the thr and ser site.9.Chromatin immunoprecipitation(ChIP)assay kit was used to exam the acetylation status in the promoter regions of GluN2B gene.ResultsPart Ⅰ:Simvastatin Enhances Spatial Memory and Long-Term Potentiation in Hippocampal CA1 through the reducing FPP.1.In comparison with control mice,SV mice spent less time to reach the hidden platform on days 5-6 after training,and SV mice stayed a longer time in platform quadrant,the alternation ratio in SV mice was significantly increased.2.The EPSP slopes were significantly increased in SV mice compared to controls,the values of PPR in SV mice were lower than those in control mice,which means the SV administration is likely to enhance the presynaptic glutamate release.3.The same protocol induced increase of EPSP slopes in SV mice,termed LTP-augmentation,which was NMDA dependent.The sub-threshold HFS could produce a stable LTP in SV mice,termed LTP-facilitation,which was NMD A independent.4.In comparison with control mice,the phosphorylation level of ERK and Akt were increased in 30d-SV-mice.5.The LTP-augmentation and LTP-facilitation in 30d-SV-mice were associated with the increasement of a7nAChR activity and ERK pathway,the LTP-facilitation was associated with the enhancement of Akt pathway,which could be abolished by FPP.6.The supplement of FPP by applying FOH in SV mice could block the SV-enhanced spatial memory,glutamate release and LTP.Part Ⅱ:Simvastatin enhances NMDA receptor GluN2B expression and phosphorylation of GluN2B and GluN2A through reducing FPP.1.In comparison with control mice,both 5d-SV-mice and SV-slices had an increasement in the density of NMDA-evoked inward currents,which could be blocked by the supplement of FPP.FTI could mimic the enhancing this effect.2.In comparison with control mice,the phosphorylation of GluN2B,GluN2A and a7nAChR was increased in 5d-SV-mice and SV-slices,which could be blocked by the supplement of FPP.FTI could mimic the enhancing this effect.3.The levels of GluN2B protein and mRNA were increased in 5d-SV-mice,and could be blocked by FPP,FTI could enhance the expression of GluN2B.SV incubated for 2 hours had no effect on the expression of GluN2B/2A.4.SV and FTI enhanced NMDA currents and phosphorylation of GluN2B and GluN2A were effected by Src signaling,and the upregulated expreesion of GluN2B.5.SV treatment for 5 days could increase the acetylation of histones H3K9 and H3K27 in GluN2B,which could be blocked by the supplement of FPP.FTI could mimic the enhancing this effect.Part Ⅲ:Simvastatin enhances activity and trafficking of α7 nicotinic acetylcholine receptor in hippocampal neurons through reducing FPP.1.The treatment of hippocampal slices with SV induced a time-and dose-dependent increase in the amplitude of ACh-evoked inward currents(IACh),without changes in the agonist sensitivity and the kinetics of desensitization.2.The treatment of hippocampal slices with SV could dose-dependent enhance the level of a7nAChR protein on the cell membrane without changes in the level of total α7nAChR.3.In comparison with control mice,SV incubation induced increases of ACh-evoked inward currents and the trafficking to membrane,which could be abolished by FPP.FTI could mimic this increasement.4.In comparison with control mice,the treatment with SV enhanced phosphorylation of PKC,which could not be changed by FPP.The SV-enhanced phosphorylation level of CaMKII could be abolish by FPP,which was NMDAR dependent but not PKC dependent.SV had no influence of PKA pathway,FTI could mimic the enhancement of phosphorylation of CaMKII.5.SV-and FTI-increased a7nAChR currents were dependent on the activation of PKC and CaMKII,while the enhancing effect of FTI on a7nAChR activity is weaker than that of SV.SV-and FTI-increased a7nAChR trafficking are CaMKII-dependent,SV had no effcet on the phosphorylation of a7nAChR.SummarySV enhances NMDA receptor GluN2B expression and phosphorylation of GluN2B and GluN2A through increased histone acetylation and Src signaling in hippocampal CA1 neurons.SV enhances activity and trafficking of a7 nicotinic acetylcholine receptor in hippocampal neurons through PKC and CaMKII signaling pathways.SV through reducing FPP increases a7nAChR-related Akt and ERK2 phosphorylation,leading to LTP enhancement and spatial cognitive potentiation.Conclusion and clinical significance:The results in the present study indicate that statins through reducing FPP affect the activity or expression of NMDAR and a7nAChR,then induce hippocampal LTP-augmentation or LTP-facilitation,which regulate the spatial cognitive potentiation.it is very important for providing an effective and feasible strategy in treatment of Alzheimer’s diseas...