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. 2013 Dec 2;20(3):218–226. doi: 10.1111/cns.12190

Simvastatin Exerts Antiamnesic Effect in Aβ25‐35‐Injected Mice

Wen‐Hong Zhi 1,2, Yan‐Ying Zeng 2, Zi‐Hong Lu 1, Wei‐Jun Qu 1, Wei‐Xian Chen 2, Lei Chen 1,✉, Ling Chen 1
PMCID: PMC6493162  PMID: 24289538

Summary

Aim and Methods

Simvastatin (SV) is reported to improve cognition and slow the progression of Alzheimer's disease (AD). This study explored the mechanisms underlying the antiamnesic effect of SV in AD using behavior tests, histological examination, western blot analysis, and electrophysiological recording technique in AD model mice created by intracerebroventricular injection (i.c.v.) of Aβ25‐35.

Results

Chronic administration of SV (40 mg/kg/day) for 11 days after Aβ25‐35‐injection ameliorated the impairment of acquisition performance and probe trail test in Morris water maze task and alternation behavior in Y maze task in Aβ25‐35‐mice. Aβ25‐35‐induced apoptosis of hippocampal CA1 pyramidal cells and Aβ25‐35‐impaired high‐frequency stimulation (HFS)‐dependent long‐term potentiation (LTP) induction in hippocampal Schaffer collaterale‐CA1 synapse were rescued by SV‐treatment. SV prevented Aβ25‐35‐inhibited protein kinase B (Akt) and extracellular signal‐related kinase‐2 (ERK2) phosphorylation, which was sensitive to α7 nicotinic acetylcholine receptor (α7nAChR) antagonist MLA. SV‐induced neuroprotection was attenuated by MLA or phosphatidylinositol‐3‐kinase (PI3K) antagonist LY294002. SV‐rescued LTP induction was blocked by α7nAChR, PI3K or MAPK/ERK kinase (MEK) antagonist. Finally, the antiamnesia of SV in Aβ25‐35‐mice was attenuated by blockage of SV‐induced neuroprotection or SV‐rescued LTP induction.

Conclusion

The antiamnesia of SV in Aβ25‐35‐mice depends on its neuroprotection and synaptic plasticity improvement.

Keywords: Neuroprotection, Simvastatin, Synaptic plasticity, α7 nicotinic acetylcholine receptor, β‐amyloid peptide

Introduction

Alzheimer's disease (AD) is a neurodegenerative disorder and a common cause of dementia in the elderly. The main neuropathological characteristics in AD include deposition of β‐amyloid peptide (Aβ), plaque formation, neurofibrillary tangles and impairment of cholinergic system 1. Besides this, a cerebrovascular pathology is also a clinical landmark of the disease 2. AD patients display reduced cerebral blood flow at rest and lessened perfusion to activated areas. Statins, a direct inhibitor of 3‐hy‐droxy‐3‐methylglutaryl CoA (HMG‐CoA) reductase, are widely used for the treatment of hypercholesterolemia 3. Epidemiological evidence shows a good relevance between statins therapy and a low incidence of AD, particularly in patients with cerebrovascular disease 4. Clinical trials have proved that simvastatin (SV) can improve cognitive function of mild to moderate AD and slow the progression of AD 5, 6. Tong et al. report that SV, if initiated early in the disease process, is fully effective against both vascular and memory deficits of adult but not aged AD transgenic mice 7. The antiamnesia of SV is independent of cholesterol‐lowering effect or changes in Aβ levels or plaque load 4, 7, 8, and the underlying mechanism still remains unclear.

The α7 nicotinic acetylcholine receptors (α7nAChRs) are expressed in hippocampus where they regulate neurotransmitter release, activate intracellular signaling pathways, and influence downstream processes involved in learning and memory 9. It has been proved that α7nAChR may be one of the most likely targets of Aβ‐neurotoxicity 9, 10. Lovastatin can up‐regulate α7nAChR in cultured neurons, which is independent of changes in cholesterol levels 11. Furthermore, blockade of α7nAChR by cholinesterase inhibitors (ChEIs) is prevented by application of mevastatin and lovastatin 12. These reports indicate that statins may modulate α7nAChR at the receptor level.

Aβ containing the 11 amino acids (25–35) (Aβ25‐35) retains the ability to self‐aggregate and has been proved to be neurotoxic in vitro and in vivo 13, 14. An animal model of AD‐type amnesia created by intracerebroventricular injection (i.c.v.) of Aβ25‐35 has been widely used to analyze the behavioral and neuropathological consequences of Aβ‐neurotoxicity in vivo 10, 14, 15, 16. Previous studies show that injection (i.c.v.) of Aβ25‐35 in mice causes impairment in learning and memory ability and neuronal loss within 1 or 2 weeks after administration, which can be reversed by cholinergic agonist 10, 14, 15. Here, we studied the effects of a commonly used statin drug, simvastatin which can easily pass through blood–brain barrier 17, on Aβ25‐35‐induced deficits in spatial memory behavioral, hippocampal morphological, electrophysiological, and biochemical changes, and the role of α7nAChR in SV‐action. The study demonstrated that the administration of SV in Aβ25‐35‐mice exerted a potent antiamnesic effect, which was mediated through SV‐induced neuroprotection and SV‐improved synaptic plasticity. The results also showed that α7nAChR‐mediated phosphatidylinositol‐3‐kinase (PI3K)‐protein kinase B (Akt) and extracellular signal‐related kinase (ERK) signaling pathways were involved in the beneficial effect of SV in AD.

Materials and methods

Experimental Animals

Male mice (ICR, Oirental Bio Service Inc., Nanjing, China), weighing 20–25 g at the beginning of the experiment, were used in the study. The animals were housed in a light controlled room under a 12‐h light–dark cycle starting at 7:00 AM and kept at a temperature of 23°C. They received food and water ad libitum. Experiments were approved by the Nanjing Medical University Animal Care and Use Committee. Care of animals conformed to standards established by the National Institutes of Health and all efforts were made to minimize animal suffering and to reduce the number of animals used.

Preparation of an Animal Model of Alzheimer's Disease

“Aggregated” Aβ25‐35 (Sigma Chemical Co., St. Louis, MO, USA) was obtained by incubating at 37°C for 4 days and then diluted to the final concentration with saline just before the experiment. Alzheimer's disease mouse model was prepared by injection (i.c.v.) of “aggregated” Aβ25‐35 as previously report 14. After mice were anesthetized with 2% chloral hydrate (20 mL/kg), they were placed in a sterotactic device (Kopf Instruments, Tujunga, CA, USA). A 26‐gauge single guide cannula (Plastics One, Roanoke, VA, USA) was implanted into the right lateral ventricle (0.3 mm posterior 1.0 mm lateral, and 2.5 mm ventral to bregma). After surgery, a 28‐gauge dummy cannula (Plastics One) was inserted into each guide cannula. Aβ25‐35 (9 nmol/3 μL/mouse) was injected with a stepper‐motorized micro‐syringe (Stoelting, Wood Dale, IL, USA) at a rate of 0.5 μL/min. Control mice were given an equal volume of vehicle. The injection site was confirmed in preliminary experiments. Neither insertion of the needle nor injection of the saline had a significant influence on survival, and behavioral responses or cognitive functions.

Drug Administration

Simvastatin tablets (Zocor, Merck Sharp & Dohme Limited, Hoddesdon, Herts, UK) were consecutively intragastrically administered for 11 days. SV was once daily administered at a dose of 40 mg/kg/day and was firstly administered at 24 h after Aβ25‐35‐injection. α7nAChR antagonist methyl‐lycaconitine (MLA) (0.1 nmol/mouse, i.c.v.), MAPK/ERK kinase (MEK) inhibitor U0126 (0.3 nmol/mouse, i.c.v.) and PI3K inhibitor LY294002 (0.3 nmol/mouse, i.c.v.) were dissolved in DMSO, and then in 0.9% saline with a final concentration of 1% DMSO. These antagonists were injected 30 min before SV‐administration. Control mice were given an equal volume of vehicle.

Concerning the difference in physiologic and metabolic processes between human and rodent, the dosage of SV used here is higher than that taken by human subjects if the dosage is expressed per unit of body weight 18. This issue also exists in previous studies reporting the neuroprotection or antiamnesic effect of SV against Aβ‐toxicity and the concentrations of SV used in animal studies are different from the dosage used when treating human patients 7, 8. In this study, 40 mg/kg/day SV was used because this dosage has been used in other studies examining systemic or central effects of SV in mice and chronic administration of SV at this dosage improves memory in adult AD transgenic mice 7, 19, 20. Other concentrations of antagonists were chosen as previously reported 15.

Behavior Analysis

Morris water maze task was performed on day 5 to day 9 after Aβ25‐35‐injection. In water maze task, a black plastic pool (diameter = 120 cm) was prepared, with water temperature maintained at 20 ± 1°C. Swimming paths were monitored with a video camera (AXIS‐90 Target/2; Neuroscience). Mice were given 90 seconds to reach the hidden platform which was put 1 cm below the water surface. If the mouse did not find the platform within 90 seconds, the trial was terminated and the mouse was put on it for another 30 seconds. Starting position was chosen pseudorandomly and each mouse was trained with three trials per day. All parameters were recorded and analyzed where appropriate. The probe trial was performed on day 10 after Aβ25‐35‐injection in which the platform was removed from the maze, and the percent time spent in each quadrant was assessed. Each group contained eight mice.

Y maze task was performed on day 8 after Aβ25‐35‐injection in which the maze was made of black painted wood. Each arm was 40 cm long, 13 cm high, 3 cm wide at the bottom, 10 cm wide at the top, and converged at an equal angle. Each mouse was placed at the end of one arm and allowed to move freely through the maze during an 8‐min session. The series of arm entries were recorded visually and arm entry was considered to be completed when the hind paws of the mouse were completely placed in the arm. Alternation was defined as successive entries into the three arms on overlapping triplet sets. The percentage alternation was calculated as the ratio of actual to possible alternations (defined as the total number of arm entries minus two), multiplied by 100. Each group contained eight mice.

Histological Examination

Histological examination of hippocampal CA1 region was assessed on day 11 after Aβ25‐35‐injection. After performing Morris water maze task, mice were anesthetized and perfused with ice‐cold phosphate‐buffered saline (PBS) followed by 4% para‐formaldehyde. Brains were removed and immersed in fixative (4°C overnight), and then processed for paraffin embedding. Coronal sections (5 μm) were cut from the level of hippocampus for toluidine blue and Hoechst staining. In toluidine blue staining, the pyramidal cells in hippocampal CA1 region were identified using a conventional light microscope (DP70, Olympus Optical Co., LTD, Tokyo, Japan) with a 40 × objective. In Hoechst staining, the slices were stained with Hoechst‐33342 and Hoechst‐positive cells were counted using a fluoresce‐microscope (Olympus PD70) with a 40 × objective. Density of surviving neurons or Hoechst‐positive cells (die cells) was counted in six sections per mouse and expressed as the number of cells per one mm length along the hippocampal CA1 pyramidal layer 21.

Western Blot Analysis

Western blot analysis was performed on day 5 after Aβ25‐35‐injection. After mice were decapitated, the hippocampus was taken quickly and then was homogenized in a lysis buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadate, 1% Triton X‐100, 0.5% sodium deoxycholate, 1 mM phenyl‐methylsulfonyl fluoride and protease inhibitor cocktail (Complete; Roche, Mannheim, Germany). Protein concentration was determined with BCA Protein Assay Kit (Pierce, Rochford, IL, USA). Total proteins (20–40 mg) were separated using SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to a polyphorylated difluoride (PVDF) membrane. The membranes were incubated with 5% bovine serum albumin or 5% nonfat dried milk in Tris‐buffered saline containing 0.1% Tween 20 (TBST) for 60 min at room temperature, and then were incubated with a mouse monoclonal antiphosphor Akt antibody (1:1000, Cell Signaling), a mouse monoclonal antiphosphor ERK1/2 antibody (1:1000, Cell Signaling) at 4°C overnight. After being washed with TBST for three times, the membranes were incubated with an HRP‐labeled secondary antibody, and developed using the ECL detection Kit (Amersham Biosciences, Piscataway, NJ, USA). Following visualization, the blots were stripped by incubation in stripping buffer (Restore, Pierce Chemical Co, Rockford, IL,USA) for 5 min, reblocked for 60 min with 5% nonfat dried milk at room temperature, then incubated with antitotal ERK1/2 (1:1000, Cell Signaling), Akt (1:1000, Cell Signaling). Western blot bands were scanned and analyzed with the image analysis software package, National Institutes of Health Image. Hippocampal samples were collected from the hemisphere of eight mice as a set of western blot analysis.

Electrophysiological Analysis

Electrophysiological recording was performed on day 9 and day 10 after Aβ25‐35‐injection. After performing Y maze task, mice were decapitated and the brains were rapidly removed and the coronal brain slices (400 μm) were cut using a vibrating microtome (Microslicer DTK 1500, Dousaka EM Co, Kyoto, Japan) in ice‐cold oxygenated (95% O2/5% CO2) modified artificial cerebrospinal fluid (mACSF) composed of (in mM) NaCl 126, CaCl2 1, KCl 2.5, MgCl2 1, NaHCO3 26, KH2PO4 1.25, and D‐glucose 20. After one hour recovery, hippocampal slices were transferred to a recording chamber.

For recording field excitatory postsynaptic potential (fEPSP), slices were perfused continually with the oxygenated mACSF at the temperature of 30 ± 1°C. A glass microelectrode with the resistance of 4–5 MΩ filled with 2 M NaCl was inserted into the stratum radiatum region of CA1 area. fEPSP was generated by stimulating the Schaffer collateral/commissural pathway using a stimulator (SEN‐3301, Nihon Kohden, Tokyto, Japan). Stimulus pulses (0.1 ms duration) were delivered every 15 seconds. Signals were obtained using an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA, USA), sampled at 20 kHz and filtered at 10 kHz, and the output was digitized with a Digidata 1200 converter (Axon Instruments). The basal synaptic transmission was recorded using the stimulation intensity yielding the half‐maximal fEPSP slope for a given slice. Long‐term potentiation (LTP) was induced by high‐frequency stimulus (HFS, 100 pulses at 100 Hz) with the same stimulation intensity as pre‐HFS. Single pulse recording resumed immediately following HFS and continued for 60 min. A series of fEPSPs slope post‐HFS (55–60 min) 20% larger than pre‐HFS (15–20 min) baseline and lasting for over 60 min was scored as LTP.

Data Analysis

Data are expressed as means ± SEM and were analyzed with pClamp (Axon Instruments) and State7 software (StataCorp LP, College Station, TX, USA) software. ANOVAs followed by the Bonferroni's post hoc test was used for statistical analysis with the significant level set at P < 0.05.

Results

Effect of SV on Aβ25‐35‐Impaired Cognition

The effect of SV on long‐term memory in Aβ25‐35‐mice was explored using Morris water maze task. As shown in figure 1A, the escape‐latency to reach the hidden platform was prolonged in Aβ25‐35‐mice when compared to that in control mice (P < 0.01 on the 5th day after training). The prolongation of escape‐latency in Aβ25‐35‐mice was markedly attenuated by administration of SV (P < 0.01 on the 5th after training). Here, the swimming speed was unaffected by either Aβ25‐35‐injection or SV‐administration (P > 0.05).

Figure 1.

Figure 1

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Effect of SV on behavioral performance in Aβ25‐35‐mice. (A) Morris water task shows that the escape‐latency for Aβ25‐35‐mice (43.27 ± 6.64 seconds, on the 5th day after training) was longer than that for control mice (15.60 ± 4.10 seconds, on the 5th day after training) (P < 0.01). After SV‐treatment, the latency on the 5th day after training for Aβ25‐35‐mice was 23.91 ± 4.32 seconds (P < 0.01 vs. vehicle‐treated Aβ25‐35‐mice). (B) Probe trial shows that Aβ25‐35‐mice spent less time in the target quadrant than did control mice. SV treatment markedly increased the percentage time in training‐quadrant of Aβ25‐35‐mice. **P < 0.01 versus control mice, ## P < 0.01 versus vehicle‐treated Aβ25‐35‐mice. (C) Spontaneous of alternation in Y maze task markedly decreased in Aβ25‐35‐mice. Administration of SV increased the percentage of alternation in Aβ25‐35‐mice. **P < 0.01 versus control mice, ## P < 0.01 versus vehicle‐treated Aβ25‐35‐mice. Each group data contained eight mice.

After the hidden‐platform was removed from the pool, Aβ25‐35‐mice were not able to remember the location of the platform and spent significantly less time in the target quadrant than did the control mice (P < 0.01). Notably, after treatment with SV, Aβ25‐35‐mice presented better performance in the probe trail test than did saline‐treated Aβ25‐35‐mice (P < 0.01) (Figure 1B).

Spontaneous alternation behavior, which is regarded as a measure of short‐term memory, was investigated using Y maze task on day 8 after Aβ25‐35‐injection. Figure 1C shows that Aβ25‐35‐mice displayed significantly impaired memory, which was rescued by SV‐administration. In contrast, the number of arm entries did not change among all the experimental groups, demonstrating that general locomotor activity was not affected by Aβ25‐35‐injection or SV‐administration (P > 0.05). These results demonstrate that SV improves Aβ25‐35‐impaired long‐term and short‐term memory.

Effect of SV on Aβ25‐35‐Induced Apoptosis of Pyramidal Cells

We then explored whether SV had protection on hippocampal CA1 pyramidal cells, which is related to the cognitive function, on day 11 after Aβ25‐35‐injection. Figure 2A shows that the number of pyramidal cells was reduced approximately 34.02 ± 2.01% by Aβ25‐35‐injection (P < 0.01). After administration of SV, the surviving pyramidal cells in Aβ25‐35‐mice markedly increased (P < 0.05). The neuronal survival in control mice was not affected by SV‐administration. Figure 2B shows that there were more apoptotic cells (Hoechst‐positive cells) in Aβ25‐35‐mice, which could be reduced by administration of SV. These results demonstrate direct protection of SV against Aβ25‐35‐neurotoxicity in vivo.

Figure 2.

Figure 2

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Neuroprotective effect of SV on Aβ25‐35‐induced neuronal death. (A) The death of pyramidal cells in hippocampal CA1 region was examined using Toluidine blue staining. Healthy CA1 pyramidal neurons showed a round cell body with a plainly stained nucleus. Bar graph represents the number of surviving pyramidal cells. **P < 0.01 versus control mice, # P < 0.05 versus vehicle‐treated Aβ25‐35‐mice. (B) Representative images of Hoechst staining in hippocampal CA1 region of control (left), Aβ25‐35‐mice (middle) and SV‐treated Aβ25‐35‐mice (right). Each group data contained eight mice.

Involvement of α7nAChR in SV‐Increased Akt and ERK Phosphorylation in Aβ25‐35‐Mice

There is evidence that Aβ25‐35‐induced neurotoxicity in hippocampal CA1 pyramidal cells is mediated through the reduction of Akt and ERK phosphorylation 22, 23. As shown in Figure 3A, SV rescued the reduction of phosphorylation level of Akt (p‐Akt) in hippocampus of Aβ25‐35‐mice. Besides this, the phosphorylation level of hippocampal ERK2 (p‐ERK2) was lower in Aβ25‐35‐mice, which was rescued by SV (Figure 3B). It was also found that administration of α7nAChR antagonist MLA significantly attenuated SV‐improved p‐Akt and p‐ERK in Aβ25‐35‐mice (Figure 3A, B). Here, the neuroprotection of SV in Aβ25‐35‐mice was markedly blocked by administration of MLA or PI3K inhibitor LY294002. In contrast, administration of MEK inhibitor U0126 did not have such effect (Figure 3C). These results indicate that α7nAChR‐mediated PI3K/Akt signaling pathway is likely involved in SV‐induced neuroprotection against Aβ25‐35‐toxicity.

Figure 3.

Figure 3

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Involvement of α7nAChR‐PI3K/Akt signaling pathway in SV‐induced neuroprotection in Aβ25‐35‐mice. (A and B) p‐Akt (A) and p‐ERK (B) decreased in Aβ25‐35‐mice, which could be rescued by SV‐treatment. Administration of α7nAChR antagonist MLA markedly blocked SV‐action in Aβ25‐35‐mice. The densitometric values for p‐Akt and p‐ERK2 were first normalized by the protein amounts of Akt and ERK2, and then normalized by the basal values of p‐Akt/Akt and p‐ERK2/ERK2 in control mice, respectively. **P < 0.01 versus control mice; ## P < 0.01 versus vehicle‐treated Aβ25‐35‐mice, ++ P < 0.01 versus SV‐treated Aβ25‐35‐mice, + P < 0.05 versus SV‐treated Aβ25‐35‐mice. (C) SV‐neuroprotection on hippocampal CA1 pyramidal cells in Aβ25‐35‐mice was attenuated by antagonism of α7nAChR and PI3K. In contrast, the neuroprotection of SV was unaffected by MEK inhibitor. **P < 0.01 versus control mice; ## P < 0.01 versus vehicle‐treated Aβ25‐35‐mice; ++ P < 0.01 versus SV‐treated Aβ25‐35‐mice.

Involvement of α7nAChR‐Mediated PI3K/Akt and ERK Signaling Pathways in SV‐Rescued LTP in Aβ25‐35‐Mice

We then examined the effect of SV on the basal transmission of Schaffer collaterale‐CA1 synapse in Aβ25‐35‐mice. Input/output (I/O) curve shows that the slope of fEPSP in slices obtained from Aβ25‐35‐mice significantly decreased compared to that from control mice (P < 0.01), indicating the impaired synaptic transmission. After administration of SV, Aβ25‐35‐induced synaptic dysfunction was markedly rescued (P < 0.01, Figure 4A).

Figure 4.

Figure 4

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Effect of SV on long‐term potentiation (LTP) induction in Aβ25‐35‐mice. (A) Slopes of EPSP are plotted against stimulus intensity ranging from 0.1 to 1.1 mA. The correlation coefficient (b) of the regression line for I/O curve in Aβ25‐35‐mice (b = 0.88) was markedly smaller than that in control group (b = 1.31) (P < 0.01). After SV‐treatment, the correlation coefficient in Aβ25‐35‐mice significantly increased (b = 1.27). Data for each point consisted of 14 slices derived from eight mice. (B and C) HFS (denoted by↑) induced LTP in control mice (B) but failed to induce LTP in Aβ25‐35‐mice (C). (D) After treatment with SV, HFS induced LTP in slices obtained from Aβ25‐35‐mice.

We also found that HFS evoked a stable potentiation of fEPSP slope in control slices (143.29 ± 8.81% at 60 min post‐HFS, n = 14 slices/eight mice), but failed to induce the potentiation of fEPSP slope in Aβ25‐35‐mice slices (102.13 ± 7.54% at 60 min post‐HFS, n = 14 slices/eight mice, Figure 4B, C). After administration of SV, LTP could be induced in Aβ25‐35‐mice (145.17 ± 6.01% at 60 min post‐HFS, n = 15 slices/eight mice, Figure 4D). It was also found that after administration of MLA, LY294002, or U0126, the slopes of fEPSP markedly decreased and LTP could not be induced in slices from SV‐treated Aβ25‐35‐mice (Figure 5). Collectively, our results indicate an involvement of α7nAChR‐mediated PI3K/Akt and ERK signaling pathways in SV‐rescued LTP in Aβ25‐35‐mice.

Figure 5.

Figure 5

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Involvement of α7nAChR and PI3K signaling pathway in SV‐rescued LTP induction in Aβ25‐35‐mice. (A) SV‐enhanced fEPSP in Aβ25‐35‐mice was markedly blocked by MLA, U0126, or LY294002. *P < 0.05 versus SV‐treated Aβ25‐35‐mice, **P < 0.01 versus SV‐treated Aβ25‐35‐mice; stimulation intensity was 0.5 mA. (B) LTP could not be induced in Aβ25‐35‐mice by coadministration of SV and MLA. (C) SV‐rescued LTP induction in Aβ25‐35‐mice was markedly inhibited by administration of U0126. (D) Administration of LY294002 inhibited LTP induction in SV‐treated Aβ25‐35‐mice.

Involvement of α7nAChR‐Mediated PI3K/Akt and ERK Signaling Pathways in Antiamnesic Effect of SV in Aβ25‐35‐Mice

As shown in figure 6, the antiamnesic effects of SV on the prolongation of escape‐latency and decrease of swimming time in the target quadrant in Morris water maze task were markedly attenuated by application of MLA or LY294002. In SV‐treated Aβ25‐35‐mice, the increase in alternation behavior in Y maze task was also attenuated by MLA or LY294002. Here, it was noted that application of U0126 also blocked the antiamnesic effect of SV but exerted less effect on SV‐action than did MLA or LY294002.

Figure 6.

Figure 6

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Effect of α7nAChR, PI3K, and MEK inhibitors on antiamnesic effect of SV in Aβ25‐35‐mice. (A) The histogram shows the latency on the 5th day after training in Morris water maze task. Following administration of MLA, LY294002 or U0126, the latency in SV‐treated Aβ25‐35‐mice was significantly prolonged. **P < 0.01 versus control mice; ##P < 0.01 versus vehicle‐treated Aβ25‐35‐mice, ++ P < 0.01 versus SV‐treated Aβ25‐35‐mice. (B) The percentage time in training quadrant in SV‐treated Aβ25‐35‐mice was decreased by administration of MLA, LY294002 or U0126. **P < 0.01 versus control mice; ##P < 0.01 versus vehicle‐treated Aβ25‐35‐mice, ++ P < 0.01 versus SV‐treated Aβ25‐35‐mice, + P < 0.05 versus SV‐treated Aβ25‐35‐mice. (C) SV‐improved percentage of alternation in Aβ25‐35‐mice in Y maze task was also markedly attenuated by MLA or LY294002 or U0126. **P < 0.01 versus control mice; ##P < 0.01 versus vehicle‐treated Aβ25‐35‐mice, ++ P < 0.01 versus SV‐treated Aβ25‐35‐mice, + P < 0.05 versus SV‐treated Aβ25‐35‐mice.

Discussion

A chronic cerebral hypoperfusion is supposed to play an important role in neurodegeneration and amnesia in AD. The decrease in cerebral blood flow has been correlated with AD progression and with response to therapy 24, 25. These reports support the notion that statins, a kind of broadly used cholesterol‐lowering drugs, have therapeutic potential in AD 26. The present results showed that administration of SV for 11 days could improve the cognitive deficits, decrease the neuronal apoptosis and rescue LTP induction in Aβ25‐35‐mice (Figure 1, 2, 4). The neuroprotection of SV against Aβ‐toxicity was sensitive to α7nAChR‐cascaded PI3K/Akt signaling pathway (Figure 2). The rescue of LTP in SV‐treated Aβ25‐35‐mice depended on α7nAChR‐PI3K/Akt and ERK signaling pathways (Figure 5).

Chronic SV‐treatment improves the behavior performance in transgenic AD model mice independent of decreasing brain Aβ level or plaque load 7, 8. Moreover, an enhancement of spatial learning and memory in normal mice by SV‐treatment is consistent with this concept 8. A variety of mechanisms have been proposed to explain how statins display favorable effects in AD, such as increased expression of Akt, endothelial nitric oxide synthase and c‐Fos or Egr‐1 7, 8. This study found that administration of SV, a lipophilic statin that is able to transverse the blood brain barrier, markedly attenuated the hippocampal neuronal death induced by Aβ25‐35‐injection (Figure 2), first providing in vivo evidence of direct neuroprotection of SV against Aβ‐toxicity.

The down‐regulation of α7nAChRs in hippocampus and cortex is the most initial disruption of cholinergic system in AD, which correlates well with Aβ‐induced neurotoxicity 27. Activation of α7nAChRs has been proved to ameliorate Aβ‐induced hippocampal neuronal death 9, 15. It is reported that Aβ1‐40 prevents the stimulation of sympathetic α7nAChR to cause the release of nitric oxide in parasympathetic nitrergic nerves and subsequent vasodilatation, which can be reversed by mevastatin and lovastatin 28. Here, administration of α7nAChR antagonist significantly decreased the surviving cell in hippocampal CA1 area in SV‐treated Aβ25‐35‐mice (Figure 3). Combined with these reports, it is indicated that α7nAChR may be involved in the protection of SV against Aβ‐neurotoxicity. Cholesterol is a very abundant component of the membrane where AChR is located 29. SV reduces cholesterol level by inhibiting HMG‐CoA reductase activity. However, there is evidence that the effects of stains on α7nAChR are likely independent of lipid‐lowering action. For example, Guan's research group report that stains treatment can upregulate α7nAChR mRNA and protein in cultured neurons and astrocytes, but pretreatment of lovastatin fails to inhibit the effect of cholesterol on α7nAChR expression 11, 30. Besides this, statins prevent cholinesterase inhibitors (ChEIs)‐induced inhibition in α7nAChR and this effect is seen on concurrent administration of statins (lovastatin and mevastatin) with ChEIs, which suggests that statins protect α7nAChR function directly at the receptor level 12. As discussed above, SV may upregulate α7nAChR expression or modulate α7nAChR function directly which are impaired upon Aβ‐toxicity. More experiments are needed to confirm whether (and how) SV modulates α7nAChR.

Aβ‐neurotoxicity in hippocampal CA1 pyramidal neurons is mediated through down‐regulating PI3K/Akt and ERK signaling pathways which play an important role in cell growth, survival and proliferation 22, 23. α7nAChR, in particular, contributes to activation of PI3K/Akt pathway, which is important for the neuroprotection of α7nAChR against Aβ‐toxicity 15, 23. This study found that Aβ25‐35‐induced decrease of p‐Akt and p‐ERK was markedly rescued by SV, which was sensitive to α7nAChR antagonist. Moreover, the antagonists of α7nAChR and PI3K, but not of MEK markedly attenuated SV‐improved surviving cell in Aβ25‐35‐mice (Figure 3). These results indicate that SV may target α7nAChR to activate PI3K/Akt and ERK, but only α7nAChR‐PI3K/Akt signaling pathway is involved in the neuroprotection of SV against Aβ‐toxicity. On the other hand, although statins are reported to activate PI3K‐Akt and ERK pathways 31, 32, in vitro evidence shows that the neuroprotection of pretreatment with SV against Aβ‐induced toxicity is not able to activate Akt or ERK2 33. This discrepancy may be due to the difference in AD model types and dosage or time of SV‐treatment. Of course, besides α7nAChR‐PI3K/Akt pathway, the neuroprotection of SV may be mediated through other mechanisms such as decreasing Aβ‐induced intracellular calcium rise, accumulation of reactive oxygen species and caspase‐3 activity 33.

Synapse damage occurs during the early stage of AD, which is correlative with cognitive decline 34. This study showed that LTP could be induced in SV‐treated Aβ25‐35‐mice (Figure 4), implying that SV rescues Aβ‐impaired hippocampal synaptic plasticity. Activation of ERK signaling pathway in hippocampus is important for LTP induction 35. Here, HFS failed to induce LTP in Aβ25‐35‐mice if they were coadministrated with SV and U0126 (Figure 5), which indicates that ERK signaling pathway is responsible for SV‐rescued synaptic plasticity that was impaired by Aβ25‐35‐injection. Several research groups have shown that Aβ‐induced blockade of α7nAChR can depress synaptic transmission and impair LTP induction 9, 10. There is evidence that PI3K/Akt pathway is important for hippocampal LTP induction and the level of p‐Akt in the hippocampus is in parallel with spatial memory formation 36. Li's research group reports that SV‐enhanced hippocampal LTP in C57BL/six mice depends on the activation of Akt 37. Here, administration of α7nAChR or PI3K antagonist significantly blocked LTP in SV‐treated Aβ25‐35‐mice (Figure 5), suggesting that α7nAChR and PI3K/Akt pathway are probably involved in SV‐rescued synaptic plasticity. As discussed above, SV‐increased p‐Akt may be mediated through α7nAChR. Besides this, SV enhances hippocampal LTP through inhibiting farnesylation to augment the recruitment of PI3K activity 38. Although pre‐treatment with SV prevents Aβ‐induced synapse damage in vitro 39, in vivo study reports that chronic SV‐treatment did not affect synaptic markers PSD‐95, synaptophysin, or the NMDA receptor subunit NR2B in synaptosomal P2 fractions from cortex and hippocampus in adult and aged AD mice 7. More experiments are needed for exploring the mechanisms underlying SV‐rescued synaptic plasticity in AD.

In summary, this study showed that blockage of α7nAChR, PI3K, and MEK markedly attenuated SV‐improved behavior performance in Aβ25‐35‐mice (Figure 6), indicating that SV improves Aβ‐induced deficit in learning and memory through direct neuroprotection and rescue of synaptic plasticity. α7nAChR is a potential target of SV and SV may exert its antiamenisc effect through modulating PI3K/Akt and MAPK/ERK signaling pathways. This study provides a possible cellular basis for the beneficial effect of SV on Aβ‐impaired cognition function.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgments

We thank Dr. Edith Hamel and Dr. Xin‐Kang Tong for the helpful discussion on the manuscript. This work was supported by Fonds de recherche Santé‐National Natural Science Foundation of China Collaboration (812111370), National Natural Science Foundation of China (31271206) and Science and Technology Project of Jiangsu Province (BK2011029).

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