Introduction

Alzheimer's disease (AD) is a progressive neurodegenerative disorder commonly leading to dementia in the elderly, which accounts for 60%–80% of all cases. In the global aging trend, the number of people with AD will increase to more than 100 million worldwide by 20501. Although there have been several efforts to clarify the causes of this disease and explore effective pharmacologic therapies, no treatment has demonstrated potency in slowing the progression of AD2.

Extracellular acetylcholine (ACh) is a key structural element in functional neural networks3,4. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are two major forms of cholinesterases in the mammalian brain. AChE plays a dominant role in halting cholinergic neurotransmission, but the physiological function of BuChE is not completely understood5. However, in the AD brain, AChE activity decreases from mild to severe stages, and BuChE activity is unchanged or even increased. BuChE might replace AChE in hydrolyzing brain ACh during AD progression6,7.

To date, cholinesterase inhibitors have been used as a first-line symptomatic therapy for AD3. Recent studies have shown that AChE and BuChE participate in AD progression. AChE accelerates the assembly of Aβ into amyloid fibrils; the peripheral binding site of AChE might be involved in amyloid formation8,9,10. BuChE activity has been associated with β-amyloid plaques in a transgenic AD mouse model11. Preclinical studies have indicated that the dual cholinesterase inhibitor rivastigmine exhibits a neuroprotective effect in fetal rat primary cortical cultures11,12. It is possible to identify a dual cholinesterase inhibitor that inhibits the activity of AChE and BuChE and simultaneously induces a neuroprotective effect.

The β amyloid peptide (Aβ) is a major component of extracellular senile plaques, which is a characteristic hallmark of AD13. Several studies have suggested that the Aβ1–42 fragment is highly cytotoxic to neurons; this fragment has been widely used in in vivo and in vitro research to induce memory impairment and neuron damage14,15,16. Aβ causes mitochondrial dysfunction in neurons, leading to energy supplement impairment and mitochondria-mediated apoptosis17,18. Numerous signaling pathways are involved in Aβ-induced neurotoxicity, including Wnt signal transduction pathways, the PI3K/Akt/mTOR pathway, the AMP-active protein kinase pathway and the sirtuin pathway19,20,21,22,23.

DL0410 ((1,1′-([1,1′-biphenyl]-4,4′-diyl)bis(3-(piperidin-1-yl)propan-1-one)dihydrochloride, Figure 1), an acetylcholinesterase/butyrocholinesterase inhibitor, is a new synthetic compound belonging to phthalazinone units, which exhibits a different chemical structure among cholinesterase inhibitors, such as rivastigmine, donepezil, and tacrine. DL0410 was selected from more than 100 000 compounds using high-throughput screening assays for AChE and BuChE inhibitors. In a previous study, we showed that DL0410 binds to the active site of AChE, similar to donepezil, and the safety and efficacy of this compound were also confirmed using a series of experiments24. It has also been demonstrated that DL0410 improves cognitive deficits and reverses the plaque load in APP/PS1 transgenic mice25. In the present study, an Aβ1–42-induced amnesia mouse model and an AD cellular model were used to investigate the protective effects and to explore the neuroprotective mechanisms of DL0410.

Figure 1
figure 1

The structure of the candidate drug DL0410.

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Materials and methods

Reagents

DL0410 (chemical purity was higher than 99% based on HPLC) was obtained from the National Center for Pharmaceutical Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences (Bejing, China). Donepezil hydrochloride was purchased from the Jinan Chenghui Shuangda Chemical Industry Limited Company (Shandong, China). Rivastigmine was purchased from the Nantong Baihua Bio-pharmaceutical Company (Jiangsu, China). Hoechst 33342, acetylthiocholine iodide (ASCh), S-butyrylthiocholine chloride (BuSCh), tetraisopropyl pyrophosphoramide (iso-OMPA), and 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich (St Louis, USA). A CellTiter 96® AQueous One Solution Cell Proliferation Assay kit was purchased from Promega (Madison WI, USA). Rhodamine 123 (Rh123) was purchased from Dojindo Laboratory (Kumamoto, Japan). An Alexa Fluor 488-conjugated secondary antibody was purchased from Invitrogen (CA, USA).

The Aβ1–42 peptide was purchased from Sangon Biotech (Shanghai, China) and dissolved in sterile 0.1% DMSO at a concentration of 1 mg/mL, followed by aggregation through incubation at 37 °C for 7 d prior to use. A human Aβ1–42 ELISA Kit was purchased from Shanghai ExCell Biology, Inc. Human plasma was purchased from the Beijing Red Cross Blood Center. Primary antibodies against GAPDH, N-terminal kinase (JNK), and p-JNK were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies against brain-derived neurotrophic factor (BDNF), p-Akt, cAMP-responsive element binding protein (CREB) and p-CREB were purchased from Epitomics (Burlingame, CA, USA). The anti-amyloid-β precursor protein (β-APP) antibody [Y188] (ab32136) was purchased from Abcam (Cambridge, UK). Anti-Aβ1–40/42 (AB5076) was purchased from Millipore (MA, USA). An anti-rabbit IgG HRP-linked antibody (#7074) and an anti-mouse IgG HRP-linked antibody (#7076) were purchased from CST (Danvers, MA, USA).

An AChE inhibitory activity assay

Cholinesterase inhibitory activity was measured using Ellman's method with slight modifications26. The principle of this assay is that AChE hydrolyzes ASCh and generates choline iodide. Choline iodide reacts with DTNB as a thiol chromogenic agent to produce a yellow compound called TNB, which can be quantitatively measured through colorimetry. The quantity of choline iodide indicates the response to AChE activity.

AChE was extracted from rat brains, and the cerebral blood vessels were removed. Subsequently, 0.9% saline solution (20 mg/mL) was added, followed by centrifugation at 800×g for 10 min at 4 °C. The resulting supernatant was collected and used in an AChE inhibitory activity assay. Five serial dilutions of the samples were measured to inhibit AChE activity. In the AChE reaction system, the assay is performed in 96-well plates using 0.05 mol/L phosphate-buffered saline (PBS) and a Spectra Max M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The reaction system comprised 10 μL of test compounds, 30 μL of 0.05 mol/L PBS, 20 μL of AChE, 60 μL of 3.75 mmol/L ASCh, and 80 μL of 0.25 mg/mL DTNB. After a 60-min incubation at 37 °C, the absorbance intensity of the system was measured at 412 nm. Donepezil and rivastigmine were used as reference compounds.

A BuChE inhibitory activity assay

The experimental principle of this assay is similar to the assay above, where BuChE hydrolyzes BuSCh and generates choline iodide, which reacts with DTNB to produce TNB. TNB can be quantitatively measured through colorimetry. The quantity of choline iodide is indicated as the response to BuChE activity.

In this assay, BuChE was derived from human plasma and diluted 200 times in 0.05 mol/L PBS. The assay was performed in 96-well plates using a Spectra Max M5 microplate reader. The reaction system comprised 10 μL of test compounds, 40 μL of BuChE, 70 μL of 7.5 mmol/L BuSCh and 80 μL of 0.25 mg/mL DTNB. After incubation at 37 °C for 60 min, the absorbance intensity of the system was quantified at a wavelength of 412 nm. Rivastigmine and iso-OMPA were used as reference compounds.

Animals and treatments

Male ICR mice (18–22 g, Vital River Co, Beijing, China) were housed 4–5 per cage with free access to food and water under a 12/12 h day/night cycle. All experimental procedures in the present study were performed in accordance with institutional guidelines and ethics, and approval was obtained through the Laboratories Institutional Animal Care and Use Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College.

The mice were randomized into six groups: sham group, Aβ1–42-treated group, rivastigmine 2 mg/kg group27, DL0410 1 mg/kg group, DL0410 3 mg/kg group, and DL0410 9 mg/kg group. Rivastigmine and DL0410 were dissolved in distilled water and administered to the mice through oral gavage once daily for 3 d, followed by injection with Aβ1–42. After receiving the Aβ1–42 injection, the mice were continuously treated with DL0410 or rivastigmine for 11 d.

Chloral hydrate powder was dissolved in 0.9% saline (1 g in 10 mL solution), and each mouse was anesthetized with an intraperitoneal (ip) injection at a dose of 400 mg/kg of body weight and placed on a stereotaxic frame. Aggregated Aβ1–42 was implanted into the right cerebral ventricle according to the following coordinates: DV=−2.3 mm, ML=−1.0 mm and AP=0.4 mm caudal to bregma (2 μg in 2 μL 0.1% DMSO for each injection). The sham-operated animals were injected with the same amount of 0.1% DMSO in an identical manner.

Morris water maze test

Three days after receiving the Aβ1–42 injection, the Morris water maze test was performed as previously described28. The experimental apparatus comprised a circular water tank (100 cm diameter, 35 cm height) containing water at 23±1 °C. A platform of 4.5 cm in diameter was placed at the midpoint of one quadrant (1 cm below the water surface). Each mouse received two training periods per day for five consecutive days. The latency and swim distance to escape from the water maze (finding the submerged escape platform) were calculated for each trial. In the probe trial, we recorded the number of times that the mice crossed the quadrant in the platform. All data were recorded using a computerized video system.

Passive avoidance test

The passive avoidance task was initiated 9 d after the Aβ1–42 injection. The passive avoidance apparatus comprised an illuminated chamber attached to a darkened chamber containing a metal floor to deliver a mild electric shock29. A guillotine door separated the two compartments. The mice were placed into the illuminated chamber, facing away from the door of the dark chamber, and acclimated for 1 min. As soon as the mouse entered the dark chamber, the door was slid back into place, triggering an electric shock. The latency and error scores (the duration of time and number of times needed to enter the darkened chamber) were recorded. The retention test was conducted at 24 h later, placing the mouse in the illuminated chamber and using the same protocol but without the electric shock. The upper time limit was set at 300 s.

Immunohistochemistry analysis

After the behavioral experiments, the mice were anesthetized through an intraperitoneal (ip) injection of 10% chloral hydrate (0.04 mL/10 g), followed by transcardial perfusion with 50 mL of saline solution and 50 mL of 4% paraformaldehyde30. The brains were removed and fixed in 0.05 mol/L phosphate buffer (pH 7.4) containing 4% paraformaldehyde overnight. Each tissue block was embedded in paraffin, and the sections were cut to the desired thickness (3-μm thickness) using a microtome. The tissue sections were used for Aβ1–40/42 and BDNF detection. The results were observed using microscopy.

The tissue sections were blocked in 10% normal serum with 1% BSA in Tris-buffered saline (TBS) for 2 h at room temperature. The sections were subsequently incubated with primary anti-Aβ1–40/42 antibody or anti-BDNF antibody overnight at 4 °C. The sections were rinsed in TBS containing 0.025% Triton X-100 with gentle agitation and subsequently incubated in 0.3% distilled H2O2 in TBS for 15 min. The secondary antibody was applied to the slide at a dilution recommended by the manufacturer (in TBS with 1% BSA) and incubated for 1 h at room temperature. The labeling was visualized using 0.04% H2O2 and 0.05% 3,3'-diaminobenzidine. The results were calculated using ImageJ software 1.50i.

An AChE activity assay

The mouse brains were homogenized in a glass Teflon homogenizer containing 9 volumes of 0.9% saline solution and subsequently centrifuged at 800×g for 10 min at 4 °C. The resulting supernatant was collected and used in the AChE activity assay. AChE activity was detected using an AChE inhibitory activity assay kit according to the manufacturer's instructions.

A BuChE activity assay

Blood samples were collected from the mice in sodium heparin tubes and centrifuged at 4000×g for 10 min at 4 °C. Plasma was collected from the samples for use in a BuChE activity assay. BuChE activity was assessed using a BuChE inhibitory activity assay kit, according to the manufacturer's instructions.

Cell culture and treatments

We used stably co-transfected human neuroblastoma SH-SY5Y cells expressing APP-Swedish mutation and wild-type human APP (APPsw-SY5Y cells), as previously reported31,32. In the absence of copper, the Aβ overexpression cell line exhibited no neurotoxicity in the culture process; the presence of copper triggered the toxicity of Aβ. In this AD cellular model, copper was used as a stimulator for Aβ-mediated neurotoxicity.

The cells were cultured in DMEM/F12 supplemented with 10% FBS, 1×105U/L penicillin, and 100 mg/L streptomycin at 37 °C in a humidified, 5% CO2 incubator. Subsequently, 200 μg/mL G418 was added into the medium to maintain the genotypically stable cell strains.

The cells were incubated with 10 μmol/L donepezil, 10 μmol/L rivastigmine, and 1, 3, and 10 μmol/L DL0410 for 2 h, followed by treatment with 200 μmol/L copper for 24 h. The cells and medium were subsequently collected to investigate the effects of DL0410.

A cell viability assay

After treatment, the medium was discarded and replaced with 100 μL of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) solution according to the manufacturer's protocol. After a 2 h incubation at 37 °C, the absorbance at 490 nm was measured using a Spectra Max M5 microplate reader.

Mitochondrial membrane potential detection

Rh123 was used to detect changes in mitochondrial membrane potential (Δψm) in APPsw-SY5Y cells. After treatment as described above, APPsw-SY5Y cells were incubated with 10 μmol/L Rh123 for 30 min. The nucleic acid dye Hoechst 33342, used to identify cell nuclei, was added at a final concentration of 10 μmol/L at 10 min after the Rh123 incubation.

Fluorescent images and intensities were acquired and analyzed using a Cellomics Arrayscan VTI HCS Reader (Thermo Fisher Scientific Cellomics, Pittsburgh, PA, USA). The Hoechst 33342- and Rh123-stained images were acquired using 386/23 nm excitation and 460/40 nm emission and 485/20 nm excitation and 535/50 nm emission wavelengths, respectively.

Western blot assay

Cells from the cerebral cortices of the mice were lysed in RIPA lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 20 mmol/L EDTA, 50 mmol/L sodium fluoride, 1 mmol/L sodium orthovanadate, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS)]. The RIPA buffer was supplemented with complete protease inhibitor cocktail tablets (Roche Applied Science, Mannheim, Germany). The homogenates were centrifuged at 13 000×g for 20 min to obtain the desired supernatant.

Total protein concentrations were determined using the BCA method (CoWin Bioscience Co, Beijing, China). Equal amounts (30 μg) of protein samples were separated on 12% SDS-polyacrylamide gels and transferred to PVDF membranes (Millipore, MA, USA). The membranes were blocked with 5% BSA and 0.05% Tween-20 in TBS for 2 h at 37 °C, followed by overnight incubation with primary antibodies and incubation with HRP-conjugated secondary antibodies. Protein was detected using ChemiGlow Western Blotting Detection Reagents (CoWin Bioscience Co, Beijing, China). The results were quantified using Quantity One software (Bio-Rad, PA, USA).

An ELISA assay

The Aβ1–42 in the culture medium was measured using a sensitive and specific ELISA assay according to the manufacturer's instructions (ExCell Biology, Shanghai, China). A total of 100 μL of sample was added to each well, followed by 50 μL of antibody. The solution was incubated at room temperature for 2 h. After several washes, an enzyme-conjugated secondary antibody (100 μL) was added to each well, and the ELISA plate was incubated at room temperature for 1 h. After additional washes, the substrate (100 μL) was added, followed by incubation at room temperature for 15 min. The reaction was stopped after adding stop buffer (100 μL); the absorbance at 490 nm was detected on a Spectra Max M5 microplate reader. A standard curve with known amounts of Aβ1–42 was also generated. The Aβ1–42 level was calculated according to the standard curve.

Statistical analysis

Statistical analyses were performed using SPSS software (version 16.0, SPSS Inc, Chicago, IL, USA). Group differences in the escape latency and search distance in the Morris water maze test were analyzed using two-way ANOVA with repeated measures. Other data were analyzed using one-way ANOVA followed by Tukey's post hoc test. All data are expressed as the mean±SEM. Statistical significance was defined as P<0.05 for all tests.

Results

The inhibitory effects of DL0410 on cholinesterase activity in vitro

The obtained IC50 values indicated that DL0410 exhibited AChE inhibitory activity similar to the non-competitive AChE inhibitor donepezil and had substantially greater inhibitory activity than rivastigmine. DL0410 also showed a potent BuChE inhibitory effect, with an IC50 value of 3.96 μmol/L. DL0410 exerted potent cholinesterase inhibitory effects in vitro in a concentration-dependent manner (Table 1).

Table 1 IC50 values of DL0410 as AChEI-BuChEI. Mean±SEM. n=3.
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The effect of DL0410 on the cell viability of APPsw-SY5Y cells subjected to copper

An AD cellular model comprising APPsw-SY5Y cells stimulated with copper was employed to evaluate the neuroprotective effects of DL0410. As shown in Figure 2, treatment with 200 μmol/L of copper for 24 h resulted in 51.60% cell death (P<0.01 vs control). Pretreatment with various concentrations of DL0410 markedly increased cell viability (DL0410 1 μmol/L: 12.87% increase; DL0410 3 μmol/L: 18.20% increase; DL0410 10 μmol/L: 24.85% increase; P<0.01) compared with the Cu2+ induced APPsw-SY5Y cell damage model (Cu2+ model), and this increase was concentration-dependent. DL0410 showed better protection against Aβ-induced neurotoxicity than donepezil and rivastigmine at the same concentration (10 μmol/L).

Figure 2
figure 2

DL0410 improved the cell viability of APPsw-SY5Y cells subjected to copper. The results were obtained from 3 independent experiments, and the data are expressed as the mean±SEM. **P<0.01 vs control. ##P<0.01 vs the Cu2+ model.

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The effect of DL0410 on Aβ1–42-induced memory impairment

The effect of DL0410 on spatial memory was investigated via Morris water maze performance. MWM acquisition training tests were performed for five consecutive days, and the latency times were recorded. Aβ1–42-treated mice showed more escape latency compared with vehicle-treated mice (P<0.01 vs sham, Figure 3A). The DL0410 treatment showed less latency than Aβ1–42-treated mice (P<0.01 vs Aβ model, Figure 3A). We also observed that mice administered Aβ1-42 had significantly longer latency time (P<0.001 vs sham, Figure 3B) and search distances (P<0.01 vs sham, Figure 3C) than vehicle-treated mice. The DL0410 treatment significantly decreased the latency (P<0.01 or P<0.001 vs Aβ model, Figure 3B) and search distance (P<0.01 vs Aβ model, Figure 3C). In the probe trial, compared with vehicle-treated mice, Aβ1–42-treated mice showed reduced crossing numbers in the target quadrant (P<0.05 vs sham, Figure 3D). DL0410-treatment increased crossing numbers where the platform was located (P<0.05 vs Aβ model, Figure 3D). DL0410 had beneficial effects in attenuating spatial learning deficits in Aβ1–42-treated mice.

Figure 3
figure 3

The effects of DL0410 on learning and memory capabilities in an Aβ1–42-induced amnesia mouse model. The data are expressed as the mean±SEM. n=15. (A) DL0410 shortened the latency time to the hidden platform during Morris water maze performance. (B) DL0410 decreased the latency to the platform during Morris water maze performance in the 5th training day in another independent experiment. (C) DL0410 decreased the search distance to the platform in the Morris water maze. (D) DL0410 increased crossing numbers in the target quadrant in the probe trial. (E) DL0410 prolonged the latency to enter the dark compartment in the passive avoidance test. (F) DL0410 decreased the number of errors in the passive avoidance test. *P<0.05, **P<0.01 vs sham. #P<0.05, ##P<0.01 vs the Aβ model.

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The effect of DL0410 on memory impairment was also evaluated using passive avoidance tasks. Statistical analyses indicated that mice administered Aβ1–42 showed significant memory deficits. The Aβ1–42-treated mice had shorter latencies in entering the dark compartment (P<0.01 vs sham, Figure 3E) during the retention trial and made more errors during the acquisition trial (P<0.05 vs sham, Figure 3F). DL0410 treatments of 1, 3, and 9 mg/kg significantly prolonged the latency (P<0.01 vs Aβ model, Figure 3E) and decreased the error number (P<0.01 vs Aβ model, Figure 3F), indicating that DL0410 remarkably protected mice from Aβ1–42-induced memory deficits in a dose-dependent manner at dosages of 1 to 9 mg/kg.

The effects of DL0410 on Aβ levels in an Aβ1–42-induced amnesia mouse model and on APPsw-SY5Y cells

The effects of DL0410 on Aβ levels in the Aβ1–42-induced amnesia mouse model were evaluated. Previously, we designed this test to detect the Aβ level in the cortex region to ensure that the injected exogenous Aβ would be distributed through the brain. Immunohistochemical assays showed a large number of Aβ1–40/1–42 deposits in the cortices of Aβ1–42-treated mice (Figure 4Ab), but no detectable Aβ1–40/42 deposits were observed in sham-treated mice (Figure 4Aa). Compared with Aβ1–42-treated mice, the DL0410 treatment reduced the areas of Aβ deposits in the cortices in a dose-dependent manner (Figure 4Ad, 4Ae, 4Af). Western blot results revealed that 9 mg/kg DL0410 significantly decreased β-APP protein levels in the cerebral cortices of Aβ1–42-treated mice (P<0.05 vs Aβ model, Figure 4B).

Figure 4
figure 4

DL0410 inhibited the level of Aβ in the Aβ1–42-induced amnesia mouse model and in APPsw-SY5Y cells. (A) DL0410 decreased Aβ1–40/42 deposits in mouse cerebral cortices: (a) sham group, (b) Aβ group, (c) Aβ+rivastigmine (2 mg/kg) group, (d) Aβ+DL0410 (1 mg/kg) group, (e) Aβ+DL0410 (3 mg/kg) group, and (f) Aβ+DL0410 (9 mg/kg) group. (B) The percentage of Aβ1–40/42 positive cells in the cortex. The data are expressed as the mean±SEM. n=5. **P<0.01 vs sham. #P<0.05, ##P<0.01 vs model. (C) The effects of DL0410 on Aβ1–42 production in vitro. APPsw-SY5Y cells were incubated with Cu2+ 200 μmol/L for 24 h to induce Aβ1–42 production; the amounts of Aβ1–42 in the culture media were measured through an ELISA assay. The results were obtained from 3 independent experiments, and the data are expressed as the mean±SEM. **P<0.01 vs control. #P<0.05, ##P<0.01 vs the Cu2+ model. (D) DL0410 suppressed β-APP expression in the Aβ1–42-induced amnesia mouse model. The expression of β-APP in the cerebral cortex was determined using the Western blot assay. The data are expressed as the mean±SEM. n=5. **P<0.01 vs sham. #P<0.05 vs the Aβ model. The results were obtained from 3 independent experiments, and the data are expressed as the mean±SEM. **P<0.01 vs control. #P<0.05, ##P<0.01 vs the Cu2+ model.

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APPsw-SY5Y cells were used to examine the effects of DL0410 on Aβ levels in vitro. An ELISA assay was applied to detect extracellular Aβ1–42 levels. As shown in Figure 4C, treatment with copper significantly increased Aβ1–42 production compared with untreated controls (P<0.05 vs control). Pretreatment with 10 μmol/L DL0410 markedly reduced the levels of extracellular Aβ1–42 by 19.05% (P<0.05 vs Cu2+ model), and DL0410 decreased copper-stimulated Aβ1–42 production in a concentration-dependent manner.

The effects of DL0410 on CREB and BDNF expression in an Aβ1–42-induced amnesia mouse model

CREB phosphorylation was detected using a Western blot assay, and hippocampal BDNF levels were assessed through immunohistochemistry. As shown in Figure 5, the Western blot results revealed that the DL0410 treatment significantly increased the ratio of p-CREB/CREB in the cerebral cortices of Aβ1–42-treated mice (P<0.05 vs Aβ model, Figure 5A). In addition, DL0410 increased the number of BDNF-positive neurons in the cortices of Aβ1–42-treated mice (Figure 5B).

Figure 5
figure 5

The effects of DL0410 on CREB and BDNF expression in an Aβ1–42-induced amnesia mouse model. (A) The effect of DL0410 on p-CREB and total-CREB expression in the cerebral cortices of Aβ1-42-treated mice. The data are expressed as the mean±SEM. n=5. *P<0.05, vs sham. #P<0.05 vs the Aβ model. (B) The percentage of positive BDNF cells in the cortex. (C) DL0410-normalized, Aβ1-42-induced decreases in BDNF levels in mouse cortices: (a) sham group, (b) Aβ group, (c) Aβ+rivastigmine (2 mg/kg) group, (d) Aβ+DL0410 (1 mg/kg) group, (e) Aβ+DL0410 (3 mg/kg) group, and (f) Aβ+DL0410 (9 mg/kg) group. The data are expressed as the mean±SEM. n=5. *P<0.05, **P<0.01 vs sham. #P<0.05 vs the Aβ model.

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The effects of DL0410 on cholinesterase activity in an Aβ1–42-induced amnesia mouse model

The effects of DL0410 on brain AChE and plasma BuChE activity were detected. As shown in Figure 6A, AChE activity in the Aβ1–42-treated group was significantly increased (P<0.05 vs sham). Lower AChE activity was observed in the rivastigmine- and DL0410-treated groups compared with the Aβ1–42-treated group, but these differences were not statistically significant. Aβ1–42-treated mice also showed significantly increased BuChE activity (P<0.05 vs sham, Figure 6B), whereas rivastigmine and the DL0410 treatments decreased BuChE activity (P<0.01 vs Aβ model, P<0.001 vs Aβ model, Figure 6B).

Figure 6
figure 6

The effects of DL0410 on cholinesterase activity in an Aβ1–42-induced amnesia mouse model. (A) Effects of DL0410 on brain AChE activity in the Aβ1–42-induced amnesia mouse model. The data are expressed as the mean±SEM. n=5. *P<0.05 vs sham. (B) DL0410 decreased plasma BuChE activity in the Aβ1–42-induced amnesia mouse model. The data are expressed as the mean±SEM. n=5. *P<0.05 vs sham. ##P<0.01 vs the Aβ model.

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The effect of DL0410 on mitochondrial function in copper-treated APPsw-SY5Y cells

The protective effect of DL0410 on the mitochondrial function in copper-treated APPsw-SY5Y cells was investigated using Rh123 fluorescent dye, and the Δψm was monitored as the fluorescence intensity of Rh123. When the cells were exposed to 200 μmol/L copper for 24 h, the mitochondrial membranes were depolarized, and the fluorescence intensity increased by 211.50% (P<0.01 vs control). Pretreatment with DL0410 reduced the fluorescence intensity in a concentration-dependent manner (P<0.05 vs Cu2+ model, Figure 7B), suggesting that DL0410 protects APPsw-SY5Y cells by reversing copper-induced Δψm loss.

Figure 7
figure 7

The effect of DL0410 on mitochondrial membrane potential in APPsw-SY5Y cells exposed to copper. (A) Representative photographs of Rh123 staining in different groups. (B) Quantitative analysis of Rh123 fluorescence intensity among the groups. The results were obtained from 3 independent experiments, and the data are expressed as the mean±SEM. **P<0.01 vs control. #P<0.05 vs the Cu2+ model.

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The effects of DL0410 on the apoptosis-related Akt/JNK signaling pathway in copper-treated APPsw-SY5Y cells

The effects of DL0410 on the Akt/JNK pathway were also assessed using a Western blot assay. Copper-treated APPsw-SY5Y cells showed a significantly increased ratio of p-JNK/JNK (P<0.05 vs control, Figure 8A) and a remarkably decreased ratio of p-Akt/Akt (P<0.01 vs control, Figure 8B). In contrast, the DL0410 treatment decreased the phosphorylation of JNK and increased the phosphorylation of Akt (P<0.05 vs Cu2+ model, Figure 8). These data indicated that DL0410 might attenuate cell apoptosis through the regulation of the Akt/JNK pathway.

Figure 8
figure 8

DL0410 attenuated cell apoptosis through the regulation of the Akt/JNK pathway. (A) The effects of DL0410 on p-JNK and total JNK expression in copper-treated APPsw-SY5Y cells. (B) The effects of DL0410 on p-Akt and total Akt expression in copper-treated APPsw-SY5Y cells. The results were obtained from 3 independent experiments, and the data are expressed as the mean±SEM. *P<0.05, **P<0.01 vs control. #P<0.05 vs the Cu2+ model.

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Discussion

The results of the present study demonstrated that DL0410 improved learning and memory dysfunction in Aβ1–42-treated mice and reduced mitochondria-mediated APPsw-SY5Y cell apoptosis. The mechanism of these beneficial effects might reflect cholinesterase inhibition, Aβ production inhibition and CREB/BDNF pathway and the Akt/JNK pathway regulation.

The “amyloid hypothesis” suggests that the abnormal deposition of amyloid-β peptide in the brain plays a key role in AD pathogenesis, as the extracellular Aβ accumulation is a pathological hallmark of AD33,34. Aβ has neurotoxicity effects on cholinergic neurons and the learning function35,36,37,38. In addition, cognitive function improvement is the primary goal of AD clinical treatment, and cholinesterase inhibitors have been broadly established as a first-line AD symptomatic therapy39. In vitro data demonstrated that DL0410 has potent AChE and BuChE inhibitory effects compared with clinically approved medications, such as donepezil and rivastigmine, and the DL0410 treatment decreased extracellular Aβ1–42 levels and increased cell viability in copper-induced toxicity in APPsw-SY5Y cell lines. To investigate the neuroprotective effect of DL0410 on cognitive impairment and cholinesterase deficits in vivo, we established an acute intracerebroventricular injection Aβ1–42 mouse model and copper-treated APPsw-SY5Y cell model31,32. In vivo data suggested that the intracerebroventricular infusion of Aβ1–42 resulted in significant learning and memory impairment, and DL0410 significantly improved behavioral performance in a dose-dependent manner. Furthermore, DL0410 exerted an inhibitory effect on AChE and BuChE activities. To evaluate whether the behavioral improvement observed in the DL0410-treated mice was correlated with changes in Aβ metabolism, we analyzed the presence of Aβ deposits and β-APP protein levels in the brains of Aβ1–42-treated mice. We showed that the DL0410 treatment attenuated Aβ1–42-induced Aβ overproduction in the mouse brain. The results from in vivo and in vitro studies indicated that the regulation of Aβ production represents a potential neuroprotective mechanism of DL0410 in AD therapy.

CREB is a widely expressed nuclear transcription factor that plays a critical role in learning and memory. The expression of phosphorylated CREB in transgenic mice and disease patients is reduced, leading to a reduction in the BDNF level. Hence, the CREB/BDNF signaling pathway might act as a key modulator in the pathophysiology of AD40,41,42. To further examine the molecular mechanism underlying the improvement of AD impairment through DL0410, we assessed the effects of DL0410 on the phosphorylation of CREB and the expression of BDNF protein in the brains of Aβ1–42-treated mice. According to the present study, DL0410 (9 mg/kg) significantly increased the phosphorylation of CREB in the cerebral cortices of Aβ1–42-treated mice. DL0410 increased the number of BDNF-positive neurons in the mice cortices. This in vivo mechanism study indicated that the protective effect of DL0410 might be associated with the up-regulation of the above signaling pathway.

Mitochondria play a critical role in neuronal energy supply and synaptic plasticity43,44,45. Many studies have suggested that mitochondrial dysfunction is a common feature in AD neurons46. It has also been reported that mitochondria-derived reactive oxygen species enhance amyloidogenic amyloid precursor protein processing, whereas Aβ leads to mitochondrial functional impairment47. In the present in vitro study, when cells were treated with copper for 24 h, the mitochondrial membranes were depolarized, and DL0410 significantly alleviated mitochondrial dysfunction through improvements in mitochondrial membrane potential.

We further investigated the molecular components involved in the mitochondria- mediated apoptosis pathway. Based on in vivo experiments, DL0410 increased CREB activity in the brains of Aβ1–42-treated mice. Studies have confirmed that the activation of CREB is positively regulated through Akt48,49,50,51,52. Akt plays important roles in preventing cell apoptosis53. The activity of JNK is negatively regulated through Akt, whereas JNK activation is involved in the mitochondria-mediated apoptosis pathway42,43. We observed that APPsw-SY5Y cells exposed to copper exhibited increased levels of p-JNK and decreased levels of p-Akt. DL0410 significantly increased the phosphorylation of Akt and suppressed the phosphorylation of JNK. The results of the present study suggested that DL0410 attenuates Aβ-induced (stimulated through copper) apoptosis via the regulation of the Akt/JNK pathway.

In conclusion, the present study examined the therapeutic potential of DL0410 in Aβ-induced cognitive impairment and neuronal damage. The results demonstrated that DL0410 exhibited potent inhibitory effects on cholinesterase, improved learning and memory dysfunction in rodents, and exhibited neuroprotective effects both in vitro and in vivo. The Akt/JNK pathway was involved in the neuroprotective effects of DL0410. These results indicated that DL0410 is a potential pharmaceutical candidate for AD therapy.

Author contribution

Guan-hua DU and Ai-lin LIU designed the experiment and revised the manuscript; Dan ZHOU, Wei ZHOU, Jun-ke SONG, Zhang-ying FENG, and Ran-yao YANG performed the experiments; Jun-ke SONG helped to edit several figures and analyse data; Song WU and Lin WANG supplied the DL0410 compound; Dan ZHOU and Wei ZHOU analyzed the data.