p-Hydroxybenzyl Alcohol Prevents Memory Deficits by Increasing Neurotrophic Factors and Decreasing Inflammatory Factors in a Mice Model of Alzheimer’s Disease

Abstract

p-hydroxybenzyl alcohol (HBA) is one of the major components of Gastrodia elata Blume (GEB) phenolic compound. HBA has been reported to have a protective effect on amyloid-β (Aβ) induced cell death. However, the systemic effects and the detail molecular mechanism of HBA in Alzheimer’s disease (AD) animal models is not clear. In this study, we revealed the protective effects and the potential mechanisms of HBA on the impairments of cognitive function induced by soluble Aβ oligomers. Our results showed that HBA prevented neuronal cells death in a dose-dependent manner. The working memory and the spatial memory were significantly lower in AD model mice. HBA treatment prevented the memory deficits of the AD mice. HBA treatment significantly prevented the decreased spine density and decreased expression levels of synaptic proteins induced by Aβ₄₂. In addition, both mRNA levels and protein levels of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in the Aβ₄₂-treated mice were decreased, the decreases were prevented by HBA treatment. The expression levels of TNF-α and IL-1β were increased by Aβ₄₂ treatment and the increase can be prevented by the HBA treatment. Moreover, HBA prevents the decreases in the level of nuclear erythroid 2 p45-related factor 2 (Nrf2) induced by Aβ₄₂ in hippocampal. Thus, we predict that HBA might prevent Aβ₄₂ oligomer-induced synapse and cognitive impairments through multiple targets including increasing Nrf2, increasing neurotrophic factors and decreasing inflammatory factors. Our study provided novel insights into the cellular mechanisms for the protective effects of HBA on AD.

Keywords

Alzheimer’s disease brain-derived neurotrophic factor glial cell line-derived neurotrophic factor inflammatory factors memory deficits nuclear erythroid 2 p45-related factor 2 p-hydroxybenzyl alcohol

INTRODUCTION

Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by neuroinflammation, synaptic dysfunction, cognitive deficits, memory loss, and extensive neuronal death [1 –4]. Currently, there is still lack of effective treatment for AD. As the global elderly population increases, AD poses a serious threat to human health.

There is growing evidence shows that the accumulation of amyloid-β peptide (Aβ) is an important contributor to the pathogenesis of AD [5]. Aβ amyloidogenesis is a complex process, including monomers, oligomers, protofibrils, and mature fibrils [6]. Soluble oligomeric Aβ (SO Aβ) in cerebrospinal fluid is considered as a biomarker of AD [7]. SO Aβ could also induced synapse loss and decreased learning and memory when injected into the mice brain [8 –11]. Therefore, preventing SO Aβ induced neurotoxicity is proposed to be a valuable therapeutic strategy for the treatment of AD.

p-hydroxybenzyl alcohol (HBA) is believed to be one of the major phenolic compounds of Gastrodia elata Blume (GEB) which has been used as a folk medicine [12]. 4-hydroxybenzyl alcohol is also a metabolic product of E. coli which is the cleavage product produced during the biosynthesis of the thiazole moiety of thiamine from tyrosine [13]. It has been reported that HBA has therapeutic potential in several central nervous system diseases, such as ischemic brain damage, epilepsy, and scopolamine-induced amnesia [14, 15]. HBA provided significant neuroprotecive effects on MCAO-induced injury through inhibiting lipid peroxidation and increasing endogenous antioxidant proteins [16]. HBA has been reported to attenuate the production of TNF-α to play an anti-inflammatory effect in lipopolysaccharide (LPS)-activated macrophage cells [17, 18]. HBA is also helpful in attenuating cognitive impairments in a mouse model of scopolamine-induced amnesia [19]. It has been demonstrated that HBA has a protective effect against Aβ-induced death of the BV-2 microglial-derived cell [20]. However, the systemic effects of HBA in AD animal models in vivo are not clear and the cellular mechanisms of HBA against Aβ neurotoxicity has not been well characterized. Therefore, we evaluated the protective effects of HBA on recognition memory and spatial memory in mouse model of AD and investigated the potential mechanisms of the beneficial effects of HBA in AD. Moreover, the disruption of synapse formation plays an important role in the memory deficits of AD [21]. Mounting evidence demonstrated that Aβ₄₂-induced synaptic loss in the hippocampus occurs at the early stage of AD [22]. However, the protective effects and the underlying mechanisms of HBA in Aβ oligomer-induced synaptic injury is unknown. In this study, we have evaluated the protective effects of HBA against Aβ oligomer-induced synaptic impairments both in cultured hippocampal neurons and hippocampal tissues, and then we provided the underlying mechanisms of the beneficial effects of HBA in AD.

MATERIALS AND METHODS

Aβ oligomer preparation

Preparation of soluble Aβ₄₂ as described previously [23]. Briefly, the synthesized Aβ₄₂ was dissolved in ice-cold 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (Sigma-Aldrich), vortexed and aliquoted for freezing. Aβ₄₂ was spin-vacuumed just prior to the experiment, dissolved in HFIP solution (final concentration: 10% (v/v) HFIP) and incubated at room temperature for 20 min. The solution was centrifuged at 14,000 g for 15 min at 4°C and the supernatant was collected. A 50 μM Aβ₄₂ solution was obtained after the HFIP was completely evaporated and were kept at room temperature under constant stirring for 48 h. And then the tube was transferred to refrigerator and maintained at 4°C.

SH-SY5Y cell culture and cell viability assay

The neuroblastoma SH-SY5Y cell line has been used extensively to neuron-like behavior in response to toxins in researches on AD and neurotoxicity [24]. SH-SY5Y cells purchased from American Type Culture Collection (Manassas, VA, USA) were cultured as described previously [25]. SH-SY5Y cells is maintained in a complete growth medium: Dulbecco’s Modified Eagle Media (DMEM) and F-12 mixture containing 10% fetal bovine serum (FBS), supplemented with 1% glutamine and 1% penicillin-streptomycin (Gaithersburg, MD, USA). SH-SY5Y cells were seeded onto a 96-well plate at a density of 1×10⁴ cells/well in 100 μl of medium and incubated at 37°C. When cells grew to about 80%, HBA (p-hydroxybenzyl alcohol) was added to 96-well plate, making the final concentration was 20, 40, 80, 120, and 160 μM respectively. 2 h later, Aβ₄₂ oligomers (1 μM) were added for 24 h. Cell viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colormetric assay (Sigma-Aldrich, St. Louis, MO, USA). After incubation, 5 μl of MTT (5 mg/ml) was added to each well and the cells were cultured for another 4 h, then 100 μl 10% SDS was added to each well to dissolve the formazan. The color reaction, generated by the reduction of tetrazine of MTT by mitochondria, was measured spectrophotometrically at wavelength 490 nm with a reference at 655 nm using the microplate reader (Thermo Fisher, USA). The cell viability was calculated by dividing the optical density of the treated group by that of the control group.

Animals

Two-month-old ICR mice or 1-day-old ICR mice were used in this study. The animals were housed in a temperature-controlled animal facility with a 12-h light/dark cycle. Experiments were approved by the Animal Care and Use Committee of the Ningbo University (Ningbo, China). All procedures followed the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1996).

Primary hippocampal neuronal cultures and transfection

Primary neuronal cultures from postnatal 1-day-old ICR mice were prepared as previous reported [23]. Briefly, the tissue was chopped and digested in 20 mg/ml papain for 20 min at 37°C. Then, the dissociated cells (7×10⁵ cells/cm²) were then plated on poly-D-lysine (100 μg/ml) pre-coated coverslips in Dulbecco’s modified Eagle media (Invitrogen) containing 10 % FBS (Invitrogen) and 10 % F-12 (Invitrogen). Cultures were maintained in an incubator with 5% CO₂ at 37°C for 24 h. And then the medium was changed to Neurobasal medium (Invitrogen) supplemented with 2% B27, 1% glutamine. Half of the medium was replaced with Neurobasal medium containing 2% B27, 1% glutamine every three days. At the 5^th day in vitro (DIV5), cytosine arabinofuranoside (Invitrogen) was added at a final concentration of 2 μM to decrease glial cell growth. The neurons were transfected with farnesylated enhanced green fluorescent protein (F-GFP) and GFP-actin by Lipofectamine 2000 (Invitrogen) at DIV5. At DIV15, neurons were incubated with 120 μM HBA for 2 h before 0.5 μM soluble Aβ₄₂ oligomers were added for another 3 h.

Confocal imaging and analysis

The morphology of spine was measured by confocal imaging. After drug treatments, the neurons were maintained in a recording chamber with extracellular solution (148.00 mM NaCl, 3.00 mM KCl, 3.00 mM CaCl₂, 10.00 mM HEPES, and 8.00 mM glucose, pH 7.3) at room temperature. Living neurons were captured by a Fluoview 1000 confocal microscope (Olympus, Tokyo, Japan) using a×60 oil objective, 1024×1024 resolution, at an excitation wavelength of 488 nm. To measure spine density, images were acquired at DIV15 in 2-D stack and were analyzed by using Fluoview-1000 software. All lengths of the secondary dendritic branches were measured by tracing their extension and spines were counted. For all analysis, images were analyzed blind to treatments and data were collected from at least three independent experiments.

Animal surgery

Two-month-old male ICR mice were anesthetized by intraperitoneal administration of sodium pentobarbital (50 mg/kg) before they were placed in a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA). Then the pipe (Shenzhen RWD Life Science) was buried into bilateral hippocampal regions of the mice using the following coordinates: AP -1.7 mm from bregma; ML ± 1.0 mm from the midline; DV -1.5 mm from pia mater. After 7 days postoperative recovery, experimental mice were given three consecutive injections Aβ (0.5 μg/mouse/day), control mice were given an equal volume of saline. HBA was dissolved in 0.9% sterile saline at a concentration of 1 mM and was intragastric administrated at a final concentration of 5, 15 mg/kg once a day for 18 days. HBA treatment was given 1 h before and continued throughout the behavioral tests.

Behavior tests

Novel object recognition test

The novel object recognition (NOR) task, consisted of a familiarization phase and a test phase, was carried out in an open-field arena (60×60×15 cm) on the 11^th to 12^th day after the first injection. On the first day, they were familiarized with two identical copies of a sample object for 5 min. On the second day, one of the objects was replaced by a novel one with a different shape and color, and the animals were also allowed to explore the arena for 5 min. To ensure the absence of olfactory cues, the open-field arena and the objects were cleaned thoroughly. Exploration was defined as sniffing or touching the objects with the nose or forepaws at the distance. The discrimination indexes, the ratio of the amount of time spent exploring any one of the two objects (training session) or the novel object (retention session) over the total time spent exploring both objects, was used to measure the cognitive function of animals.

Morris water maze

Morris water maze (MWM) was performed as described [23, 26]. Briefly, the equipment included a pool with a diameter of 110 cm that was filled with opaque water at approximately 22 ± 1 °C. Spatial memory is assessed by recording the latency time for the animal to escape from the water onto an escape platform (8 cm in diameter) during the place navigation phase. At the learning time, mice were given 90 s to find the hidden platform which was 1 cm below the water surface. A place navigation test of the Morris water maze consisted of four trials (interval 20-30 min) each day during the 14^th day to the 17^th day after surgery. The time that it took for an animal to reach the platform (latency period) was recorded. On the 18^th day, the platform was removed from the maze. A probe trial was conducted to measure the trajectories and entries of mice to the target quadrant with a video tracking system (Ethovision XT).

Tumor necrosis factor-alpha (TNF-α) assay

TNF-α levels in hippocampal tissues were assayed using a commercial mouse TNF-α ELISA Kit (LiankeBio, China). The hippocampus tissues were homogenized in RIPA buffer containing 0.1% PMSF (Solarbio, China) and hold for 1 h to be sufficiently lysed. The lysates were centrifuged at 13,200 rpm for 30 min at 4°C and the supernatant fraction was used for test. For these analyses, 20 μl of the supernatant from the hippocampal homogenate was used to assay TNF-α levels. The content of TNF-α was detected based on measures of absorbance at 450 nm/well and 570 nm/well in a 96 well plate reader (Thermo Scientific, USA). TNF-α levels were expressed as ng TNF-α/mg protein in the sample.

Interleukin-1Beta (IL-1β) assay

IL-1β levels in hippocampal tissues were assayed using a commercial mouse IL-1β ELISA Kit (LiankeBio, China). For these analyses, 20 μl of the supernatant from the hippocampal homogenate (as described for TNF-α estimation) was used to assay IL-1β levels. In this case, the content of IL-1β was analyzed based on measures of absorbance at 450 nm/well and 570 nm/well in a 96 well plate reader (Thermo Scientific, USA). IL-1β levels were expressed as ng IL-1β/mg protein in the sample.

Reverse transcription-quantitative PCR

Total RNA from hippocampal tissues were isolated using trizol reagent (Thermo Fisher Scientific, Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions, and 0.5 μg total RNA was used for reverse transcription reaction using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO CO., LTD. Life Science Department OSAKA JAPAN). Quantitative PCR (qPCR) was conducted using a Mx3005P Multiplex Quantitative PCR system (Aglient Stratagene, USA), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and nuclear factor-E2-related factor2 (Nrf2), mRNA levels were assessed by real-time PCR, with β-actin used as an internal control. PCR amplification was done by denaturation at 95°C for 10 s, and annealing and extension at 60°C for 15 s for 40 cycles. The relative expression level of BDNF, GDNF, and Nrf2 was calculated using the ΔΔCq method. qPCR analysis was conducted using primers as follows: β-actin Forward 5’-AACAGTCCGCCTAGAAGCAC-3’, Reverse 5’-CGTTGACATCCGTAAAGACC-3’, BDNF Forward 5’-GCCTTCATGCAACCGAAGTA-3’, Reverse 5’-TGAGTCTCCAGGACAGCAAA-3’, GDNF Forward 5’-AGAGGGGCAAAAATCGGG-3’, Reverse 5’-CCGCTGCAATATCGAAAGATCA-3’, Nrf2 Forward 5’-TCTGTGTACGGTTCTGCCTG-3’, Reverse 5’-CAGCTTTCTATACTGGCCGC-3’.

Western blot

The hippocampus was homogenized in RIPA buffer containing 0.1 % PMSF (Solarbio, China) and hold for 1 h to be sufficiently lysed. The lysates were centrifuged at 13,200 rpm for 30 min at 4°C and the supernatant fraction was used for protein analyses. The protein concentration in supernatant fraction was determined using the BCA protein assay kit (CWbio, China). Equal amounts of soluble protein (50 μg) were separated by 10% SDS-PAGE and transferred onto poly-vinylidene fluoride (PVDF) membranes (0.45 μm, Millipore, USA). After blocking with 5% fat-free milk (Solarbio, China) for 2 h, membranes were incubated with rabbit anti-synaptotagmin (1 : 5000, Millipore, USA), rabbit anti-post synaptic protein 95 (PSD95, 1 : 5000, Cell Signaling, USA), rabbit-synaptophysin (1 : 5000, Millipore, USA), rabbit- Glutamate receptor subunit 1(GluR1, 1 : 5000, Millipore, USA), rabbit anti-Nrf2 (1 : 1000, Cell Signaling, USA), rabbit anti-BDNF (1 : 1000, abcom, UK) and rabbit anti-GDNF(1 : 1000, abcom, UK), and mouse anti-β-actin (1 : 5000, 4A biotech, China) at 4 °C overnight. Then incubate it with chemical-dye conjugated anti-rabbit antibody or chemical-dye conjugated anti-mouse antibody, respectively. Target bands were detected and quantified with an automatic chemiluminescence imaging system (Tanon 5200, Shanghai).

Statistical analyses

Data are presented as mean±SEM. All data were analyzed using one-way ANOVA with post hoc comparisons, with the exception of the data of the place navigation test of the water maze trials, which were analyzed by two-way repeated-measures ANOVA with post hoc comparisons. p <0.05 was considered statistically significant.

RESULTS

HBA prevents the death of SH–SY5Y cells caused by oligomeric Aβ₄₂ in a dose-dependent manner

To investigate whether HBA has neuronal protective effects, we use MTT to measure the cell viability in oligomeric Aβ₄₂ and/or HBA-treated cells. The cell viability was obviously decreased in the cells cultured with Aβ₄₂ oligomers (p <0.001, Fig. 1A). HBA (20-160 μM) alone treatment did not has any influence on the cell viability of SH-SY5Y cells (Fig.1B). However, it prevented Aβ₄₂ oligomers-induced cell death in a dose-dependent manner (Fig. 1A). 20 μM HBA pre-treatment did not prevent Aβ₄₂ oligomers-induced cell death in SH-SY5Y cells, while 40-160 μM HBA incubation significantly increased the number of survival cells when compared with cells treated by Aβ₄₂ oligomers (p <0.05).

Fig.1

HBA prevents the death of SH-SY5Y cells caused by oligomeric Aβ₄₂ in a dose-dependent manner. A) The cell viability in oligomeric Aβ₄₂ and/or HBA-treated SH-SY5Y cells. ^***p <0.001 versus the control group, ^#p <0.05 versus the Aβ₄₂-treated group, ^###p <0.001 versus the Aβ₄₂-treated group. B) The cell viability in HBA-treated alone SH-SY5Y cells. n=18. HBA, p-hydroxybenzyl alcohol.

Administration of HBA reverses cognitive deficit of oligomeric-Aβ₄₂-treated mice

Spatial memory and recognition memory deficits were early impairments of AD [26]. Therefore, we use the Morris water maze and new object recognition task to examine the effect of HBA on Aβ₄₂-induced working memory and spatial memory deficits, respectively.

The discriminant indicators are used to evaluate the cognitive functions of animals in novel object recognition tasks. During the training period, there was no significant difference in the discrimination index of all these groups exposed to two identical subjects (Fig. 2A). In the retention session (replaced one of the original objects with a new object), the discrimination indexes of Aβ₄₂-treated mice were significantly lower than that of control mice (p <0.001, Fig. 2B). Two different doses of HBA were used to treat mice in this experiment (5 and 15 mg/kg). HBA alone treatment did not change the discrimination indexes of the mice. However, the treatment of HBA significantly prevented the decreased discrimination indexes induced by Aβ₄₂ (p <0.001, Fig. 2B). These results indicate that HBA might reverse Aβ₄₂-induced working memory deficits.

Fig.2

Effects of HBA on oligomeric-Aβ₄₂-induced cognitive deficits in the new object recognition test in mice. A) Quantitative comparison of the discrimination index of the object in the training session. B) Quantitative comparison of the recognition index in the retention session. ^***p <0.001 versus the control group, ^###p <0.001 versus the Aβ₄₂-treated group, n=7. HBA, p-hydroxybenzyl alcohol.

To further investigate whether HBA prevents spatial memory deficits produced by injection of Aβ₄₂ oligomers into hippocampal regions of the mice, we examined memory performance in a Morris water maze test in mice treated with Aβ₄₂ oligomers in the presence or the absence of HBA. Two-way ANOVA for repeated-measures revealed significant changes in drug effect [F(5, 111)=7.47, p <0.001, Fig. 3A]. The escape latency of Aβ-treated mice on day 3 and 4 was significantly prolonged compared with that of the control mice (p <0.01). The escape latency of 5 or 15 mg/kg HBA alone treated mice did not significantly different from that of the control mice. However, 5 or 15 mg/kg HBA treatment prevented the prolongation of latency induced by Aβ (p <0.001, Fig. 3A). In the probe test, after the hidden platform was removed from the target quadrant, the Aβ₄₂-treated mice spent shorter time in the target quadrant compared with the control mice (p <0.001). Injection of HBA reversed the decrease of time in the target quadrant of the Aβ-treated mice (p <0.01). However, treatment of HBA alone at the dose of 5 or 15 mg/kg did not has effect on the time in the target quadrant (Fig. 3B). Moreover, we measured the numbers that the mice swam cross the original place where the platform is. The numbers of the target platform crosses were decreased in the Aβ-treated mice (p <0.05). Injection of HBA reversed the decreased crosses over the target platform of Aβ-treated mice (p <0.05). However, treatment of HBA alone did not have effect on numbers of the target platform crosses (Fig. 3C). We did not observe significant difference in velocity among the six groups in the water maze task (Fig. 3D), indicating that the differences in latency, time and number of crosses over the target platform among the groups were not caused by the differences in velocity. Taken together, these data suggested that HBA prevented Aβ-induced impairments in spatial learning and memory.

Fig.3

Effects of HBA on oligomeric-Aβ-induced spatial learning and memory deficits in the water maze tests in mice. A) The escape latency of the control, oligomeric Aβ₄₂-treated group, and/or HBA-treated group (5, 15 mg/kg) during the four training days. Oligomeric Aβ₄₂ overall increased escape latency, which was reversed by HBA (5 or 15 mg/kg). ^***p <0.001 versus the control group, ^##p <0.01 versus Aβ₄₂-treated group, ^###p <0.001 versus the Aβ₄₂-treated group. n=7. B) Quantitative comparison of the time in the target quadrant. ^***p <0.001 versus the control group, ^##p <0.05 versus Aβ₄₂-treated group, n=7. C) Quantitative comparison of the number of target platform crosses. ^*p <0.05 versus the control group, ^#p <0.05 versus Aβ₄₂-treated group, n=7. D) Swimming speed of the mice in the control, Aβ₄₂ oligomer, and/or HBA-treated groups. No significant difference was found among the six groups of mice. HBA, p-hydroxybenzyl alcohol.

HBA treatment prevents the synapse formation impairment induced by Aβ₄₂ in the hippocampal neurons and hippocampal tissue

To investigate the role of Aβ₄₂ and HBA in synapse formation, the expression levels of the synaptic proteins synaptotagmin, synaptophysin, PSD 95, and GluR1 were measured in the hippocampal of Aβ₄₂ and/or HBA treated mice. The expression levels of synaptotagmin, synaptophysin, PSD 95, and GluR1 were significantly decreased in the hippocampal of Aβ₄₂ treated mice (p <0.05). 5 and 15 mg/kg HBA treatment significantly prevented the decreased expression levels of synaptotagmin, synaptophysin, PSD 95, and GluR1 induced by Aβ₄₂ (p <0.05, Fig. 4).

Fig.4

HBA treatment prevented the decreased expression levels of synaptic proteins in the hippocampal of the Aβ₄₂-treated mice. A) Sample western-blot plot of synaptotagmin and β-actin in the control, oligomeric Aβ₄₂-treated and/or HBA-treated mice (5, 15 mg/kg) and quantitative comparison of hippocampal synaptotagmin levels in different groups. B) Sample western-blot plot of PSD 95 and β-actin in the control, oligomeric Aβ₄₂-treated mice, and/or HBA-treated mice (5, 15 mg/kg) and quantitative comparison of hippocampal PSD 95 levels in different groups. C) Sample western-blot plot of synaptophysin and β-actin in the control, oligomeric Aβ₄₂-treated and/or HBA-treated mice (5, 15 mg/kg) and quantitative comparison of hippocampal synaptophysin levels in different groups. D) Sample western-blot plot of GluR1and β-actin in the control, oligomeric Aβ₄₂-treated and/or HBA-treated mice (5, 15 mg/kg) and quantitative comparison of hippocampal GluR1 levels in different groups. ^*p <0.05 versus the control group, ^**p <0.01 versus the control group, ^***p <0.001 versus the control group, ^#p <0.05 versus the Aβ₄₂-treated group, ^##p <0.01 versus the Aβ₄₂-treated group, ^###p <0.001 versus the Aβ₄₂-treated group n=7. HBA, p-hydroxybenzyl alcohol; PSD95, post synaptic protein 95; GluR1, Glutamate receptor subunit 1.

To further confirm the role of HBA on synapse formation, dendritic spine density at DIV15 was quantified. Application of oligomeric Aβ₄₂ potently decreased the spine density (Fig. 5A). 120 μM HBA pre-treatment did not alter spine density, but it significantly prevented the Aβ₄₂ oligomers induced spine loss. The spine density in the Aβ₄₂ oligomers and 120 μM HBA co-treated group was significantly higher than that in the group treated with Aβ₄₂ oligomers alone (p <0.01, Fig. 5B) and was not significantly different from that in the control group (Fig. 5B). These results suggested that HBA prevented dendritic spine loss caused by Aβ₄₂ oligomers in hippocampal neurons.

Fig.5

HBA prevented Aβ₄₂-oligomer-induced spine loss at DIV15. A) Spine morphology was revealed by co-transfection of F-GFP with GFP-actin. Representative images of neurons treated with vehicle, 0.5 μM soluble Aβ₄₂ oligomers, 120 μM HBA + Aβ₄₂ oligomers, 120 μM HBA. Scale bar: 20 and 5 μm. B) Quantitative comparison of the density of dendritic spine. ^**p <0.01 versus the vehicle-treated group, ^##p <0.01 versus the Aβ₄₂-treated group, n = 15. HBA, p-hydroxybenzyl alcohol.

HBA treatment prevents the decreased mRNA level and protein level of BDNF and GDNF in the hippocampal of the Aβ₄₂-treated mice

To investigate the underlying mechanisms of the protective effects of HBA on neurotoxicity, we measured both the mRNA levels and protein levels of BDNF and GDNF. Both the mRNA levels and protein levels of BDNF and GDNF were significantly lower in the oligomic Aβ₄₂-induced mice than those of the control mice (p <0.05, Fig. 6). 5 and 15 mg/kg HBA alone treatment did not have effect on mRNA levels and protein levels of BDNF and GDNF. However, it prevented the decreased mRNA levels and protein levels of BDNF and GDNF induced by Aβ₄₂ (p <0.05, Fig. 6).

Fig.6

HBA prevented the decreased levels of BDNF and GDNF by Aβ₄₂-oligomer in hippocampal tissues. A) HBA prevented the decreased expression level of BDNF by Aβ₄₂-oligomer in hippocampal tissues. B) HBA prevented the decreased expression level of GDNF by Aβ₄₂-oligomer in hippocampal tissues. C) Sample western-blot plot of hippocampal BDNF and quantitative comparison of its levels in the control, oligomeric Aβ₄₂-treated group, and/or HBA-treated mice (5, 15 mg/kg). D) Sample western-blot plot of hippocampal GDNF and quantitative comparison of its levels in the control, oligomeric Aβ₄₂-treated group, and/or HBA-treated mice (5, 15 mg/kg). ^*p <0.05 versus the control group, ^***p <0.001 versus the control group. ^#p <0.05 versus the Aβ₄₂-treated group, ^##p <0.01 versus the Aβ₄₂-treated group, ^###p <0.001 versus the Aβ₄₂-treated group, n=7. HBA, p-hydroxybenzyl alcohol; BDNF, brain-derived neurotrophic factor; GDNF, glial cell line-derived neurotrophic factor.

HBA prevents the increased levels of TNF-α and IL-1β in hippocampal of the Aβ₄₂-oligomer treated mice

To investigate whether HBA executed its effects through decreasing inflammatory factors, we measured the content of TNF-α and IL-1β in the hippocampal tissues of the experimental mice. The concentrations of TNF-α and IL-1β were significantly higher in the oligomic Aβ₄₂-induced mice compared to those of the control mice (p <0.001, Fig. 7). HBA alone treatment did not alter the levels of TNF-α and IL-1β. However, HBA treatment significantly prevented Aβ₄₂ oligomers-induced increase in the levels of TNF-α and IL-1β (p <0.01, Fig. 7).

Fig.7

HBA prevents the increase of the levels of TNF-α and IL-1β induced by Aβ₄₂-oligomer in hippocampus. A) HBA prevented the increased levels of TNF-α induced by Aβ₄₂-oligomer in hippocampal tissues. B) HBA prevented the increased levels of IL-1β induced by Aβ₄₂-oligomer in hippocampal tissues. ^***p <0.001 versus the control group, ^##p <0.01 versus the Aβ₄₂-treated group, ^###p <0.001 versus the Aβ₄₂-treated group, n=7. HBA, p-hydroxybenzyl alcohol; TNF-α, Tumor necrosis factor-alpha; IL-1β, Interleukin-1β.

HBA prevents the decreases in the level of Nrf2 induced by Aβ₄₂-oligomer in hippocampal

To investigate the whether Nrf2 is involved in the protective effects of HBA, we measured both the mRNA level and protein level of Nrf2. Both the mRNA level and protein level of Nrf2 was significantly lower in the oligomic Aβ₄₂-induced group than those of the control group (p <0.05, Fig. 8). 5 and 15 mg/kg HBA alone treatment did not alter the level of Nrf2, but it prevented the decreased expression level of Nrf2 induced by Aβ₄₂ (p <0.01, Fig. 8).

Fig.8

HBA treatment prevented the decreased expression level of Nrf2 in the hippocampal of the Aβ₄₂-treated mice. A) Quantitative comparison of Nrf2 mRNA levels in the hippocampal neurons in different groups. B) Sample western-blot plot of hippocampal Nrf2 and quantitative comparison of its level in the control, oligomeric Aβ₄₂-treated group, and/or HBA-treated mice (5, 15 mg/kg). ^*p <0.05 versus the control group, ^***p <0.001 versus the control group, ^##p <0.01 versus the Aβ₄₂-treated group, ^###p <0.001 versus the Aβ₄₂-treated group, n=7. HBA, p-hydroxybenzyl alcohol; Nrf2, nuclear erythroid 2 p45-related factor 2.

DISCUSSION

In present study, using mouse and cellular models of AD, we provide additional evidence for the neuroprotective effects of HBA on learning and memory deficits in the brains of AD mice and for the prevention of spine loss in cellular model of AD. We provided a new perspective on the potential mechanism of HBA for AD. HBA might improve cognitive function and prevent synapse damage through multiple ways including increasing Nrf2, increasing neurotrophic factors, and anti-inflammation effects.

First of all, our results showed that HBA can prevent AD-like phenotypes induced by Aβ₄₂ oligomers in a dose-dependent manner in SH-SY5Y cells. In addition, HBA is effective in Aβ₄₂ oligomers-induced impairments of recognition and spatial memory in mice. Aβ₄₂ oligomers are considered to be the main pathological factors of AD. Numerous reports have described that the injection of Aβ₄₂ oligomers into rodent hippocampus may result in concomitant neuronal damage and impairments of cognitive function [11]. We demonstrate that Aβ₄₂ oligomers cause spatial memory and recognition memory deficits in mice, which is consistence with previous studies [23]. Chronic systemic administration of HBA for 18 days (5 mg/kg and 15 mg/kg) attenuated cognitive impairments induced by oligomeric Aβ₄₂, suggesting that HBA might be developed to treat AD.

It has been demonstrated that synaptic impairments, rather than neuronal degeneration are synchronized with the impairments of cognitive function, suggesting that synaptic impairments may play a central role in the pathogenesis of AD [27 –30]. In the present study, we studied the effect of HBA and Aβ₄₂ oligomers on the synapse formation. We found that both the spine density and the expression of synaptic proteins were decreased by Aβ₄₂ oligomers. HBA treatment prevent the decrease of synaptic proteins and loss of spines. This result suggests that HBA also produced the neuroprotective effects on the impairments of synapse formation induced by oligomeric Aβ₄₂.

Neurotrophic factor might be involved in the protective effects of HBA on synapse. BDNF is neurotrophins that is highly expressed in the brain and has been widely accepted that its function in regulating synapses, with structural and functional effects [31]. GDNF also has been reported to plays an important role in cognitive impairment [32]. Our results show that Aβ₄₂ oligomers exhibit a strong inhibitory effect on the expression levels of BDNF and GDNF in the hippocampus, and HBA prevents this decrease in BDNF and GDNF induced by Aβ₄₂ oligomers. This result suggests that the increasing expression levels of BDNF and GDNF may be related to improvement of the synaptic and cognitive function by HBA.

Anti-inflammation effects might also involve in the neural protective effects of HBA. Inflammation is an underlying component in AD and its associated neuropathology [33]. Moreover, long-term use of anti-inflammatory drugs is associated with the reduced risk of developing AD [2]. Inflammatory signals triggered by TNF-α and IL-1β play key roles in the normal formation and regulation of memory and plasticity [34]. High expression of TNF-α and IL-1β in the brain directly inhibits hippocampal synaptic plasticity [34 –36]. In present study, we found that content of TNF-α and IL-1β was significantly increased by oligomeric Aβ₄₂. Pre-treatment with HBA protected neurons against Aβ₄₂-induced increase of TNF-α and IL-1β. Thus, HBA may exert neural protective effects through decreasing the inflammation factors.

HBA might also play its beneficial role in AD via its antioxidant effect. In this study we found that expression levels of Nrf2 is decreased in AD model mice which is consistent with the previous study that Nrf2 was decreased in the AD model mice [37, 38]. HBA treatment prevented the decreased expression level of Nrf2 in AD. It has been reported that Nrf2 can translocate into nucleus where it functions as a strong transcriptional activator of antioxidant response element (ARE)-responsive genes such as NAD(P)H: quinone oxidoreductase (NQO1) and heme oxygenase-1 (HO-1) [39]. Our result is consistent with previous study that HBA show its protective role through activating Nrf2 and anti-oxidant effects [14]. Beside the important role of Nrf2 in anti-oxidation, Nrf2 has also been show to play a key role in expression of inflammation factors and neurotrophic factors [40 –43]. Nrf2 can attenuate the inflammatory response through preventing the overproduction of inflammatory mediators such as TNF-α and IL-1β via ARE dependent or independent pathway [43 –45]. It has been reported that Nrf2 is recruited to transcription regulatory regions of proinflammatory cytokine genes and blocking transcription activity [45]. Nrf2 signal pathway also has been reported to take part in the regulation of the expression of neurotrophic factors, such as BDNF and GDNF [43, 46]. Knockdown of Nrf2 will decrease BDNF and GDNF levels in the hippocampus [42, 43]. It has been reported that GDNF and BDNF gene expression can be increased partly via Nrf2/ARE pathway [14 , 46]. Thus, HBA might increase the expression of neurotrophic factors and decrease inflammatory factors through increasing the content of Nrf2. However it is possible HBA can reduce inflammatory factors and increase neurotrophic factors through other pathway independent of Nrf2 [15, 47]. The relationship between Nrf2, neurotrophic factors, and inflammatory factors in HBA with the AD model mice needs further study. It should be mentioned that HBA is a safe derivative of traditional herbal medicine GEB and it can cross the blood-brain barrier [48]. Therefore, HBA may prevent AD progression at early stage of AD pathogenesis and thus has great potential to become a new AD treatment.

Footnotes

ACKNOWLEDGMENTS

This research was supported by the National Natural Science Foundation of China (Grant No. 81771166 and No. 81471398), Zhejiang Provincial Natural Science Foundation of China (Grant No. LY16H090001), Natural Science Foundation of Ningbo (Grant No. 2015A610211), Ningbo municipal innovation team of life science and health (Grant No. 2015C110026), and the K.C. Wong Magna Fund in Ningbo University.

Authors’ disclosures available online ().

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p-Hydroxybenzyl Alcohol Prevents Memory Deficits by Increasing Neurotrophic Factors and Decreasing Inflammatory Factors in a Mice Model of Alzheimer’s Disease

Abstract

Keywords

INTRODUCTION

MATERIALS AND METHODS

Aβ oligomer preparation

SH-SY5Y cell culture and cell viability assay

Animals

Primary hippocampal neuronal cultures and transfection

Confocal imaging and analysis

Animal surgery

Behavior tests

Novel object recognition test

Morris water maze

Tumor necrosis factor-alpha (TNF-α) assay

Interleukin-1Beta (IL-1β) assay

Reverse transcription-quantitative PCR

Western blot

Statistical analyses

RESULTS

HBA prevents the death of SH–SY5Y cells caused by oligomeric Aβ42 in a dose-dependent manner

Administration of HBA reverses cognitive deficit of oligomeric-Aβ42-treated mice

HBA treatment prevents the synapse formation impairment induced by Aβ42 in the hippocampal neurons and hippocampal tissue

HBA treatment prevents the decreased mRNA level and protein level of BDNF and GDNF in the hippocampal of the Aβ42-treated mice

HBA prevents the increased levels of TNF-α and IL-1β in hippocampal of the Aβ42-oligomer treated mice

HBA prevents the decreases in the level of Nrf2 induced by Aβ42-oligomer in hippocampal

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

HBA prevents the death of SH–SY5Y cells caused by oligomeric Aβ₄₂ in a dose-dependent manner

Administration of HBA reverses cognitive deficit of oligomeric-Aβ₄₂-treated mice

HBA treatment prevents the synapse formation impairment induced by Aβ₄₂ in the hippocampal neurons and hippocampal tissue

HBA treatment prevents the decreased mRNA level and protein level of BDNF and GDNF in the hippocampal of the Aβ₄₂-treated mice

HBA prevents the increased levels of TNF-α and IL-1β in hippocampal of the Aβ₄₂-oligomer treated mice

HBA prevents the decreases in the level of Nrf2 induced by Aβ₄₂-oligomer in hippocampal