Material and chemicals

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, H4034), L-glutamine (G-8540) and D-glucose (G-7528) were purchased from Sigma (St. Louis, MO, USA). Minimum essential medium (MEM, LM 007 − 01) and Hank’s balanced salt solution (HBSS, LB 003 − 01) were bought from Welgene (Kyungsan, South Korea). Donor horse serum, heat inactivated (HS, S0900-500) was obtained from Biowest (Nuaillé, France). Penicillin streptomycin (LS0202-02) was acquired from Gibco BRL (Rockville, MD, USA). Phosphorylated (p)-tropomyosin receptor kinase (Trk) A (cat. #9141), TrKA/B (cat. #4619), extracellular signal-regulated kinase (ERK1/2, cat. #9102), p-ERK1/2 (cat. #9101), c-Jun N-terminal kinases (JNK, cat. #9252), p-JNK (cat. #9251), p38 (cat. #9212), p-p38 (cat. #9101), HO-1 (cat. #70081), p-Akt (cat. #9271), Akt (cat. #9272), p-glycogen synthase kinase-3β (p-GSK-3β, cat. #9336), GSK-3β (cat. #9315), p-IkBα (cat. #2859), IkBα (cat. #4812), p-NF-κB (cat. #3033), NF-κB (cat. #4764) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against glyoxalase 1 (GLO-I, cat. sc0133144), glyoxalase 2 (GLO-II, cat. sc271786), receptor for advanced glycation end products (RAGE, cat. sc365154), glyceraldehyde-3-phosphate dehydrogenase, (GAPDH, cat. sc32233), silent mating type information regulation 2 homologue (Sirt)-1 (cat. sc135792), Sirt-2 (cat. sc28298), Sirt-3 (cat. sc365175), Sirt-4 (cat. sc135797), Sirt-5 (cat. sc271635), and Sirt-7 (cat. sc365344) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). p-TrkB (cat. ab229908), p-Tau (cat. ab32057), Tau (cat. ab254256), TPH1 (cat. ab52954), TPH2 (cat. ab288067), nerve growth factor (NGF, cat. ab66459), thioredoxin reductases1 (TXNRD1, cat. ab124954), Trx (cat. ab273877), glucocorticoid receptor (GR, cat. ab183127), and ionized calcium binding adaptor molecule-1 (Iba-1, cat. ab16588) antibodies were purchased from Abcam (Cambridge, UK). BDNF (cat. A11028) antibody was received from Abclonal. APP (cat. 14974982) and amyloid β-protein (Aβ) oligomers (oAβ, cat. AHB0052) were obtained from Invitrogen (MA, USA).

Animals and study design

ICR mice (7-weeks-old, male) were acquired from Orient Bio (Gyeonggi-do, Korea) and placed in a standard temperature-(22 ± 2 °C) with 65% humidity and a 12 h/12 h light/dark cycle. The Animal Care Committee of the Center of Animal Care and Use at Gachon University (GIACUC-R2020008) reviewed and approved all experimental guidelines. One week after the acclimation period, the ICR mice were divided into five groups: control (CON, n = 8), 25 mg/kg of MGO (n = 8), 30 mg/kg of MGO (n = 8), 65 mg/kg of MGO (n = 6–8), and 65 mg/kg of MGO + 40 mg/kg of Trp (MGO + Trp, n = 8). Trp (40 mg/kg) was oral administered to experimental ICR mice for 2 or 3 weeks. MGO (25, 30, or 65 mg/kg) and Trp (40 mg/kg) were administered to experimental ICR mice for 2 or 3 weeks via rectal injection [30% v/v glycerol in phosphate-buffered saline (PBS, pH 7.4)] (see Figs. 1A and 8A, and 9A).

Fig. 1

Depression-like behavior and working memory loss in mice. (A) Schematic diagram of the experimental plan. (B) The total distance, time spent and speed in the open field test (OFT). (C) The immobility time in the tail suspension test (TST) chamber (D) The immobility time in the forced swimming test (FST) chamber (E) The percentage of sucrose consumption in the sucrose preference test (SPT). (F) The time spend in the opened arms from elevated plus maze (EPM) test (H) The latency to reach the target in the barnes maze (BM) test after training 3 days. (H) The percentage of recognition index in novel object tool in the novel objective recognition test (NORT). (I) The number of total arm entries and the summary of the percentage of alteration in Y-maze test. All results obtain from behavior tests was calculated using SMART3.0 SUPER PACK. The values are presented as mean ± SEM (n = 6–8). #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. control group (CON)

Open field test (OFT)

The OFT was conducted in an open plastic box (45 cm × 45 cm × 45 cm), as reported by Ueno et al. with modifications [35]. The box zone was divided into 24 grids of 11.25 × 11.25 cm. The mice were individually placed in the center of an open plastic box and allowed to observe freely, after which a video was recorded for 5 min. An open plastic box was cleaned with 70% ethanol. The total distance, time spent, and speed in the central zone(s) was analyzed using SMART3.0 SUPER PACK (Panlab; Harvard Apparatus, Barcelona, Spain).

Tail suspension test (TST)

TST was performed as mentioned by Ueno et al. with a bit of change [35]. The TST was conducted in a TST chamber (60 cm length, 60 cm height, 11.5 cm depth, and 15 cm width), and the mice were suspended using painless tape-based fixation. Before recording, all the mice were adapted to the TST chamber for 2 min. The mice were individually placed in the TST chamber, and videos were recorded for 4 min. Next, the immobility of the mice was analyzed using the SMART3.0 SUPER PACK.

Forced swimming test (FST)

The FST was performed as reported by Kang et al. with slight modifications [35]. It was performed in an FST chamber (50 cm height × 20 cm diameter) filled with water (at a level of 30 cm) set at room temperature (25 ± 1 °C). Before recording, all mice were pre-adapted to the FST chamber for 2 min and then placed in the FST chamber, after which the video was recorded for 4 min. The immobility of the mice was evaluated using the SMART3.0 SUPER PACK.

Sucrose preference test (SPT)

SPT was performed as reported by Luo et al. with slight modifications [36]. The mice were adapted to 2% sucrose solution (w/v) were placed in each cage for 2 days, and then deprived of water and food for 16 h, followed by SPT. After 48 h, two bottles with 2% sucrose solution (20 ml) and tap water (20 ml) were freely given. Three hours later, sucrose and water consumption (ml) were recorded. The percentage of sucrose preference was calculated as sucrose consumption / (sucrose consumption + water consumption).

Elevated plus maze (EPM) test

The EPM test was analyzed as described in previous report [37]. The comprised of open arms (35 cm × 5 cm) and closed arms (35 cm × 5 cm × 15 cm) that crossed from a central area (5 cm × 5 cm). The floor and walls were painted black, the entire maze was elevated to a height of 45 cm above floor. The mice were individually placed in the center of EPM tool and allowed to observe freely, after which a video was recorded for 5 min. The total distance and time spent in open arms were analyzed using SMART3.0 SUPER PACK.

Barnes maze (BM) test

The BM test was constructed from black polyethylene and consist of circular are (45 cm diameter) with 12 circular holes (4.5 cm diameter) as see in Fig. 1G. The maze was elevated 50 cm above the floor. The escape box (35 cm × 25 cm × 15 cm) was constructed black polyethylene. Mice were placed circular central zone and then trained to reach the escape box for 4 days. The mean latency time to reach the target was analyzed using SMART3.0 SUPER PACK.

Novel objective recognition test (NORT)

Multiple experiments began with an acquisition trial designed to acquire mice with two similar objects. This was followed by a probe trial in which a familiar object was substituted with a novel object to assess recognition memory. After each trial, the box and objects were thoroughly cleaned to eliminate olfactory cues from preceding mice. Plastic toys of various shapes were used as objects in this examination. Object exploration was defined as beginning to sniff the object from a maximum distance of 1 cm or less, excluding climbing and sitting on the object from exploratory activities. Recognition memory was gauged by comparing the time spent exploring the novel object to the time spent exploring the familiar object. The training trial lasted 5 min, after which the mice were returned to their home cages at 10-minute intervals. Subsequently, the probe trial, which lasted for 3 min, involved replacing one familiar object with a novel one, and the time spent exploring each object was recorded.

Y-Maze test

The mice were positioned at the terminus of one arm in a symmetric Y-maze featuring arms 40 cm in length, 8 cm in width, and 20 cm in height. The mice were allowed to explore the apparatus freely for 5 min. An overhead camera captured the sequence of arm entries (e.g., ACBCABCBCA). An alteration was registered when the mouse accessed three different arms within a triad of overlapping triplet sets (e.g., in the ACBCABCBCA sequence, five alternations were tallied). Spontaneous alternation was determined by counting the instances when the mouse sequentially entered each of the three arms of the maze, divided by the maximum potential alternations. The maximum number of alternations was calculated as the total number of arm entries minus two.

Measurement of Trytophan (Trp), 5-hydroxytryptophan (5-HTP), and serotonin (5-HT) levels in the plasma

Trp, 5-HTP, and 5-HT levels were analyzed as described by Fuertig et al. [38] with slight modifications. Briefly, mouse plasma was mixed with 0.1% formic acid and acetonitrile (ACN), vortexed, and mixed within 30 s for 5 min. The reaction samples were centrifuged, vortexed with 0.1% formic acid, and injected into the Agilent LC 1100 series LC-MS/MS system (CA, USA). Chromatography was performed using an Agilent ZORBAX Extend-C18 column (1.0 × 150 mm, 3.5 μm) operated at 30 °C. The solvent system consisted of mobile phase A [100% ACN)] and B (0.1% formic acid in distilled water) at a ratio of 1:1 (v/v, %). To detect tryptophan, 5-HTP, and serotonin in the analytes, the MS/MS system was operated in positive (ESI+), negative (ESI-), and multiple reaction monitoring modes.

Measurement of neurotransmitter levels in brain tissues

The dopamine (DA), epinephrine (EPI), and 5-HT levels were analyzed using the protocol reported by Planchez et al. [39]. After the sacrifice of mice, the whole brain was removed and homogenized in 0.1 M perchloric acid (PCA) (10 mg/µL). The samples were then centrifuged at 12,000 rpm for 30 min. The supernatant was filtered using 0.2 μm filters and injected into the HPLC system (Waters Corp., MA, USA). The samples (20 µL) were analyzed using a Kromasil C18 column (150 mm × 4.6 mm, 5 μm) with an electrochemical detector. The mobile phase consisted of 2% citric acid, 2% K2HPO4, 1 mM EDTA, 1.2% MeOH, and 7 mg/mL sodium octyl sulfate. The detector conditions were + 0.008 V and a 0-100 nA sensitivity range. The flow rate was maintained at 1.0 ml/min.

Measurement of Trp levels in cell medium and extracts

Trp levels were analyzed according to a previously reported method [40]. N2a cells were seeded in a 60 Φ dish, washed with cold PBS, centrifuged, and extracted with ice-cold 50% methanol/50% piperazine –N (PIPES)-EDTA at -20 °C for 5 to 10 min. After the cell extracts were separated by centrifugation, the samples were injected into the HPLC system. The samples were analyzed using a Sepax HP-C18 column (250 mm × 4.6 mm, 5 μm) with 15 mM potassium phosphate and 2.7% ACN as an isocratic mobile phase at a detection wavelength of 220 nm. The flow rate was 0.8 mL/min, and the column temperature was maintained at 30 °C.

Neurite outgrowth assay

N2a cells were seeded into a 6-well plate (4.0 × 105 cells/well) and incubated for 4 h. After incubation, the cells were treated with or without MGO (500 µM) and tryptophan-free medium for 48 h. IncuCyte® ZOOM Imaging System was used to evaluate the neurite outgrowth, neurite length, and cell morphology of treated or untreated cells.

Field excitatory postsynaptic potential (fEPSP) activity

fEPSP activity was performed as described in previous report [41]. The prepared the organotypic hippocampal tissue on micro-electrode array was tested as shown below. Bipolar electrical stimulation was applied to stratum radiatum region of CA (Cornu Ammonis) 2 and CA3 for inducing the Schaffer collateral and commissural pathways. The intensity of bipolar stimulation was set at 80 µA, 120 µs per phase which was optimized level to offer 40–65% of the maximum hippocampal tissue response. Theta-burst stimulation (TBS) consisted of 300 biphasic pulses, 3 trains at 100 Hz for 1 s (train interval for 5 min). Each experiment for inducing LTP was planned with total 100 min protocol. There were 40 min of test recording of field excitatory postsynaptic potential (fEPSP) at one stimulation per minute (baseline; first 10 min), 10 min of TBS, and 50 min of post-TBS fEPSP measurement. The point for inducing Schaffer collateral and commissural pathways was chosen by morphological structure of hippocampal tissue and appropriate response by bipolar electrical stimulation. While the experiments were processing, the slices were continuously perfused with fresh aCSF at the rate of 3 ml/min. MGO and CNQX were treated alone or together with aCSF 10 min after recording. Unrefined analog MEA signal and acquired fEPSPs level from triggering amplitudes over 40 µV were digitized by MC Rack from Multi Channel Systems. And customized MATLAB (v.7.0.1, Mathworks. Inc.) was used for removing stimulus artifacts and integrating the evoked field potential trajectory.

Primary hippocampal neuron culture

Timed pregnant (TP) 17 days (SD) rats were purchased from Koatech (Korea). The hippocampal tissue was enzymatically dissociated using 0.25% trypsin solution (Gibco). The dissociated hippocampal tissue was washed with 1x HBSS (Gibco) and resuspended in a neurobasal medium containing 2% B27, 2-mM L-glutamine, and 1% penicillin-streptomycin (Gibco). Hippocampal neurons were seeded in 96-well plates or 18-mm coverslips pre-coated with 0.1 mg/ml poly-L lysine (Sigma). Hippocampal neurons were plated in 96-well plates at a density of 2 × 104 cells/well or on 18-mm coverslips at a density of 3 × 104 cells/coverslip, respectively. Half of the culture medium was replaced every 3–4 days. Primary hippocampal neurons were maintained in a 5% CO2 humidified incubator at 37 ℃ for 14 days. At DIV 11–13, primary hippocampal neuron media was changed with or without tryptophan before treatment with 500 µM of MGO. WST-1 (Roche, Basel, Switzerland) was used to measure the metabolic activity of viable cells. WST-1 levels were determined according to the manufacturer’s instructions. After incubation with MGO for 24 h, WST-1 reagent was added to the wells. The primary hippocampal neurons were incubated at 37 ℃ in 5% CO2 for 2 h. The absorbance of the reaction medium was measured at 450 nm using a Vector X4 multilabel plate reader (Perkin Elmer).

Spine density analysis

Hippocampal neurons were fixed in 4% paraformaldehyde (PFA) solution for 20 min and washed with 1x PBS. The fixed hippocampal neurons were permeabilized in PBS containing 0.1% Triton X-100 for 15 min and blocked with 1% bovine serum albumin (BSA) for 45 min. The hippocampal neurons were stained with primary antibodies for 1 h, followed by incubation with fluorescence-conjugated secondary antibodies or phalloidin 488 (Thermo Fisher Scientific) for 2 h. The hippocampal neurons were washed with PBS and mounted on glass slides. Images of the hippocampal neurons were captured using a Zeiss LSM 700 confocal microscope with a 20x objective. Hippocampal neurons were fixed in a 4% PFA solution for 20 min at room temperature and washed using 1x PBS. Fixed hippocampal neurons were permeabilized in PBS containing 0.1% Triton X-100 for 15 min and blocked 1% BSA for 45 min at room temperature. Hippocampal neurons were stained with primary antibody for 1 h at room temperature followed by microtubule-associated protein 2 (MAP2; 1:200, mouse-IgG; Sigma-Aldrich). Secondary antibodies were goat anti-mouse 594 (1:200; Invitrogen, UK) and cytopainter phalloidin 488 (1:200; Invitrogen, UK), and incubated for 2 h at room temperature. The hippocampal neurons were washed with PBS and mounted on the slide glass. Images of hippocampal neurons were captured by a Zeiss LSM 700 confocal microscope with a 20x objective. The 20x images were taken with 1024 × 1024 pixels. Dendritic spines were analyzed with Zen 2.3 SP1 software. Z-stack images were collected at 20 μm intervals and then compressed into a 2D image with maximal intensity projection.

Cytokines levels, NAD+ and catalase (CAT) activity measurement

The cytokines including interleukin (IL)-6, -10, and tumor necrosis factor-α (TNF-α) from mouse plasma were analyzed using enzyme-linked immunosorbent assay (ELISA) kits (R&D system, Minneapolis, MN, USA). For measuring the NAD+ or CAT activity, the mouse brain tissue was homogenized in buffer, and then quantified using a colorimetric or quantification BioVision assay kit according to the manufacturer’s instructions.

Western blot analysis

N2a cells and brain tissues lysed in PRO-PREP™ protein extraction solution (iNtRON, Seoul, Korea) at -20 °C for 24 h. After the cell lysates were separated by centrifugation, protein concentration was determined using the Bradford assay. Proteins (30–50 µg) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes using a Trans-Blot® Turbo™ blotting system. The membranes were then blocked using 5% skim milk for 1 h and incubated overnight at 4 °C with primary antibodies. After overnight incubation, the membranes were washed and incubated with secondary antibodies at room temperature (25 °C) for 1 h. The bands were detected using a ChemiDoc™ XRS + imaging system (Bio-Rad, CA, USA).

Quantitative real-time PCR

Total RNA was extracted from cultured N2a cells using TRIzol® reagent (Invitrogen, CA, USA). A total of 1 µg of RNA was reverse transcribed to complementary DNA using the PrimeScript™ RT reagent Kit (Takara Bio, Otsu, Japan), according to the manufacturer’s protocol. Quantitative polymerase chain reaction (qPCR) was performed using SYBR® Premix Ex Taq™ (Takara Bio) on an Mx3005P qPCR System (Agilent Technologies). The Supplementary Material lists the primer sequences (Supplementary Table S1). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a housekeeping gene.

Immunochemistry (IHC) analysis

IHC assay was performed in described as described in previous report [42]. The mice were anaesthetized and then perfused with cold saline. The brain in 4% PFA was fixed for 1 day at 4 °C and incubated in 30% sucrose solution for 3 days at 4 °C. The coronal Sect. (25 μm) were taken by using cryostat (Cryotome, Thermo Electron Corporation) and stored at 4 °C. Each section washed with 0.2% Triton X-100 in PBS and then incubated in blocking solution including 0.5% BSA and 3% goat serum in 0.4% PBS with Tween 20). The prepared sections were incubated with primary antibody (1: 300; cat. NBP2-16908, Novus Biological, CO, USA) overnight at 4 °C. Afterward, the sections were washed and then AlexaFluor 555 Donkey anti-rabbit IgG secondary antibody (1: 500; A-31572, Invitrogen, MA, USA) was incubated for 1 h at room temperature. The stained sections were mounted onto slides using Fluoromount™ aqueous mounting medium (Sigma-Aldrich) with DAPI Vector Laboratories, CA, USA). The stained slides were observed under a laser scanning confocal microscope (Nikon A1+) and analyzed using the NIS-Elements imaging software. Iba-1stained brain sections were analyzed by region of interest (ROI) intensity ratio (%), and the microscopy magnification set as 4× – 10×.

Histological analysis

Paraffin blocks of large intestine, small intestine, and brain tissues were fixed in 4% PFA. After fixation, all tissues were dehydrated by exposure to ethanol, washed with xylene, embedded in paraffin blocks, and sectioned to 5 μm thicknesses. Sections were stained using a hematoxylin and eosin (H&E) staining kit (Sigma-Aldrich). For immunohistochemical analysis, sections were incubated in 0.1% protease K in phosphate-buffered saline (PBS) for antigen retrieval and incubated in 3% H2O2 in PBS for 15 min. Sections were incubated with 1% normal horse serum in phosphate-buffered saline (PBS) for 20 min. After blocking, the sections were incubated with GR (1: 100, Abcam, UK), TPH1 (1: 100, Abcam, UK) and TPH2 (1: 100, Abcam, UK) over night in a shaker at 4 °C. The sections were washed and incubated with biotinylated respective IgG secondary antibodies at room temperature (25 °C) for 1 h. The sections were then rinsed with PBS and incubated with VECTASTAIN® ABC reagent for 30 min. The antibody expression was detected using 3,3-diaminobenzidine (DAB). The stained tissue slides were observed and photographed using a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan) at 10× magnification, and detected intensity was calculated using the ImageJ software (NIH, Bethesda, MD).

Molecular docking

For protein preparation, the 3D crystal structure of sirt-3 (4V1C) and sirt-5 (5XHS) were retrieved from the protein data bank and prepared by removing water molecules and ligands. To prepare the ligand for docking, the 3D chemical structure of MGO was acquired from PubChem. The MGO was then uploaded to Discovery Studio, and the structure was modified by adding hydrogen and cleaning the geometry. The prepared proteins and ligands were uploaded to AutoDock Vina. Then, hydrogen was added, and the torsion parameter was set following grid box settings at 126, 126, and 126 Å for X, Y, and Z, respectively. The final output files from AutoGrid were saved in the PDBQT format. The obtained PDBQT file was used for docking analysis. A Lamarckian genetic algorithm (LGA) was selected as the best conformer. All other parameters needed for the docking analysis were used at default settings in AutoDock. Docking visualization between the protein and ligand was performed using the Discovery Studio Visualizer.

Statistical analysis

All data are presented as the mean ± standard error of the mean. All statistical analyses were performed using Prism 5.0 (GraphPad Software Inc., CA, USA), SPSS software (version 25.0; IBM SPSS Statistics Inc., IL, USA), and t-tests. Statistical comparisons between control and experimental groups were performed using Bonferroni’s test for multiple comparisons and one-way analysis of variance. Tukey’s post hoc comparison tests after one-way ANOVA were used to calculate the statistical significance of the cell viability assay and spine density analysis. Differences were considered statistically significant at p < 0.05.