Honokiol Alleviates Cognitive Deficits of Alzheimer’s Disease (PS1 V97L ) Transgenic Mice by Activating Mitochondrial SIRT3

Abstract

Accumulating evidence has demonstrated that mitochondrial dysfunction is a prominent early event in the progression of Alzheimer’s disease (AD). Whether protecting mitochondrial function can reduce amyloid-β oligomer (AβO)-induced neurotoxicity in PS1_V97L transgenic mice remains unknown. In this study, we examined the possible protective effects of honokiol (HKL) on mitochondrial dysfunction induced by AβOs in neurons, and cognitive function in AD PS1_V97Ltransgenic mice. We determined that HKL increased mitochondrial sirtuin 3 (SIRT3) expression levels and activity, which in turn markedly improved ATP production and weakened mitochondrial reactive oxygen species production. We demonstrated that the enhanced energy metabolism and attenuated oxidative stress of HKL restores AβO-mediated mitochondrial dysfunction in vitro and in vivo. Consequently, memory deficits in the PS1_V97L transgenic mice were rescued by HKL in the early stages. These results suggest that HKL has therapeutic potential for delaying the onset of AD symptoms by alleviating mitochondrial impairment and increasing hyperactivation of SIRT3 in the pathogenesis of preclinical AD.

Keywords

Alzheimer’s disease amyloid-β protein cognitive dysfunction mitochondria sirtuin 3 (SIRT3)

INTRODUCTION

Much evidence has accumulated to indicate that amyloid-β (Aβ) and Aβ oligomers (AβOs) play a decisive role in the pathogenesis of Alzheimer’s disease (AD) [1]. Despite tremendous efforts to uncover the underlying pathology of AD in recent years, no effective treatment that can slow the progress of AD has been identified to date [2]. Therefore, increasingly, studies are focusing on mitochondrial dysfunction and energy metabolism damage, which have recently been shown to be associated with AD [3, 4].

Research has indicated that mitochondrial dysfunction is the earliest manifestation of AD and constitutes the major pathological change during the course of this disease [5, 6]. AβOs are closely related to mitochondrial dysfunction [7] and affect mitochondrial function in various ways [8, 9]. Hence, reviving mitochondrial function may be a plausible approach in treating AD. The key to such treatment is protecting mitochondrial function, including electron transfer, adenosine triphosphate (ATP) synthesis, and decreasing mitochondrial membrane potential (mΔψ) and production of reactive oxygen species (ROS) in AD [10].

Acetylation and deacetylation are crucial post-translational mechanisms to alter the activity of proteins. The deacetylation of mitochondrial silencing regulatory proteins (sirtuins), particularly sirtuin 3 (SIRT3), plays an important role in maintaining mitochondrial function [11]. In recent years, it has been found that the downregulation of mitochondrial SIRT3 is associated with mitochondrial dysfunction in AD [12]. Interestingly, the expression of SIRT3 in experimental mice and patients with AD continuously decreases as the disease progresses [13]. Therefore, activation of mitochondrial SIRT3 may be a new therapeutic strategy for the treatment of AD.

Honokiol (HKL, C₁₈H₁₈O₂), isolated from the bark of Magnolia officinalis, can cross the blood– brain barrier to play a therapeutic role in individuals with anxiety, epilepsy, and cerebrovascular injury [14]. In addition, HKL has been proven to prevent age-related memory and learning deficits found in senescence-accelerated (SAMP8) mice by safeguarding cholinergic neurons [15], and to attenuate neuronal apoptosis via the mitochondrial apoptosis pathway in model mice created by intra-hippocampal AβO injection [16].

To date, it is unclear how HKL enhances mitochondrial function during the early stages of AD. We hypothesized that HKL may be a candidate for improving mitochondrial function in the early stages of AD and explore this hypothesis and the underlying mechanisms in this study.

MATERIALS AND METHODS

Ethics statement

The study protocol was approved by the Ethics Committee of Capital Medical University, and every effort was made to minimize the number and suffering of animals.

Animals

Male and female PS1_V97L-transgenic (Tg) mice, aged 6 months, and their sex- and age-matched wild-type (WT) litter-mates (five males and five females in each group) were enrolled in the experiment. Data from our previous studies clearly showed that Aβ oligomers levels in the PS1_V97L 6-month-old brain were just beginning to rise and memory loss occurred at the age of 9 months [17]. Therefore, we chose PS1_V97L-Tg mice at 6 to 9 months of age to reflect preclinical AD. The mice were divided into four groups (each group, n = 10): WT-control group, WT-HKL group, PS1_V97L-Tg control group, and PS1_V97L-Tg HKL intervention group. These mice were housed under standard conditions. PS1_V97L-Tg mice expressing human PS1 harboring the V97L mutation were generated as previously described [18]. WT littermates served as negative controls.

Aβ oligomer preparation

AβOs were generated as described previously [19]. Briefly, 1 mg Aβ_{1 - 42} lyophilized powder (Invitrogen, Carlsbad, CA, USA) was suspended in 1,1,1,3,3,3-hexa-fluoro-2-propanol (Sigma, St Louis, MO, USA) to a concentration of 1 mM, followed by vibration mixing to generate a homogenous suspension. Next, the peptide film solution was resuspended in dimethyl sulfoxide (DMSO) to 5 mM and sonicated for 10 min. Then, the DMSO-Aβ_1–42 solution was diluted with cold phosphate-buffered saline (PBS) containing 0.05% sodium dodecyl sulfate (SDS) to 100μM, followed by a 24-h incubation at 4°C. For higher aggregation, the peptide solution was further diluted with PBS to 11.1μM and incubated for another 2 weeks at 4°C. Oligomer preparations were centrifuged at 13,000 rpm for 10 min at 4°C prior to use.

Drug treatments

Primary cultured hippocampal neurons were divided into four groups: control, AβOs, AβOs plus HKL, and AβOs plus HKL plus SIRT3 inhibitor (AGK7) (CAS 304896-21-7, Santa Cruz, Dallas, TX, USA). Neuronal cell drug treatments were performed using 10μM HKL, 1μM AβOs, or 20μM AGK7. AGK7 was preincubated for 1 h prior to AβO treatment. The data from each group comprise three repeated independent experiments. Different drugs were given to the corresponding mice: WT-HKL group (HKL, 20 mg/kg), WT-control group (sterile saline, 20 mg/kg), PS1_V97L-Tg HKL intervention group (HKL, 20 mg/kg), and PS1_V97L– Tg control group (sterile saline, 20 mg/kg). HKL doses were chosen with reference to previous studies [20]. HKL was dissolved in sterile saline containing 10% DMSO, while control groups were administered normal sterile saline containing 10% DMSO. HKL and sterile saline were administered to the respective groups via intraperitoneal injection (i.p.) once per day for 3 months.

Primary hippocampal neuron culture

Primary hippocampal neurons were obtained from 18-day pregnant Sprague– Dawley rats (Experimental Animal Center, Military Academy of Sciences, Beijing, China). The rats were sacrificed using 10% chloral hydrate anesthesia, and the brains were removed from the fetal rats. Briefly, the hippocampi were separated under sterile conditions and digested using 0.25% trypsin at 37°C for 20 min. After filtering through a 200-Mesh filter, cells were seeded into polylysine-coated 6-well plates at a density of 4×10⁵/mL, and then cultured in a 37°C, 5% CO₂ incubator. After 24 h, the cells were incubated at 37°C with serum-free Neurobasal-A medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and B-27 supplements (Gibco, Grand Island, NY, USA). The medium was changed every 2 days. After 7 days, the cultured cells were ready to be processed.

Cell viability

Cytotoxicity was measured using a 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H- tetrazolium bromide (MTT) assay (M2128, Sigma) according to the manufacturer’s instructions. Briefly, 5×10³ cells were plated into 96-well plates and grown. At different time points, cells were incubated with MTT in serum-free medium at 37°C for 4 h. The medium was then removed and 150μL DMSO was added to the cells. After vortexing for 10 min, the absorbance at 490 nm was measured using a microplate reader.

Mitochondrial preparation

Mitochondria were isolated from primary hippocampal neurons or murine hippocampal brain tissue using the appropriate kit (Invitrogen). Briefly, neurons or brain tissues were lysed in mitochondrial fractionation lysis buffer on ice. The cell lysates were then centrifuged at 700×g for 10 min at 4°C to remove cell debris. Following centrifugation, the supernatant was transferred into a new tube and centrifuged again at 12,000×g for 5 min at 4°C. The resulting supernatant and pellet were collected as the cytosolic fraction and mitochondrial fraction, respectively.

Immunoblot analysis

Samples were prepared in sample loading buffer and proteins were separated using SDS– PAGE (Invitrogen), and then transferred to a PVDF membrane (Invitrogen) for immunoblotting. After blocking in TBS buffer containing 5% nonfat dry milk for 1 h at room temperature (20– 25°C), the membrane was then incubated with primary antibodies and softly shaken overnight at 4°C. This was accompanied by incubation with the corresponding secondary antibody for 1 h at room temperature (20– 25°C). The following antibodies were used: rabbit polyclonal antibody to acetyl Lysine (1:2,000 dilution; ab80178, Abcam, Cambridge, UK), rabbit polyclonal antibody to SIRT3 (1:2,000 dilution; ab189860, Abcam), mouse monoclonal antibody to OSCP (A-8) (1:5,000 dilution; sc-365162, Santa Cruz), rabbit monoclonal antibody to ATP5O (acetyl-K139) (1:5,000 dilution; ab214339; Abcam), rabbit monoclonal antibody to SOD2/MnSOD (1:5,000 dilution; acetyl-K68; ab137037, Abcam), rabbit polyclonal antibody to SOD2 (1:3,000 dilution; ab13533, Abcam), mouse monoclonal antibody to purified Aβ_{1 - 16} (6E10; 1:1,000 dilution; sc-803001, Santa Cruz), and mouse monoclonal antibody to Cox IV as a mitochondrial marker (1:2,000 dilution; ab33985, Abcam).

Mitochondrial membrane potential assay

JC-1kits (Jiancheng Bioengineering Institute, Nanjing, China) were used to assess the mΔψ of the neurons according to the manufacturer’s protocols. After 24 h of treatment with AβOs, HKL, or AGK7, cells were trypsinized, collected, centrifuged, resuspended in 0.5 mL DMEM and 0.5 mL JC staining solution, and then incubated for 20 min. Cells were washed twice with JC stain buffer solution, and then analyzed using flow cytometry (FCM).

Measurement of mitochondrial energy metabolism related parameters

An appropriate concentration of mitochondria (2 mg/mL) was placed in assay buffer [21]. F1FO-ATP synthase enzymatic activity and ATP production were measured using a quantitative test kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

Measurement of mitochondrial oxidative stress related parameters

An appropriate concentration of mitochondria (2 mg/mL) was placed in the assay buffer [21]. SOD synthase catalytic activity and ROS levels were measured using a quantitative test kit (Jiancheng Bioengineering institute, Nanjing, China) according to the manufacturer’s instructions.

Quantification of apoptotic cells by flow cytometry

The extent of apoptosis was measured using an annexin V-FITC/PI apoptosis detection kit (Invitrogen) according to the manufacturer’s instructions. Cells treated with HKL for 24 h were harvested, washed twice with PBS, gently resuspended in binding buffer, and incubated with annexin-V-FITC and propidium iodide (PI) in the dark for 10 min, and then detected using flow cytometry (Accuri C6, BD, Franklin Lankes, NJ, USA).

Immunohistochemistry

Rats were transcardially perfused with PBS and 4% paraformaldehyde (PFA). The brains were dissected and post-fixed for 24 h in 4% PFA and embedded in paraffin. The hippocampus was identified and used for immunohistochemical analysis. Slides were incubated in blocking solution (PBS containing 2% BSA, 1% normal goat serum, and 0.2% Triton X-100) and afterward incubated for 1 h in permeabilization buffer (10% goat serum 0.1% Triton X-100 in PBS). Sections were then incubated with rabbit polyclonal antibody to SIRT3 (1:2,000 dilution; ab189860; Abcam) at 4°C overnight, followed by washing and further incubation with secondary antibodies for 2 h in antibody solution (5% goat serum, 0.05% Triton X-100 in PBS).

Behavioral tests

Spatial memory in mice was assessed at 9 months of age using the Morris water maze. For five consecutive days, four groups of mice were trained to find a platform hidden below the water surface in a 100-cm diameter pool. Training consisted of three trials per day with an inter-trial interval of 30 s [22]. On the 6th day, retention of trained spatial memory was assessed using a probe trial in which mice performed a 60-s free-search of the pool with the platform removed. The escape latency, swimming distance, and movement path were recorded.

Statistical analysis

All data are expressed as group means±SEM. According to animal experimental design principles and similar animal research literatures [23 –25], we determined a sample size of 10 mice per group. Group differences in the escape latency and distance in the Morris water maze training task were analyzed using two-way analysis of variance (ANOVA) with repeated measures, with the factors being treatment and training day. The other data were analyzed using one-way ANOVA followed by post-hoc Bonferroni test to detect intergroup differences. GraphPad Prism software (GraphPad Software, San Diego, CA) was used to perform the statistical analysis. P values less than 0.05 were considered significant.

RESULTS

HKL improves neuronal cell viability after incubation with Aβ oligomers in hippocampal neurons

In order to verify the AβO preparation, Aβ_{1 - 42}, with a molecular weight of 4 kDa, was used as a control in immunoblotting. The AβOs consisted mainly of 9- and 12-mers; the molecular weight ranged from 30 kDa to 50 kDa (Fig. 1A). Our results suggested that the Aβ oligomers used in this experiment were acceptable for the experiments.

We used MTT chemiluminescence to measure cell viability when the primary hippocampal neuronal cells were exposed to different doses of AβOs. Low doses of AβOs (250 nM) did not cause significant cell death; the cell viability of the medium (1μM)-and high (10μM)-dose groups decreased to 80.40±3.57% (p < 0.05, n = 6) and 51.26±3.28% (p < 0.01, n = 6), respectively, as compared with the control group (Fig. 1B).

Fig.1

HKL protects against Aβ oligomer-induced toxicity in cultured rat hippocampal neurons. A) The prepared Aβ oligomers were verified using western blotting. Aβm and AβOs represent Aβ monomer (synthetic Aβ_{1 - 42}) and Aβ oligomers, respectively. B) Aβ oligomers and HKL were incubated in a cell culture dish for 24 h before the MTT assay. The effect of Aβ oligomer-induced cell viability on primary cultured hippocampus neuron. C) The protective effect of HKL on Aβ oligomer-induced cell viability. Results are presented as the mean±SD (n = 10 samples from three independent experiments). *p < 0.05 versus the control group, **p < 0.01 versus the control group, ***p < 0.001 versus the control group.

Secondly, to assess whether HKL improves neuronal cell viability following AβO stimulation, we added different doses of HKL to 1μM AβO-treated neuronal cells for 24 h. With 5μM and 10μM HKL treatments, cell viability increased to 88.02±3.79% (p < 0.05, n = 6) and 93.9±4.24% (p < 0.05, n = 6) as compared with the AβOs group, respectively (Fig. 1C).

Based on the above findings, 1μM AβOs was the concentration chosen for assaying cell toxicity, and cells were treated with 10μM HKL in the subsequent experiments.

HKL increases SIRT3 levels and its activity in AβO-induced hippocampal neurons

We examined the effect of HKL on the total lysine acetylation level in primary cultured hippocampal neurons. It was determined that different doses of HKL reduced the level of mitochondrial acetylation (Fig. 2A). This demonstrated the dose-dependent effect of HKL on neuronal cells. This result suggested that HKL may potentially activate SIRT3 in neurons. Next, we sought to determine whether HKL could also reduce the level of mitochondrial protein acetylation at different time points. Hippocampal neurons were treated with 10μM HKL and mitochondrial acetylation levels were analyzed over time. This demonstrated the time-dependent effect of HKL on neuronal cells. Peak activation was seen at 24 h (Fig. 2B).

Fig.2

HKL activates SIRT3 and deacetylates mitochondrial proteins in neurons. A) Primary cultured hippocampal neurons were treated with different doses of HKL, as indicated. Mitochondrial lysate was prepared and analyzed for lysine-acetylation using anti-acetyl lysine antibody (Ac-K). Total MnSOD levels served as a loading control. B) Neurons were treated with 10μM HKL at different time points, as indicated. C) Mitochondrial lysates from the four groups were analyzed using western blotting with the indicated antibodies. D-F) Quantification of relative acetylated (Ac) MnSOD, and acetylated OSCP and SIRT3 levels in neurons treated with Aβ oligomers and HKL. Values represent the mean±SD of each separate experiment performed in triplicate. **p < 0.01 versus AβOs group, ***p < 0.001 versus AβOs group.

Next, we tested whether HKL reduced mitochondrial acetylation and increased SIRT3 levels under the influence of AβOs. To this end, primary cultured hippocampal neurons were divided into four groups: control, AβOs (1μM), AβOs (1μM) plus HKL (10μM), AβOs (1μM) plus HKL(10μM) plus AGK7 (20μM) for 24 h. Our results showed that 20μM AGK7 is the lowest concentration that still effectively inhibits mitochondrial SIRT3. SIRT3 levels were analyzed using immunoblotting. The expression level of SIRT3 in the HKL-treated group was nearly double that of the AβOs group (Fig. 2C, F). To test whether increased SIRT3 levels were associated with increased SIRT3 activity, we analyzed acetylation of two SIRT3 substrates, manganese superoxide dismutase (MnSOD) and oligomycin sensitivity-conferring protein (OSCP). Deacetylation of MnSOD at K-68 and OSCP acetylation at K-139 by SIRT3 was demonstrated to increase its activity (Fig. 2C). We observed increased SIRT3 activity as established by reduced acetylation of MnSOD and OSCP after HKL treatment (Fig. 2C– E). However, these changes were not evident in groups in which the SIRT3 inhibitor AGK7 was added. Collectively, these data indicated that HKL is capable of activating mitochondrial SIRT3 in AβO-treated neuronal cells.

HKL attenuates ATP deficiency and mitochondrial oxidative stress via the SIRT3 pathway in AβO-treated hippocampal neurons

The activity of F1FO-ATP synthase in the HKL intervention group was double that of the AβOs group (90.5±8.4 versus 44.6±5.7 nmol/min/mg, p < 0.01; Fig. 3A). Accordingly, the production of ATP was 59.1% more than that of the AβOs group (4.36±0.18 versus 2.74±0.14, p < 0.01; Fig. 3B). In terms of mitochondrial oxidative stress, we found that the activity of SOD synthase in the HKL intervention group was 64.4% more than that of the AβOs group (13.7±1.9 versus 8.4±1.2 nmol/min/mg, p < 0.05; Fig. 3C). Accordingly, the production of ROS was also 32.7% lower during this same period (124.7±9.4 versus185.5±8.5, p < 0.05; Fig. 3D). However, AGK7 had no obvious effect on the production of ATP or ROS (Fig. 3A– D).

Fig.3

HKL enhances the production of energy (ATP) and reduces the production of ROS. A, B) The effect of HKL on F1FO ATP enzymatic activity and ATP production were examined in freshly isolated mitochondria from primary cultured hippocampal neurons in the four groups. C, D) The effect of HKL on SOD enzymatic activity and ROS production were examined in freshly isolated mitochondria from primary cultured hippocampal neurons in the four groups. Values represent the mean±SD of each separate experiment performed in triplicate. *p < 0.05 versus AβOs group, **p < 0.01 versus AβOs group.

HKL ameliorates Aβ oligomer-induced abnormalities of mitochondrial membrane potential via the SIRT3 pathway in hippocampal neurons

We tested the effects of HKL on mΔψ using FCM. We found that mΔψ was 38% lower in the AβOs group as compared with that in the control group (0.62±0.05 versus 1±0.07, p < 0.01; Fig. 4A). However, mΔψ in the AβOs+HKL group was 53.2% higher than that in the AβOs group (0.95±0.06 versus 0.62±0.05, p < 0.05; Fig. 4A). These changes were not apparent when the SIRT3 inhibitor AGK7 was added (Fig. 4A, B).

Fig.4

HKL regulates the mitochondrial membrane potential identified in neuronal mitochondria. A) Mitochondrial membrane potential (Δψm) was measured using flow cytometry in the four groups of primary cultured hippocampal neurons. B) Quantitative analysis of Δψm in neuronal cells. Values represent the mean±SD of each separate experiment performed in triplicate. *p < 0.05 versus AβOs group.

HKL rescues AβO-induced neuronal apoptosis via the SIRT3 pathway in hippocampal neurons

We verified the effect of HKL treatment on the occurrence of apoptosis in neuronal cells. We further investigated apoptosis using annexin V/PI double-staining. We found that the average apoptosis rate in the AβOs group was 3.3 times higher compared with that in the control group (19.97±2.09% versus 4.67±0.45%, p < 0.001; Fig. 5A). However, after incubation with 10μM HKL for 24 h, the average apoptosis rate in the HKL intervention group was 72.7% lower than that in the AβOs group (5.46±0.40% versus 19.97±2.09%, p < 0.001; Fig. 5A). However, the average apoptosis rate in the HKL group that received AGK7 did not demonstrate a marked reduction compared with that in the AβOs+HKL group (16.63±2.13% versus 5.46±0.40%, p < 0.01; Fig. 5A, B).

Fig.5

HKL rescues Aβ oligomer-induced neuronal apoptosis. A) Apoptosis was assessed using annexin V/PI double staining in the four groups of primary cultured hippocampal neurons. On the scattergram of the bivariate flow cytometer, the lower left quadrant shows viable cells (FITC- / PI-), the upper right quadrant indicates late apoptotic cells (FITC+ / PI+), and the lower right quadrant represents early apoptotic cells (FITC+ / PI-). B) Quantitative analysis of apoptotic neuronal cells. Values represent the mean±SD of each separate experiment performed in triplicate.***p < 0.001 versus AβOs group.

HKL improves cognitive deficits in AD (PS1_V97L) transgenic mice

Based on enhanced mitochondrial function in neurons, as evidenced by increased ATP levels and reduced oxidative stress damage in neuronal cells treated with HKL, we next investigated whether HKL prevents cognitive deficit development in PS1_V97L mice. As compared with saline-treated PS1_V97L mice, HKL-treated PS1_V97L mice performed better in the Morris water maze, as reflected by significant progressive reductions in escape latency time and distance to the platform in platform learning trials, and a shorter movement path in HKL-treated mice (Fig. 6A– C). These results suggest that HKL treatment can prevent or halt cognitive decline in the early stages of AD in PS1_V97L mice.

Fig.6

HKL improves cognitive and memory abilities in the Aβ oligomer-induced AD model (PS1_V97L) transgenic mice. A) HKL improves behavioral performance of PS1_V97L transgenic mice. Escape latency during platform trials in the Morris water maze. B) Distance to platform. (C) Representative tracing graphs of the open field test. Results are presented as the mean±SD (n = 10), *p < 0.05 versus PS1_V97L-Tg control group, **p < 0.01 versus PS1_V97L-Tg control.

HKL enhances energy metabolism and attenuates mitochondrial oxidative stress by increasing SIRT3 levels and activity in PS1_V97L mice

To elucidate whether treating PS1_V97L mice with HKL increases mitochondrial function via the SIRT3 pathway, we examined the expression levels of mitochondrial SIRT3, the activity of F1FO-ATP synthase and SOD, which are regulated by SIRT3, as well as the generation of ATP and ROS. SIRT3 levels in the PS1_V97L– Tg HKL group were 1.64 times higher than those in the PS1_V97L– Tg control group (2.96±0.26 versus 1.12±0.18, P < 0.01; Fig. 7A, B). The amount and distribution of SIRT3 in the hippocampal CA1 region of PS1_V97L mice were also investigated using immunohistochemistry; the number of blue black particles in PS1_V97L– Tg HKL mice were nearly double that of the PS1_V97L– Tg control group (28.37±2.41 versus 14.68±1.83, p < 0.05; Fig. 7E, F). The activity of F1FO-ATP and SOD acetylated by SIRT3 in HKL-treated PS1_V97L mice was significantly different from the PS1_V97L– Tg control group (Fig. 7G, I). Finally, we demonstrated that ATP production in the PS1_V97L– Tg HKL group was 57.5% higher than that in the PS1_V97L– Tg control group (3.97±0.18 versus 2.52±0.13, p < 0.01; Fig. 7H). ROS production in the PS1_V97L– Tg HKL group was 28.0% lower than that in the PS1_V97L– Tg control group (113.8±8.6 versus 158.2±9.2, p < 0.05; Fig. 7J).

Fig.7

Changes in the mitochondrial SIRT3 pathway in PS1_V97L mice. A) Mitochondrial lysates were analyzed using western blotting with the indicated antibodies. B– D) Quantification of relative SIRT3, acetylated (Ac) MnSOD, and acetylated OSCP levels in the hippocampal organization of different mice. G, I) The effect of HKL on F1FO -ATP and SOD enzymatic activity, (H, J) ATP and ROS production were examined. F) Photomicrographs show immunohistochemical staining and (E) quantification of mitochondrial SIRT3 in the CA1 regions of the hippocampus of different mice (three mice per group and seven neurons per mouse). Scale bar = 100μm. Results are presented as the mean±SD (n = 10), *p < 0.05 versus PS1_V97L-Tg control group, **p < 0.01 versus PS1_V97L-Tg control, ***p < 0.001 versus PS1_V97L-Tg control.

DISCUSSION

We previously reported a missense mutation of the PS1_V97L gene in a Chinese early-onset AD pedigree, and then cultivated transgenic mice containing this PS1_V97L gene mutation [26]. In that previous study, we detected a memory impairment in PS1_V97L mice and abnormal deposition of AβOs in the related brain parenchyma [17, 27]. These demonstrated that PS1 _V97L transgenic mice are useful in the study of the pathogenesis of AD.

In this study, we investigated whether HKL could improve the learning and memory ability of PS1_V97Lmice during the pre-symptomatic stages, and then studied the underlying mechanism.

We found that HKL could attenuate mitochondrial dysfunction by regulating the activity of the mitochondrial SIRT3 in AβO-treated primary hippocampal neuronal cells and in the Tg AD PS1_V97Lmice model, including increasing ATP production, suppressing ROS production, and regulating mΔψ. In general, HKL can attenuate early pathological features in AβO-induced animal models by activating mitochondrial SIRT3, which is involved in AβO-induced mitochondrial dysfunction during the early stage of AD.

Previous studies have reported that HKL could alleviate learning and memory impairments in SAMP8 mice by decreasing the activity of acetylcholinesterase [20], and HKL could also rescue cognitive impairment in mice that had received microinjections of AβO in the hippocampus [16]. These findings are consistent with the notion that HKL could improve cognitive performance and delay cognitive impairment in the AβOs animal model (PS1_V97L mice) used in our study. The roles of AβOs in triggering early neurodegeneration during the progression of AD have been demonstrated previously [1, 28]. In our previous studies, we have demonstrated that AβOs were present from 6 months of age and cognitive impairment was noticeable from 9 months in PS1_V97L mice [17]. In this study, HKL was administered from 6 months to 9 months of age, which could represent the preclinical stage of AD. The 3-month administration of HKL in the early stages of AD effectively improved cognitive impairment and delayed progression of the condition.

Mitochondrial dysfunction is one of the early pathological features of AD, comprising reduced mitochondrial respiration, decreased respiratory enzyme activity, increased oxidative stress, and energy metabolism dysfunction [8, 29]. AβOs are closely related to mitochondrial dysfunction [30] and affect mitochondrial function in a variety of ways, including via the interaction of Aβ with several mitochondrial proteins, such as cyclophilin D (CyPD), OSCP and Aβ-binding alcohol dehydrogenase [31, 32], which is consistent with our findings that AβOs induced mitochondrial dysfunction (ATP deficiency, mitochondrial oxidative damage, and mΔψ loss in vivo and in vitro). Furthermore, our findings provide evidence of a definite molecular target; we conclude that HKL can activate mitochondrial SIRT3 to regulate mitochondrial function. Therefore, we have shown that HKL can delay the onset of learning and memory impairment in PS1_V97L mice by employing in vivo and in vitro AD models.

SIRT3 deregulation is linked to mitochondrial dysfunction in AD [33], and mitochondrial SIRT3 expression is decreased in an APP/PS1 mouse model of AD [34]. In the present study, we found that HKL treatment increased SIRT3 levels, and this was linked to reduced acetylation of MnSOD and OSCP [35], while the deacetylation of MnSOD can enhance the scavenging of mitochondrial ROS [36]. Similarly, OSCP loss can regulate mitochondrial F1FO-ATP synthase, which can regulate mitochondrial ATP production [32]. Oxidative stress plays a key role in the common pathophysiology of AD and myelinated hippocampal neurons fibers in AD brain easily degenerate when energy is deficient [13, 37]. These changes may be related to the cytotoxicity of Aβ and AβOs, which is related to the pathology and clinical symptoms of AD [38]. Furthermore, our results showed that HKL can ameliorate abnormal ROS synthesis and improve ATP production in the presence of AβOs, which is consistent with the finding that HKL can reverse cardiac myocyte hypertrophy [39]. Nevertheless, the use of HKL as a treatment in an AD model has not been described previously.

In addition, we found that HKL treatment prevented a decrease in mΔψ and maintained it at almost the same level as that of the control group. It is considered a prelude to apoptosis when the mΔψ dissipates [40]. This is because mitochondrial F1FO-ATP synthase is involved in maintaining the stability of mΔψ [32]. Furthermore, the intrinsic apoptotic path is triggered by the mitochondrial pathway [41]. Impaired mitochondrial function affects the expression level of apoptosis-related proteins, such as cytochrome C, cell lymphoma/leukemia-2, and cleaved caspases 3 and 9, and ultimately accelerates the process of cell apoptosis [42]. Protecting mitochondrial function plays a key role in controlling cell apoptosis [43]. In this study, we observed that HKL ameliorates apoptosis in AβO-treated cells. Our study suggests that improving mitochondrial function using HKL can prevent neuronal apoptosis during the initial stages.

In summary, we found that mitochondrial dysfunction plays an important role in AD development. We also found that mitochondrial dysfunction is mediated by activation of mitochondrial SIRT3, followed by upregulation of the OSCP-associated mitochondrial F1FO-ATP synthase and the antioxidant enzyme, MnSOD. The findings of this study may provide a new target for the early treatment of AD.

Footnotes

ACKNOWLEDGMENTS

This study was supported by the Key Project of the National Natural Science Foundation of China (81530036); the National Key Scientific Instrument and Equipment Development Project (31627803); Mission Program of Beijing Municipal Administration of Hospitals (SML20150801); Beijing Scholars Program; Beijing Brain Initiative from Beijing Municipal Science & Technology Commission (Z161100000216137); CHINA-CANADA Joint Initiative on Alzheimer’s Disease and Related Disorders (81261120571) and Beijing Municipal Commission of Health and Family Planning (PXM2017_026283_000002).

Authors’ disclosures available online ().

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Honokiol Alleviates Cognitive Deficits of Alzheimer’s Disease (PS1 V97L ) Transgenic Mice by Activating Mitochondrial SIRT3

Abstract

Keywords

INTRODUCTION

MATERIALS AND METHODS

Ethics statement

Animals

Aβ oligomer preparation

Drug treatments

Primary hippocampal neuron culture

Cell viability

Mitochondrial preparation

Immunoblot analysis

Mitochondrial membrane potential assay

Measurement of mitochondrial energy metabolism related parameters

Measurement of mitochondrial oxidative stress related parameters

Quantification of apoptotic cells by flow cytometry

Immunohistochemistry

Behavioral tests

Statistical analysis

RESULTS

HKL improves neuronal cell viability after incubation with Aβ oligomers in hippocampal neurons

HKL increases SIRT3 levels and its activity in AβO-induced hippocampal neurons

HKL attenuates ATP deficiency and mitochondrial oxidative stress via the SIRT3 pathway in AβO-treated hippocampal neurons

HKL ameliorates Aβ oligomer-induced abnormalities of mitochondrial membrane potential via the SIRT3 pathway in hippocampal neurons

HKL rescues AβO-induced neuronal apoptosis via the SIRT3 pathway in hippocampal neurons

HKL improves cognitive deficits in AD (PS1V97L) transgenic mice

HKL enhances energy metabolism and attenuates mitochondrial oxidative stress by increasing SIRT3 levels and activity in PS1V97L mice

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

HKL improves cognitive deficits in AD (PS1_V97L) transgenic mice

HKL enhances energy metabolism and attenuates mitochondrial oxidative stress by increasing SIRT3 levels and activity in PS1_V97L mice