Dose-Dependent Neuroprotection and Neurotoxicity of Simvastatin through Reduction of Farnesyl Pyrophosphate in Mice Treated with Intracerebroventricular Injection of Aβ 1-42

Abstract

Background:

Simvastatin (SV) has been reported to improve dementia and slow progression of Alzheimer’s disease (AD), however there are conflicting reports.

Objective & Methods:

Intracerebroventricular injection of aggregated Aβ_1-42 in mice (Aβ_1-42-mice) caused spatial cognitive deficits, long-term potentiation (LTP) impairment, and death of hippocampal pyramidal cells. The present study focused on exploring the dose-dependent effects of SV (10–80 mg/kg) on Aβ_1-42-impaired spatial memory and the underlying mechanisms.

Results:

The treatment of Aβ_1-42-mice with SV for continuous 15 days could attenuate the spatial cognitive deficits and recover the LTP induction in a “U” type dose-dependent manner. The death of pyramidal cells in Aβ_1-42-mice was significantly reduced by the SV-treatment at 20 mg/kg, but not at a dose of 10 or 40 mg/kg, even was aggravated at a dose of 80 mg/kg. Hippocampal NMDA receptor (NMDAr) NR2B phosphorylation (phospho-NR2B) was elevated in Aβ_1-42-mice, which was further dose-dependently increased by SV-treatment. Replenishment of isoprenoid farnesyl pyrophosphate (FPP) by applying farnesol (FOH) could abolish the SV-increased phospho-NR2B in Aβ_1-42-mice, but had no effect on the Aβ_1-42-enhanced phospho-NR2B. NMDAr antagonist blocked the neurotoxicity of Aβ_1-42 and SV (80 mg/kg) in Aβ_1-42-mice, whereas FOH only inhibited SV (80 mg/kg)-neurotoxicity. The SV-treatment in Aβ_1-42-mice corrected the decrease in hippocampal Akt phosphorylation. The PI3K inhibitor abolished the SV (20 mg/kg)-neuroprotection in Aβ_1-42-mice.

Conclusion:

SV-treatment in Aβ_1-42-mice exerts dose-dependent neuroprotection and neurotoxicity by reducing FPP to enhance the phosphorylation of NR2B and Akt.

Keywords

Amyloid–β 1–42 hippocampus long-term potentiation memory NMDA receptor simvastatin

INTRODUCTION

Statins, inhibitors of 3-hydroxy-3-methyl-glytaryl-coenzyme A (HMG-CoA) reductase, are widely used to treat cardiovascular diseases. Alzheimer’s disease (AD) has been established as a progressive compromise not only of the neurons but also of the cerebral vasculature [1], suggesting the neuroprotection of statins against AD. In the general population, the use of statins, but not of non-statin cholesterol-lowering drugs, is associated with a lower risk of AD [2]. Recently, statin use was associated with a significantly lower risk of dementia in the elderly patients in Taiwan [3]. The treatment with simvastatin (SV) has been reported to improve cognitive function in AD patients [4] and decrease risk for AD, which were unrelated to its cholesterol-lowering effects [5]. However, there are conflicting reports showing that statins have no effect in preventing AD or dementia [6], even some statin users suffer memory loss that is ameliorated by withdrawal from statin treatment [7]. Possible reasons for such discrepancies may include differences in the dose of statins or the stage of disease at which statins are administered. The ability of SV crossing blood-brain barrier is higher than that of atorvastatin and rosuvastatin [8]. The dose of SV for treating AD is always a highly controversial topic. The soluble species of amyloid-β (Aβ) are well known to be detrimental to neuronal function and memory [9]. Li et al. [10] reported that the administration of SV (50 mg/kg/day) for 3 months in 11-month-old female Tg2576 mice reversed learning and memory deficits. The chronic administration of SV (20 mg/kg/day) in mice can enhance presynaptic glutamate release and long-term potentiation (LTP) induction in hippocampal CA1 region, which leads to the spatial cognitive potentiation [11]. The treatment with SV (40 mg/kg/day) can rescue cognitive deficits in adult, but not aged, AD mice over-expressing the Swedish and Indiana mutations of human amyloid-β protein precursor (AβPP mice, line J20) [12]. The treatment with SV (20 mg/kg/day) failed to normalize memory in 12-month-old AβPP mice [13]. The spatial cognitive deficits and hippocampal LTP impairment in mice produced by intracerebroventricular (i.c.v.) injection of Aβ_25 - 35 could be rescued by SV (40 mg/kg/day) by enhancing the protein kinase B (Akt) phosphorylation [14]. Recently, our study has reported that SV (20 mg/kg/day) can enhance the Akt phosphorylation to prevent the Aβ_25 - 35-impaired survival of newborn neurons in hippocampal dentate gyrus [15]. However, the dose-dependent benefits of SV in protecting against AD have not yet been reported.

Soluble Aβ oligomers from three distinct sources (cultured cells, AD cortex, and synthetic peptide) enhance selectively the activation of NMDA receptor (NMDAr) [16]. The Aβ-injection (i.c.v.) is able to increase NMDA-activated currents in the hippocampal CA1 pyramidal cells through increasing the phosphorylation of NMDAr NR2B [17]. In mouse hippocampal slices and human neuronal cells, the SV treatment increases the surface distribution of the NMDAr NR2B subunit leading to the augmentation of synaptic NMDAr components [18]. Glutamate excitotoxicity is believed to be a main mechanism contributing to progressive neuronal loss in AD [19]. The noncompetitive NMDAr antagonist memantine is licensed for use in moderate to severe AD [20]. The chronic treatment with statin in rats increases level of NMDAr [21]. In addition, statins can prevent the conversion of HMG-CoA to mevalonate resulting in reduction of non-sterol intermediates, known as isoprenoids [22]. SV has a greater effect on reducing the level of isoprenoid farnesyl-pyrophosphate (FPP) [23] that serves as lipid attachments for the small GTPase superfamilies [24]. Ras is known to negatively regulate NMDAr activation [25]. Therefore, whether the increased NMDAr activation by the SV-treatment can enhance the Aβ-neurotoxicity should be an importantsubject.

Aβ_1-42 rapidly forms aggregates pos–sessing a high aggregation propensity in terms of monomer consumption and oligomer formation [26]. Intrahippocampal or cerebroventricular injection of aggregated Aβ_1-42 causes spatial cognitive deficits measured by Morris water maze [27]. Thereupon, the present study focused on examining the dose-dependent effects of SV (10–80 mg/kg) on Aβ_1-42-impaired spatial memory and exploring the underlying molecular mechanisms. The findings in this study indicate that the treatment of Aβ_1-42-mice with SV through enhancing NMDAr function exerts the dose-dependent neuroprotection and neurotoxicity.

MATERIAL AND METHODS

Experimental animals

The present study was approved by Animal Care and Ethical Committee of Nanjing Medical University. All animal handling procedures followed the guidelines of Institute for Laboratory Animal Research of the Nanjing Medical University. Male mice (ICR, Oriental Bio Service Inc., Nanjing), weighing 30–32 g (11-12 weeks) at beginning of experiment, were used. The animals were maintained in a constant environmental condition (temperature 23±2°C, humidity 55±5% , 12:12 h light/dark cycle) in the Animal Research Center of Nanjing University. They had free access to food and water before and after all procedures.

Preparation of AD model

The Aβ_1-42 and Aβ_42 - 1 (Sigma, St. Louis, MO, USA) were prepared as described by Bouter et al. [26]. Briefly, Aβ_1-42 and Aβ_42 - 1 (Sigma, St. Louis, MO, USA) were dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Sigma Aldrich), flash-freezing in liquid nitrogen, and then lyophilizing them to completely remove the solvent. Lyophilized Aβ_1-42/_42 - 1 peptides were then dissolved in 100 mM NaOH at a concentration of 6 mg/ml, aliquoted in 50μl volumes, flash-frozen in liquid nitrogen and stored at –80°C until use.

For i.c.v. injection of soluble Aβ_1-42, mice were anesthetized with ketamine (80 mg/kg, intraperitoneal (i.p.) and xylazine (10 mg/kg, i.p.), and then placed in a stereotactic apparatus (Motorized Stereotaxic Stereo Drive; Neurostar). Freshly prepared Aβ_1-42 (0.3 nmol/2μl in 0.1 M phosphate-buffered saline) were injected into the bilateral ventricles (0.3 mm posterior, 1.0 mm lateral, and 2.5 mm ventral to bregma) using a stepper-motorized micro-syringe fitted with a 26-gauge needle at a rate of 0.2μl/min. The injection site was confirmed by injecting Indian ink in preliminary experiments. The presence of aggregated Aβ_1-42 in the hippocampus has been determined by an Aβ-specific antibody immuno-staining [28]. The mice infused with Aβ_42 - 1 or vehicle at a same volume were served as the control group.

Administration of drugs

SV (Enzo Life Sciences International) was activated by alkaline lysis [12]. SV was added to the drinking water at doses of 10, 20, 40, or 80 mg/kg daily and was given starting at 4 h after Aβ_1-42-injection. Trans, trans-farnesol (farnesol, FOH) (96%) was purchased from Sigma (Cat# 27754, St. Louis, MO, USA). FOH was mixed with olive oil and administrated by gavage at dose of 50 mg/kg daily [29]. NMDAr antagonist MK801 (Sigma, St. Louis, MO, USA) dissolved in distilled water was injected (i.p.) at 30 min prior to SV-administration [30]. The mice treated with vehicle at a same volume served as the control group.

Behavioral examination

Morris water maze task

A pool (diameter = 120 cm) made of black-colored plastic was prepared. The water temperature was maintained at 20±1°C with a bath heater, used before sessions. These behavioral tests were recorded by a video monitor (Winfast PVR; Leadtek Research Inc., Fremont, CA). On days 1-2 of training, a cylindrical black-colored platform (diameter = 7 cm) was placed 0.5 cm above the surface of water. The mouse was randomly released from four different quadrants respectively, and allowed to swim for 90 s. Latency to reach visible-platform was measured. On days 3–7 of training, the platform was moved to the opposite quadrant of visible-platform and was submerged 1 cm below the water surface. Four trials were conducted each day with intertrial interval of 30 min. Latency to reach hidden-platform was measured. If the mouse could not reach the platform within 90 s, the experimenter gently assisted it onto the platform and allowed it to remain there for 15 s. Each mouse started in one of four quadrants in a random manner. On day 8 of training, the probe trial was performed by removing the platform. The mouse was released from opposite quadrant in which the platform was located, and allowed to swim for 90 s. The percentage of swim time spent in four quadrants was determined.

Y-maze task

Y-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 for 8 min. The series of arm entries was recorded visually and arm entry was considered to be complete 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). The data obtained in Morris water maze and Y-maze was analyzed by a tracking program (TopScan lite 2.0; Clever Sys. Inc., USA). The scorers were blinded to the treatmentgroups.

Histological examination

The mice were anesthetized with i.p. injection of chloral hydrate (400 mg/kg) and perfused with 4% paraformaldehyde. Brains were removed and immersed in 4% paraformaldehyde at 4°C overnight.

Toluidine blue staining

Brains were processed for paraffin embedding and coronal sections (5-μm) were cut. The pyramidal cells in hippocampal CA1 region were identified using a conventional light microscope (Olympus DP70, Tokyo, Japan) with a 40×objective. The healthy pyramidal neurons showed a round cell body with a plainly stained nucleus.

Stereological counts of cells

Brains were transferred into 30% sucrose. After gradient dehydration, coronal sections of hippocampus (40μm) were cut by freezing microtome (Leica, Nussloch, Germany) and stained with toluidine blue. Every fourth section was obtained for consecutive analysis of cell quantification. Total number of healthy pyramidal cells within the three-dimensional optical dissectors throughout hippocampal CA1 regions was counted with the Microbrightfield StereoInvestigator software (Microbrightfield, Williston, VT, USA) [31].

Hoechststaining

Coronal paraffin sections (5μm) were incubated in Hoechst 33342 (1μg/ml, Cell Signaling Technology, Inc., Boston, MA, USA) for 2 min. After several PBS rinses the sections were coverslipped. The Hoechst-positive cells were observed using a fluorescence -microscope (Olympus DP70) with a 40×objective. Density of Hoechst-positive cells was expressed as the number of cells per mm length measured along the hippocampal CA1 pyramidal layer. Neuronal densities measured from four sections per animal were averaged to provide a single value for each animal.

Electrophysiological analysis

Mice were decapitated under deep anesthesia with isoflurane. Brains were rapidly removed and coronal brain slices (400μm) were cut using a vibrating microtome (Microslicer DTK 1500, Dousaka EM Co, Kyoto, Japan) in ice-cold cutting solution (in mM): 94 sucrose, 30 NaCl, 4.5 KCl, 1.0 MgCl₂, 26 NaHCO₃, 1.2 NaH₂PO₄, and 10 D-glucose (pH 7.4). The slices were continuously perfused with artificial cerebrospinal fluid (ACSF) oxygenated with a gas mixture of 95 % O₂/5 % CO₂ at 30±1°C for 60 min. ACSF was composed of (in mM) 124 NaCl, 2 CaCl₂, 4.5 KCl, 1 MgCl₂, 26 NaHCO₃, 1.2 NaH₂PO₄, 10 D-glucose (pH 7.4). Orthodromic stimuli were delivered using an electrically polished bipolar tungsten electrode that was placed in radiatum layer of CA1 region to stimulate the Schaffer collateral/commissural pathway. Constant current pulses (0.1 ms, 0.06 Hz) were supplied by stimulator (SEN-3301, Nihon Kohden, Tokyo, Japan). Test stimulus was set at about 50% of maximal stimulus intensity that evoked a saturated excitatory postsynaptic potential (EPSP) in each slice, to avoid deprivation of releasable transmitters. EPSP was recorded from radiatum layer of CA1 with a 5 MΩ resistance glass microelectrode that was filled with 2 M NaCl and connected to a preamplifier with a high-pass filter at 5 kHz. Signals were amplified by a differential AC amplifier (A-M Systems, model 1700, Seattle, WA, USA). The EPSPs were digitized and saved using pCLAMP system (Axon Instrument Inc., CA, USA). Stability of baseline recordings was established by delivering single pulses (4/min, 0.1 ms pulse width) for 15 min prior to collection of data. Input/output (I/O) function: EPSP slopes were evoked by testing stimulation of various intensities (0.1–0.9 mA). LTP was induced by high-frequency stimuli (HFS, 100 Hz). After delivering HFS, the EPSP slopes were recorded for a further period of 60 min. In 55–60 min after delivering HFS, if the EPSP slopes were larger 20% than baseline, LTP was determined.

Western blot analysis

Entire hippocampus was taken quickly and 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 phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Roche, Germany). Protein concentration was determined with BCA Protein Assay Kit (Pierce Biotechnology Inc., USA). Total proteins (20μg) were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyphorylated difluoride membrane. The membranes were incubated with 5% nonfat dried milk for 60 min, and then incubated with a mouse monoclonal antibody of anti-phospho-NR2B (1:1000, Abcam, Cambridge, UK) or Ser473-Akt (1:1000, Cell Signaling, USA) at 4°C overnight. The membranes were incubated with an HRP-labeled secondary antibody, and developed using the ECL detection Kit (Millipore). Following visualization, the blots were stripped by incubation in stripping buffer (Restore, Pierce) for 5 min, re-blocked with 5% nonfat dried milk for 60 min, then incubated with antibody of NR2B (1:1000, Abcam, Cambridge, UK) or Akt (1:1000, Cell Signaling). Western blot bands were scanned and analyzed with the image analysis software package, National Institutes of Health Image.

Data analysis/statistics

Data were retrieved and processed with the software pCLAMP 10.0 (Molecular Devices Inc., USA) and Micro cal Origin 6.1. The group data were expressed as the means±standard error (SEM). All statistical analyses were performed using SPSS software, version 16.0 (SPSS Inc., USA). Differences among means were analyzed using the one/two/three-factor analysis of variance (ANOVA) with or without repeated-measures, followed by post hoc LSD test, where appropriate. Differences at p < 0.05 were considered statistically significant.

RESULTS

Aβ_1-42-injection (i.c.v.) impairs spatial cognitive function in mice

Spatial cognitive behaviors were examined by Morris water maze and Y-maze tests on days 7–15 after aggregated Aβ_1-42 or Aβ_42 - 1-injection (i.c.v.). In Morris water maze, the visible-platform task is used to examine search behavior or visual acuity and the hidden-platform task is thought to reflect spatial learning and memory. As shown in Fig. 1A, the latency to reach the visible-platform showed no significant difference between control mice and Aβ_1-42-mice (F_(1,27) = 0.190, p = 0.667) or Aβ_42 - 1-mice (F_(1,27) = 0.187, p = 0.572). Repeated-measures ANOVA revealed that the escape latency to reach the hidden-platform was progressively decreased with training days in all groups (F_(4,108) = 99.119, p < 0.001), which was affected by Aβ_1-42-injection (F_(1,27) = 21.273, p < 0.001), but not by Aβ_42 - 1-injection (F_(1,27) = 0.024, p = 0.879). Aβ_1-42-mice need a longer time to find the hidden platformon training days 5–7 compared to control mice (F_(1,18) = 26.622, p < 0.001). No significant difference was found in the mean swimming speed among the 3 groups (p > 0.05, n = 10). We carried out a probe trial at 24 h after the hidden platform test, in which the swimming time spent in four quadrants (target, opposite, and adjacent quadrants) was measured, to evaluate the strength of memory trace. The swimming time spent in the target quadrant was longer than those quadrants in control mice (F_(2,27) = 3.675, p = 0.039; Fig. 1B), which was affected by Aβ_1-42-injection (F_(1,27) = 6.192, p = 0.009), but not by Aβ_42 - 1-injection (F_(1,27) = 0.098, p = 0.757). Aβ_1-42-mice showed a less swimming time in target quadrant than that in control mice (p < 0.01). Subsequently, spontaneous alternation behavior in Y-maze test, as a measure of short-term spatial memory, was affected by Aβ_1-42-injection (F_(1,27) = 11.330, p = 0.002; Fig. 1C), but not by Aβ_42 - 1-injection (F_(1,27) = 0.576, p = 0.454). In comparison with control mice, the alternation rate was significantly reduced in Aβ_1-42-mice (p < 0.01, n = 10).

SV can rescue Aβ_1-42-impaired spatial memory in a “U” type dose-dependent manner

To examine the dose-dependent effects of SV on theAβ_1-42-impaired spatial memory, SV (10, 20, 40, and 80 mg/kg per day) was administered (p.o.) starting from 4 h after Aβ_1-42-injection for consecutive 15 days. In comparison with vehicle-treated Aβ_1-42-mice, the prolongation of escape latency in Morris water maze could be reduced by the SV-treatment at doses of 20 (F_(1,27) = 42.469, p < 0.001; Fig. 2A-i) and 40 mg/kg (F_(1,27) = 27.275, p < 0.001; Fig. 2A-ii), but not at dose of 10 mg/kg (F_(1,27) = 3.574, p = 0.069), and even was further increased by the SV-treatment of 80 mg/kg (F_(1,27) = 20.818, p < 0.001). The SV-treatment at any dose failed to affect the latency reaching visible-platform (p > 0.05, n = 10) and the swim speed (p > 0.05, n = 10). In probe trial test, the swimming time in target quadrant could be increased in Aβ_1-42-mice by the SV-treatment at dose of 20 (p < 0.01, n = 10; Fig. 2B) or 40 mg/kg (p < 0.05, n = 10), but not at dose of 10 or 80 mg/kg (p > 0.05, n = 10). Additionally, the reduced alternation rate in Aβ_1-42-mice was recovered by the SV-treatment at dose of 20 (p < 0.01, n = 10; Fig. 2C) or 40 mg/kg (p < 0.05, n = 10), but not at 10 or 80 mg/kg (p > 0.05, n = 10).

Neuroprotection and neurotoxicity of SV on Aβ_1-42-impaired pyramidal cells

In order to explore the mechanism underlying dose-dependent anti-amnesic effect of SV, the pyramidal cells in hippocampal CA1 region were examined on day 16 after Aβ_1-42-injection (Fig. 3A). The stereological counts of surviving pyramidal cells showed approximately 30% reduction in Aβ_1-42-mice compared to control mice (p < 0.05; Fig. 3B). In comparison with vehicle-treated Aβ_1-42-mice, the SV (20 mg/kg)-treatment significantly attenuated the loss of pyramidal cells (p < 0.05, n = 10; Fig. 3C), whereas the SV (10 or 40 mg/kg)-treatment had no effects (p > 0.05, n = 10). In particular, the SV (80 mg/kg)-treatment increased further the Aβ_1-42-induced death of pyramidal cells (p < 0.05, n = 10). As shown in Fig. 3D, a large number of Hoechst-positive cells were observed in hippocampal CA1 region of Aβ_1-42-mice. In comparison with control mice, the number of Hoechst-positive cells was significantly increased in Aβ_1-42-mice (p < 0.01, n = 10; Fig. 3E), which was reduced by the SV-treatment at 20 mg/kg (p < 0.01, n = 10) and further increased by the SV (80 mg/kg)-treatment (p < 0.01, n = 10). However, the SV (80 mg/kg)-treatment in control mice failed to cause the death of pyramidal cells (p > 0.05, n = 10) and the increase of Hoechst-positive cells (p > 0.05, n = 10).

SV can improve Aβ_1-42-impaired LTP in a “U” type dose-dependent manner

The basal properties and LTP at Schaffer collateral-CA1 synaptic transmission were examined on days 16–18 after Aβ_1-42-injection by field potential recording. Input/output function was analyzed by plotting EPSP slopes against delivering stimulant intensities from 0.1 mA to 0.9 mA. As shown in input-output curve (Fig. 4A), the slopes of EPSPs induced by 0.5–0.9 mA stimulant intensities were reduced in Aβ_1-42-mice compared to control mice (p < 0.05, n = 10). The synaptic dysfunction in Aβ_1-42-mice could be rescued by the SV-treatment at dose of 20 mg/kg (p < 0.01, n = 10; Fig. 4B) or 40 mg/kg (p < 0.05, n = 10), but not at dose of 10 mg/kg (p > 0.05, n = 10) and 80 mg/kg (p > 0.05, n = 10). In addition, the protocol of high-frequency stimuli (HFS, 100 Hz, 1 s) induced approximately 150–160% increase in the EPSP slopes for over 60 min in control mice (n = 10; Fig. 4C), indicative a normal “LTP”. By contrast, the same protocol failed to product LTP in Aβ_1-42-mice (n = 10). In Aβ_1-42-mice, the SV-treatment at 20 mg/kg perfectly protected the LTP induction (n = 10; Fig. 4D) and at 40 mg/kg partially covered the LTP induction (n = 10; Fig. 4E), but at 10 or 80 mg/kg had no effect on the impairment of LTP (n = 10).

SV enhances dose-dependently Aβ_1-42-increased NR2B phosphorylation

To investigate the mechanisms of SV-protected LTP and SV-neurotoxicity in Aβ_1-42-mice, we further measured the level of hippocampal NR2B phosphorylation (phospho-NR2B) at 48 h after Aβ_1-42-injection. Densitometry analysis revealed that the level of phospho-NR2B in Aβ_1-42-mice was elevated (p < 0.01, n = 10; Fig. 5A) without the changes in NR2B protein compared to control mice (p > 0.05, n = 10). The SV-treatment in Aβ_1-42-mice could dose-dependently increase the level of phospho-NR2B (F_(4,45) = 10.941, p < 0.001; Fig. 5B). In addition, the SV (80 mg/kg)-treatment in control mice produced about 1.5-fold increase in the level of phospho-NR2B (p < 0.01, n = 10). To test whether SV through reducing FPP [32] enhances the phospho-NR2B [33], the farnesol (FOH, 50 mg/kg) that can convert to FPP was used at 30 min prior to the SV (80 mg/kg)-treatment. The results showed that the FOH administration could inhibit the SV-enhanced phospho-NR2B in Aβ_1-42-mice (p < 0.01, n = 10; Fig. 5A), but it did not affect the Aβ_1-42-increased phospho-NR2B (p > 0.05, n = 10).

SV-enhanced phospho-NR2B exerts neurotoxicity and anti-amnesic effects

To further determine whether SV throughenhancing phospho-NR2B aggravates the Aβ_1-42-neurotoxicity, the NMDAr antagonist MK801 (6 mg/kg) or FOH were used on days 1–5 after Aβ_1-42-injection (Fig. 6A). The administration of MK801 attenuated not only the Aβ_1-42-induced death of pyramidal cells (p < 0.05, n = 10; Fig. 6B) but also the SV (80 mg/kg)-neurotoxicity in Aβ_1-42-mice (p < 0.01, n = 10). By contrast, the administration of FOH in Aβ_1-42-mice could prevent the SV (80 mg/kg)-neurotoxicity (p < 0.01, n = 10), although it had no effect on the Aβ_1-42-neurotoxicity (p > 0.05, n = 10).

To test the involvement of SV-enhanced phospho-NR2B in the LTP induction in Aβ_1-42-mice, FOH was administrated on days 11–15 after Aβ_1-42-injection (Fig. 6A). As expected, the administration of FOH in Aβ_1-42-mice blocked the SV (40 mg/kg)-protected LTP (Fig. 6C), but it hardly affected the SV (20 mg/kg)-protected LTP.

Effects of SV-enhanced phospho-Akt on Aβ_1-42-neurotoxicity

Western blot analysis on day 7 after Aβ_1-42-injection revealed that the level of hippocampal Akt phosphorylation (phospho-Akt) was reduced compared to control mice (p < 0.01, n = 10; Fig. 7A). The treatment of SV dose-dependently increased the levels of phospho-Akt in Aβ_1-42-mice (F_(4,45) = 6.386, p < 0.001; Fig. 7B), which was sensitive to the administration of FOH (p < 0.01, n = 10). The treatment with PI3K inhibitor LY294002 on days 1–5 after Aβ_1-42-injection (Fig. 7C) blocked the SV (20 mg/kg)-neuroprotection (p < 0.01, n = 10; Fig. 7D) and SV (20 mg/kg)-protected LTP induction (Fig. 7E). However, the application of LY294002 on days 11–15 after Aβ_1-42-injection (Fig. 7F) did not affect the SV (20 mg/kg)-protected LTP induction.

DISCUSSION

The present study provides in vivo evidence that the SV-treatment in Aβ_1-42-mice through reducing farnesyl pyrophosphate exerts the dose-dependent neuroprotection and neurotoxicity, as suggested by the following results. (1) The SV-treatment in Aβ_1-42-mice could dose-dependently increase the level of phospho-NR2B, which was inhibited by the application of FOH. (2) SV (20 mg/kg) reduced Aβ_1-42-induced death of pyramidal cells and spatial cognitive deficits, which was sensitive to the application of FOH. (3) SV (40 mg/kg) failed to reduce the loss of pyramidal cells in Aβ_1-42-mice, but it could rescue their spatial cognitive deficits, which was abolished by the application of FOH. (4) SV (80 mg/kg) aggravated Aβ_1-42-induced death of pyramidal cells and spatial cognitive deficits, which was blocked by the NMDAr antagonist or FOH. However, the SV (80 mg/kg)-treatment in control mice did not cause the death of pyramidal cells in hippocampal CA1 regions (data not shown).

SV through reducing FPP enhances NR2B phosphorylation

Colocalization of Aβ and NR2B subunit is observed in hippocampal neurons [34]. Consistent with the Aβ_25 - 35-action [17], the level of hippocampal phospho-NR2B was elevated by the Aβ_1-42-injection. The SV-treatment in Aβ_1-42-mice could further increase in the level of phospho-NR2B, which was blocked by the application of FOH. However, the Aβ_1-42-induced phospho-NR2B was insensitive to the application of FOH. It is conceivable that SV through reducing production of FPP increases thephospho-NR2B. FPP serves as lipid attachments for the small GTPase superfamilies, including the well-known Ras, Rho, and Rac [24]. Ras is thought to negatively regulate NMDAr function, because inhibiting Ras effect or protein can increase NMDAr currents in hippocampal neurons [35]. H-Ras-deficient mice display increased tyrosine phosphorylation of NMDAr NR2B subunits [33]. Experimental evidence indicates that isoprenylated GTPases are involved in AD pathogenesis [36]. Therefore, further studies are needed to examine whether and how SV through reduced Ras is oprenylation enhances the phospho-NR2B.

SV through enhancing NMDAr activation aggravates Aβ_1-42-neurotoxicity

In vivo infusion of Aβ is able to increase the Ca^2 + influx across NMDAr within 20–30 min [37]. The Aβ_1-42-induced progressive neuronal degeneration has been demonstrated to be mainly by increasing Ca^2 + influx across NMDAr [38]. Multiple lines of evidence suggest that the deleterious effects of Aβ prior to neuronal loss can be mediated by NMDAr, especially NR2B-NMDAr. The NR2B-NMDAr antagonists prevent the synapse loss induced by incubation with exogenous Aβ [38]. The NR2B-NMDAr antagonists can also block other effects of exogenous Aβ to neurons including disruption of intracellular calcium homeostasis [39] and endoplasmic reticulum oxidative stress [40]. Indeed, the blockade of NMDAr by MK801 could attenuate the death of pyramidal cells in Aβ_1-42-mice. The activation of extrasynaptic NMDAr can trigger the cAMP response element-binding protein (CREB) shut-off pathway leading to cell death [41]. In addition, the inhibition of NR2B abolishes the Aβ-neurotoxicity by preventing the activation of glycogen synthesis kinase-3β [42]. One important observation in this study is that SV at dose of 80 mg/kg further increased the number of Hoechst-positive cells and the loss of pyramidal cells in Aβ_1-42-mice, which could be attenuated by the treatment with MK801 or FOH on days 1–5 after Aβ_1-42-injection. Similarly, MK801 could block the Aβ_1-42-neurotoxicity, but FOH did not. The findings give a clear indication that the high-dose SV through reducing FPP can further increases Aβ_1-42-enhanced NMDAr Ca^2 + influx, which is able to aggravate the Aβ_1-42-neurotoxicity. However, this idea is not supported by a previous study that SV protects cortical neurons from NMDA-induced excitotoxicity [43].

SV-enhanced NMDAr function improves Aβ-impaired LTP

The induction of frequency-dependent LTP at Schaffer collateral-CA1 synaptic transmission has been demonstrated to require the activation of NMDAr. Um et al. [44] reported that the Aβ-increased phospho-NR2B leads to an initial increase of surface NMDAr followed by a decline of surface NMDAr. The dysfunction of NMDAr has been observed at 72 h after Aβ_25 - 35-infusion with the decline in phospho-NR2B [17]. At the synaptic level, AD patients exhibit a decreased postsynaptic intracellular scaffold proteins, including postsynaptic density protein 95 [45], which can reduce the expression of NMDAr NR2A and NR2B subunits. The administration of SV (40 mg/kg) in Aβ_1-42-mice failed to attenuate the death of pyramidal cells, but it could rescue the LTP impairment and the spatial cognitive deficits. Interestingly, the replenishment of FPP on days 11–15 after Aβ_1-42-injection blocked the SV (40 mg/kg)-protected LTP, but it did not affect the SV (20 mg/kg)-protected LTP, indicating that the anti-amnesic effect of SV (40 mg/kg) probably depends on the increased phospho-NR2B. However, the administration of SV (80 mg/kg) in Aβ_1-42-mice failed to attenuate the LTP impairment. One possible explanation is that SV (80 mg/kg) aggravates the death of pyramidal cells in Aβ_1-42-mice resulting in a more serious synaptic impairment. On the other hand, the application of SV for 2 h in hippocampal slices from wild-type C57BL/6 mice is able to enhance LTP induction, which depends upon PI3-K/Akt activation through inhibiting FPP, but not GGPP [32]. The SV-treatment in Aβ_1-42-mice caused a dose-dependent increase in the phospho-Akt, but the application of PI3K inhibitor LY294002 on days 11–15 after Aβ_1-42-injection did not affect the SV (40 mg/kg)-protected LTP. Notably, the SV-treatment at 20–40 mg/kg could rescue the synaptic dysfunction in Aβ_1-42-mice. In cultured superior cervical ganglion cells, Aβ inhibits the choline-induced inward currents, which is attenuated by the acute treatment with mevastatin and lovastatin [46]. The application of mevastatin can alter the desensitization kinetics of α7nAChR in the plasma membrane [47]. Thus, further studies are needed to evaluate the SV effects on α7nAChR function in Aβ_1-42-mice.

SV-enhanced Akt phosphorylation prevents Aβ-induced death of neuronal cells

SV can rapidly trigger translocation of Akt to the plasma membrane of endothelial cells [48]. Consistent with the results obtained from Aβ_25 - 35-mice [14], the SV-treatment in Aβ_1-42-mice could correct the decline in the phospho-Akt, which was blocked by the application of FOH. The PI3K-Akt signaling is well known to have the neuroprotective effects against Aβ-induced cell death [49]. The SV-treatment has been reported to activate the PI3K-Akt pathway in APPswe/PS1dE9 mice [50]. The administration of PI3K inhibitor LY294002 on days 1–5 after Aβ_1-42-injection can block the neuroprotection of SV (20 mg/kg). Moreover, the replenishment of FPP by the application of FOH could abolish the SV (20 mg/kg)-neuroprotection. Although direct evidence is lacking, our results indicate that the SV-cascaded Akt signaling through reduced FPP exerts a potential neuroprotection in Aβ_1-42-mice.

CONCLUSIONS

Although the underlying mechanisms have not yet been fully elucidated, the present study clearly shows that the SV-treatment in Aβ_1-42-mice plays dose-dependently two difference roles of neuroprotection and neurotoxicity (Fig. 8). The SV-treatment (20 mg/kg) exerted a most powerful neuroprotection against Aβ_1-42-induced death of pyramidal cells and spatial cognitive deficits. By contrast, the SV-treatment (80 mg/kg) in Aβ_1-42-mice aggravated the loss of pyramidal cells. The dose of 20 mg/kg SV in mice is converted into a human dose to be approximately 1.66 mg/kg [51] that is not extremely high compared to the usual clinical dose administered to manage cholesterol level (40–80 mg per day). Therefore, the findings in the present study are very important for seeking an effective and feasible therapeutic strategy of SV in AD.

Footnotes

ACKNOWLEDGMENTS

This work was supported by National 973 Basic Research Program of China (grant number 2014CB943303) and National Natural Science Foundation of China (grant numbers 31171440, 81361120247, 81471157) to Chen L.

Authors’ disclosures available online ().

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Dose-Dependent Neuroprotection and Neurotoxicity of Simvastatin through Reduction of Farnesyl Pyrophosphate in Mice Treated with Intracerebroventricular Injection of Aβ 1-42

Abstract

Background:

Objective & Methods:

Results:

Conclusion:

Keywords

INTRODUCTION

MATERIAL AND METHODS

Experimental animals

Preparation of AD model

Administration of drugs

Behavioral examination

Morris water maze task

Y-maze task

Histological examination

Toluidine blue staining

Stereological counts of cells

Hoechststaining

Electrophysiological analysis

Western blot analysis

Data analysis/statistics

RESULTS

Aβ1-42-injection (i.c.v.) impairs spatial cognitive function in mice

SV can rescue Aβ1-42-impaired spatial memory in a “U” type dose-dependent manner

Neuroprotection and neurotoxicity of SV on Aβ1-42-impaired pyramidal cells

SV can improve Aβ1-42-impaired LTP in a “U” type dose-dependent manner

SV enhances dose-dependently Aβ1-42-increased NR2B phosphorylation

SV-enhanced phospho-NR2B exerts neurotoxicity and anti-amnesic effects

Effects of SV-enhanced phospho-Akt on Aβ1-42-neurotoxicity

DISCUSSION

SV through reducing FPP enhances NR2B phosphorylation

SV through enhancing NMDAr activation aggravates Aβ1-42-neurotoxicity

SV-enhanced NMDAr function improves Aβ-impaired LTP

SV-enhanced Akt phosphorylation prevents Aβ-induced death of neuronal cells

CONCLUSIONS

Footnotes

ACKNOWLEDGMENTS

References

Aβ_1-42-injection (i.c.v.) impairs spatial cognitive function in mice

SV can rescue Aβ_1-42-impaired spatial memory in a “U” type dose-dependent manner

Neuroprotection and neurotoxicity of SV on Aβ_1-42-impaired pyramidal cells

SV can improve Aβ_1-42-impaired LTP in a “U” type dose-dependent manner

SV enhances dose-dependently Aβ_1-42-increased NR2B phosphorylation

Effects of SV-enhanced phospho-Akt on Aβ_1-42-neurotoxicity

SV through enhancing NMDAr activation aggravates Aβ_1-42-neurotoxicity