The animal study was conducted in accordance with the institutional guidelines outlined by the Principles of Laboratory Animal Care (NIH publications No. 85-23, revised 1996) and the Korea Institute of Oriental Medicine Institutional Animal Care and Use Committee (KIOM-IACUC, reference number 24–073). Twenty-four-week-old female 5xFAD transgenic mice and age-matched wild-type C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME, United States). Mice were randomly group-housed (n = 10 per group) with free access to food (Purina Korea, Seoul, Korea) and water. Mice were kept under pathogen-free conditions at a temperature of 23 °C ± 1 °C on an alternating 12-h light and dark cycle. 2′-FL (Fuc-α1,2-Gal-β1,4-Glc; Advanced Protein Technologies Corp., Suwon, Republic of Korea) was administered orally at doses of 300, 600, and 1,200 mg/kg/day for 8 weeks through a gastric tube. The dose of 2′-FL was selected based on two previous studies, with the lowest dose (300 mg/kg/day) approximating the average amount of 2′-FL that breast-fed infants would receive (Vazquez et al., 2016; Gao et al., 2024). The positive control group was orally administered donepezil 5 mg/kg/day. Distilled water (DW) was administered to the control animals using the same feeding methods over the same period. The schematic timeline of this experiment is shown in Figure 1A.

A circular water tank (diameter, 150 cm; height, 60 cm) was filled with water to a depth of 30 cm, with an escape platform placed in one quadrant. During a 4-day training period, the mice were allowed to swim freely for 60 s without the platform to acclimatize to the environment. They then underwent four training sessions per day, with the entry point varying across trials. The escape platform, submerged 1 cm below the surface, was invisible during testing. All sessions were recorded using a video tracking system (SMART v3.0, Panlab SL, Barcelona, Spain). If a mouse located the platform within 60 s, it remained there for 15 s; otherwise, it was gently guided to the platform and allowed to stay for 20 s. During the probe test, the escape platform was removed, and the time spent in the target zone, where the platform had previously been located, was recorded over a 60-s period.

The Y-maze consisted of three arms (42 cm long, 3 cm wide, 12 cm high) arranged at 120° angles and constructed using black polyvinyl plastic. The arms were randomly labeled A, B, and C. Each mouse was placed in one arm and allowed to explore freely for 8 min. Arm entries were recorded, with a valid entry defined as the full entry of the mouse’s tail into an arm. Reentries into the same arm were also noted. Spontaneous alternation behavior was defined as successive entries into all three arms in a triplet sequence.

BV2 microglia were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS (Merck KGaA, Darmstadt, Germany) and 1% penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA, United States). Water-soluble tetrazolium salt assay was performed to screen the non-toxic dose of 2′-FL on the cell line. Cells were seeded in 96-well plates (3 × 10⁴ cells/well) and incubated at 37 °C and 5% CO2 for 24 h. Cells were treated with 2′-FL (0.5–8 mg/mL) or DW for 24 h, followed by quantification of viability levels through absorbance at 450 nm (detection wavelength) and 650 nm (reference wavelength) using a Tecan microplate reader (Männedorf, Switzerland).

All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee of Dongguk University College of Medicine (IACUC-2021-13). Pregnant E−16 female Sprague–Dawley rats were purchased from Orient Bio Inc. (Seongnam-si, Korea), maintained for 3 days with free access to food and water at room temperature (RT) under a 12-h light/dark cycle, and euthanized via cervical dislocation. The brains were harvested in precooled Hanks’ balanced salt solution (PAA, Pasching, Austria), hippocampi were meticulously dissected, and cell suspensions were prepared following a previously established methodology (Munni et al., 2023a; Munni et al., 2023b). A specific serum-free neurobasal medium supplemented with B27 was added to 24-well plates containing 12-mm coverslips coated with poly-d-lysine (PDL). Cells were seeded onto coverslips at a density of 3 × 10⁴ cells/cm² for morphometric analysis, cell viability, and immunocytochemistry to determine the required protein expression, and at a density of 6 × 10⁴ cells/cm² for synaptogenesis. The plates were then incubated at 37 °C with 5% CO2 and 95% air for 3 days in vitro (DIV3), DIV8, and DIV9. 2′-FL (0.5, 1, and 2 mg/mL), d-galactose (50 mg/mL), or vehicles were added into the culture media prior to cell seeding. Fresh medium with treatment was given to the cells in a 4-day cycle. The schematic timeline of this experiment is shown in Figure 4A.

The brain was dissected from perfused mice and post-fixed in 4% paraformaldehyde (PFA) for 8 h at 4 °C. Tissues were subsequently immersed in 30% sucrose at 4 °C until fully submerged. Coronal brain sections were prepared using a brain matrix, and tissue samples were transferred into tissue clearing solution (HRTC-001, Binaree, Daegu, Korea) and incubated at 38 °C on an orbital shaker. Incubation times varied depending on the organ (24 h for 1 mm brain sections). For visualization of amyloid beta plaques, samples were treated with thioflavin S, which emits fluorescence at 488 nm. Plaque staining was performed using 0.1% thioflavin S in 50% ethanol, incubated at 25 °C with gentle shaking at 50 rpm for 15 min, followed by three washes with 1× PBS at 4 °C for 20 min each. Samples were then incubated in 10 mL of mounting and storage solution (HRMO, Binaree, Daegu, Korea) at 38 °C for 24 h with shaking at 50 rpm to match the refractive index of the tissue. Imaging was conducted using a confocal microscope to detect thioflavin S fluorescence in the green channel. Image acquisition and analysis were performed using the Imaris software (version 9.5.1, Oxford Instruments, Abingdon-on-Thames, United Kingdom).

For immunofluorescence staining, brain tissue sections were washed with 1× PBS and then sectioned into 0.1-mm-thick sections using a vibratome (Vibratome VT1200, Leica, Wetzlar, Germany). Subsequently, the tissues were stained using Binaree Tissue Clearing for Immunostaining (HRTI-001, Amuza Inc., San Diego, CA, United States) to allow easy antibody penetration into the thick tissue. Briefly, paraformaldehyde-fixed brains were immersed in fixing solution at 4 °C overnight and incubated with tissue clearing solutions A and B at 37 °C for 24 h in a shaking incubator. Primary antibodies and concentrations were anti-beta amyloid (1:100, Abcam, Cambridge, United Kingdom), anti-phospho tau antibody (1:100, Swant, Bellinzona, Switzerland). The secondary antibody used for immunofluorescence detection was Alexa Fluor 488 AffiniPure F (ab’)2 Fregment Goat Anti-Rabbit IgG (H + L) (1:200; Jackson ImmunoResearch Laboratories, Inc.). The sliced brains were then incubated in mounting and storage solution (HRMO, Binaree, Daegu, Korea) for 24 h to match the refractive index.

For in vivo analysis, blood was collected from the retro-orbital plexus of experimental mice using EDTA-treated Pasteur pipettes. Samples were centrifuged, and plasma was separated and stored at −20 °C until analysis. Cytokine levels, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), were measured using mouse ELISA kits (Endogen Inc., Cambridge, MA, United States) according to the manufacturer’s instructions. For the in vitro analysis, BV2 cells were seeded at 2 × 10⁵ cells/well in 12-well plates and incubated for 24 h. Cells were then treated with 2′-FL for 5 h, followed by d-galactose (10 mg/mL) for an additional 22 h, based on a previously established protocol. Culture media were collected, centrifuged at 1,500 rpm for 10 min at 4 °C, and the supernatants were analyzed for TNF-α and IL-6 levels using ELISA kits (LABISKOMA, Seoul, South Korea). Absorbance was measured at 450 nm using a microplate reader (Tecan, Männedorf, Switzerland).

BV2 cells were seeded at a density of 3 × 10⁶ cells/100-mm dish and incubated for 24 h. Cells were then treated with 2′-FL for 5 h followed by d-galactose (10 mg/mL) for another 30 min. Cytoplasmic and nuclear fractions were extracted using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific), following the manufacturer’s protocol. Prepared samples (30 μg/lane) were separated by SDS-PAGE using a 5% stacking gel and a 10% resolving gel. Proteins were then transferred onto polyvinylidene fluoride membranes (Merck Millipore, Carrigtwohill, Ireland). Membranes were blocked with 5% skim milk for 2 h at RT then incubated overnight at 4 °C with primary antibodies, followed by a 1-h incubation with HRP-conjugated secondary antibodies at RT. After washing, immunoreactive bands were visualized using ClarityTM Western ECL substrate (Bio-Rad, Hercules, CA, United States), and images were captured using a ChemiDoc MP Imaging System (Bio-Rad). Densitometric analysis was performed using the Gel-Pro Analyzer version 3.1 software (Media Cybernetics, Rockville, MD, United States). Primary antibodies included rabbit anti-IκBα (Cell Signaling, Danvers, MA, United States), rabbit anti-NF-κB (Cell Signaling), mouse anti-β-actin (Sigma-Aldrich, St. Louis, MO, United States), mouse anti-PCNA (Santa Cruz Biotechnology, Dallas, TX, United States), anti-rabbit IgG HRP-linked whole antibody (Amersham ECL, Little Chalfont, United Kingdom), and goat anti-mouse IgG (Enzo Life Sciences, Farmingdale, NY, United States).

Neurons on the coverslips were rinsed twice with 1x Dulbecco’s PBS (D-PBS; Invitrogen) and fixed using a progressive fixation process with 4% paraformaldehyde and 10% methanol. Cells were permeabilized with 0.1% Triton X-100 and blocked with 2.5% goat serum in 0.2% Tween-20 in PBS, followed by two washes with 1×PBS. Subsequently, neurons were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-BDNF (Abclonal, Woburn, MA, United States), mouse anti-phospho-H2AX (Millipore, Billerica, MA, United States), mouse anti-tubulin α (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, United States), rabbit anti-GABAB receptor 1 (Abclonal), and mouse anti-glutamic acid decarboxylase (Developmental Studies Hybridoma Bank, University of Iowa). After washing five times for 5 min, the cells were treated with Alexa Fluor 488/568-conjugated goat anti-mouse/rabbit IgG (Invitrogen, Carlsbad, CA, United States) for 2.5 h. A mounting solution containing DAPI was used to secure the coverslips onto the glass slides.

Neuronal viability was assessed using the trypan blue exclusion assay. On DIV3, neurons were incubated with 0.4% (w/v) trypan blue for 25 min at 37 °C, then rinsed with 1× D-PBS. Cells were observed under a light microscope, and viability was calculated as the percentage of unstained (viable) cells relative to the total number of cells. Non-viable neurons, which take up the dye due to compromised membrane integrity, appeared dark blue, while viable neurons excluded the dye and remained clear.

ROS generation was assessed using DCFH-DA (MedChemExpress, Monmouth Junction, NJ, United States). Neurons were seeded on PDL-coated coverslips at a density of 3 × 10⁴ cells/cm² in 24-well plates. On DIV3, following d-galactose treatment, cells were incubated with 5 μM DCFH-DA for 30 min at 37 °C. After incubation, the cells were washed twice with 1 × DPBS and imaged using a fluorescence microscope at 488 nm. ROS levels were quantified by measuring DCF fluorescence intensity using ImageJ software (version 1.49; NIH, Bethesda, MD, United States).

Neurons were seeded on PDL-coated coverslips in 24-well plates at a density of 3 × 10⁴ cells/cm². On DIV3, after d-galactose treatment, cells were incubated with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) (MedChemExpress) at a concentration of 5 μg/mL for 30 min at 37 °C, then washed twice with 1 × DPBS. Fluorescence images were captured to detect JC-1 monomers (green, 488 nm) and aggregates (red, 568 nm). Mitochondrial membrane potential (MMP) was quantified by calculating the red-to-green fluorescence ratio using ImageJ software (version 1.49).

Neurons were cultured on PDL-coated coverslips (3 × 10⁴ cells/cm²) until DIV8 and then treated with d-galactose for 24 h. After treatment, the cells were washed with 1 × DPBS and incubated with N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide (FM1-43) (Invitrogen) at a final concentration of 5 μg/mL for 3 min. FM1-43 is rapidly taken up by recycling synaptic vesicles, labeling active nerve terminals. Cells were rinsed twice with 1× D-PBS to remove nonspecific membrane staining, and synaptic vesicle (SV) puncta were visualized using a fluorescence microscope (excitation at 568 nm). Fluorescence intensity of SV puncta was quantified using ImageJ software (version 1.49).

A fluorescence microscope (DFC3000G, Leica) equipped with a 1.3-megapixel Sony^® (Tokyo, Japan) CCD monochrome sensor P and Leica Application SuiteX (LasX Version 3.7.2.22383) software was used to monitor and photograph the cells. Adobe Photoshop 2020 (Adobe, San Jose, CA, United States) was used for image processing. Morphometric analysis was performed using ImageJ (version 1.49; NIH) to quantify the number and length of primary neurites, the length of the longest neurite, and the number of synaptic puncta per neuron. For synaptic puncta analysis and visualization of synaptic plasticity-related protein expression, images were acquired using the Cellsens software (version 1.18; Olympus, Tokyo, Japan) with an Olympus BX53 microscope equipped with a DP74 1/1.2-inch Color CMOS camera.