Animals and treatment

Male double transgenic APP/PS1 and Wild type (WT) mice were used. All animals were littermates with identical genetic background (C57BL/6), obtained from established breeding colonies at the Animal Facility of the Faculty of Pharmacy and Food Sciences, University of Barcelona (approval number C-0032). APP/PS1 mice express a Swedish mutation (K594M/N595L) of a chimeric mouse/human APP (mo/huAPP695swe), along with the human exon-9-deleted variant of PS1 (PS1-dE9).

Mice received intraperitoneal (i.p.) injections of either LCA (15 mg·kg−1·day−1) or saline solution (0,9% (w/v) NaCl) three times per week for 4 weeks, starting at five months of age. Animals were divided into four experimental groups: WT Saline, WT LCA, APP/PS1 Saline, and APP/PS1 LCA. Throughout the treatment period, body weight was monitored weekly to adjust drug dosage and track potential weight changes. A graphical representation of the experimental design is depicted in Fig. 1.

Fig. 1The alternative text for this image may have been generated using AI.

Graphical representation of experimental design. 5-month-old male WT C57BL6/J and APP/PS1 mice were treated i.p with 15 mg/kg of LCA, three times per week for 4 weeks. Then, animals were subjected to two different behavioral tests: MWM (for 7 days) or NORT (for 5 days). Afterwards glucose metabolism was studied through GTT or ITT. Finally, animals were sacrificed by cervical dislocation in order to obtain liver and brain samples to perform staining and biochemical techniques, or by intracardially perfusion for immunochemistry techniques. Image created in BioRender. Carrasco Pérez, M. (2026) https://BioRender.com/t88q130. License number GW28WROIWO

All animals had ad libitum access to food and water and were maintained under controlled conditions of temperature, humidity, and standard 12-h light-dark cycles, following the ethical guidelines established by the European Committee (European Communities Council Directive 2010/63/EU). Every effort was made to minimise animal suffering and reduce the number of animals used, in accordance with the experimental protocol approved by the Ethics Committee of the University of Barcelona (approval number CEEA 424/22). All procedures were conducted following the European Community Council Directive 86/609/EEC and the guidelines established by the Departament d'Agricultura, Ramaderia i Pesca of the Generalitat de Catalunya.

Behavioural testsMorris water maze

Twelve animals per group were subjected to MWM test to assess spatial long-term memory and learning abilities. This experiment was performed in a 100-cm-diameter circular pool, divided into four quadrants. An escape platform was hidden inside the pool 1 cm below the water surface, which was previously stained with liquid latex to hinder visibility. Four different spatial cues were located around the pool and remained consistent throughout the test, to foster spatial orientation. Water temperature and light intensity were maintained at 28ºC and 30Lux, respectively, for the entire procedure.

The procedure consisted of two different phases: training and test. During the training phase, each animal underwent 5 different trials per day, entering the pool from 5 different locations, for 6 consecutive days. In each trial, the animals were allowed to swim freely for 1 min to find the hidden platform. If the animal failed to find the platform within this time, it was placed on the platform for 30 s.

The test phase was performed on the seventh day. The platform was removed, and each animal was placed into the pool from a single starting point. Mice were allowed to swim freely for 1 min to reach the area where the platform used to be, assessing their long-term memory. The acquired data were analysed using SMART V3.0 (Panlab Harvard Apparatus, Germany) video tracking system, with results calculated individually for each animal.

Novel object recognition

The non-spatial recognition memory of 15 animals per group was evaluated by the Novel Object Recognition test (NORT). The test was carried out in a 40 cm diameter circular open-field box, under 30Lux of constant illumination. The test was divided into three different phases: habituation, familiarization and test.

During the habituation phase, each mouse was allowed to acclimate to the arena without any object for 10 min per session over three consecutive days. In the familiarization phase, on the fourth day, each mouse was allowed to explore two identical objects (A and A’) placed in the middle of the arena for 10 min. Finally, on the fifth day, the test phase was performed by exposing each mouse to a familiar object (A) and a novel object (B) for 10 min.

After each trial the objects and the open-field box were cleaned with 70% ethanol to eliminate olfactory cues. Every trial was recorded, and the Discrimination Index (DI) was calculated on the test phase using the following equation:

Exploration was defined as looking, sniffing or touching the object. Mice with a total exploration time lower than 5 s were removed from analyses.

Glucose and insulin tolerance test

For glucose tolerance test (GTT) and insulin tolerance test (ITT), mice (n = 15 and 12 per group, respectively) were fasted for 6–8 h before i.p. administration of glucose (1.5 g/kg) or insulin (0.75 UI/kg) for GTT and ITT, respectively. Blood glucose levels were monitored using an Accu-check® Aviva glucometer (F. Hoffmann-La Roche) with blood samples collected from the tail vein.

For GTT, blood glucose was monitored at 0, 5, 15, 30, 60, and 120 min post-glucose injection. For ITT, measurements were taken at 0, 15, 30, 45, 60, and 90 min post-insulin injection. During ITT, if blood glucose levels fell below 25 mg/dL, the test was immediately ended and animals received a rescue dose of glucose (1.5 g/kg, i.p.).

Results were expressed as mg/dL of blood glucose. The statistical analysis was performed by comparing the area under the curve (AUC) of blood glucose through the analyzed time for GTT and ITT. Moreover, time-specific statistical analysis was performed in all the time periods studied in ITT.

Dendritic spine quantification

After cervical dislocation, brains (n = 5 per group) were removed and processed according to the manufacturer’s protocol using the FD Rapid GolgiStain™ Kit (Cat #PK401, FD Neurotechnologies, Inc.). Hippocampal neurons were visualized using a Leica Thunder Imager microscope (Leica Microsystems) with a 63X objective, and five dendrite images per animal were captured for analysis.

The analysis focused on specific dendritic regions: secondary branches and terminal dendrites of the dentate gyrus (DG), as well as secondary branches of the cornu ammonis 1 (CA1) basal zone and terminal dendrites of the CA1 apical zone. For secondary branches, the analysis was performed on a 30 µm segment, starting 20 µm after the branch point. For terminal dendrites, the analysis was conducted on a 30 µm segment, beginning 20 µm after the terminal end. Images were processed using ImageJ software [42] (National Institutes of Health, Bethesda, MD, USA) and spine density was expressed as the number of spines per 30 µm of dendrite length.

Post-synaptic density protein 95 quantification

Following cervical dislocation, hippocampi were isolated and stored at −80 °C until analysis. Post-synaptic density protein 95 (PSD95) levels were measured in hippocampal samples (n = 6–7 per group) using a PSD95 ELISA Kit (LS-F7142, LifeSpan BioSciences, Inc.), according to manufacturer’s instructions. Absorbance measurements were performed using a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific). Results were normalized to total protein content and expressed as μg PSD95/μg protein.

Protein extraction

Previously obtained hippocampi by cervical dislocation (n = 6–7 per group) were homogenized in lysis buffer containing 1 M Tris–HCl (pH 7.4), 5 M NaCl, 0.5 M EDTA (pH 8), and Triton X-100 in distilled water, supplemented with protease inhibitor cocktail (118,336,170,001, Complete Mini, EDTA-free; Roche Diagnostics, Mannheim, Germany) and phosphatase inhibitor cocktail (P0044, Sigma-Aldrich, St. Louis, MO, USA). Homogenates were kept on ice for 30 min and centrifuged at 14,000 g for 10 min at 4 °C. Supernatants were collected and total protein concentration was determined using the Pierce™ BCA Protein Assay Kit (#23,225, Thermo Scientific, Rockford, IL, USA).

Western blotting

Protein samples (n = 6–7 per group) were standardized to 10 µg of protein and mixed with an equal volume of sample buffer (0.25 M Tris pH 6.8, 4% (w/v) SDS, 200 mM dithiothreitol (DTT), 20% (v/v) glycerol, and bromophenol blue). Samples were denatured at 95 °C for 5 min and separated by SDS-PAGE on 10% (v/v) polyacrylamide gels at 120 V.

Separated proteins were electroblotted into methanol-activated polyvinylidene fluoride (PVDF) membranes at 100 V for 120 min. Membranes were blocked with 5% (w/v) bovine serum albumin diluted in Tris-buffered saline (150 mM NaCl, 25 mM Tris-HCl, pH 7.6) containing 0.1% (v/v) Tween-20 (TBS-T) for 1 h at room temperature (RT). Afterwards, membranes were washed three times with TBS-T for 5 min each and incubated overnight (O/N) at 4 °C with the corresponding primary antibody (detailed in Table 1).

Table 1 Antibodies for Western Blotting and Immnunohistochemistry

Following primary antibody incubation, membranes were washed three times for 5 min with TBS-T and incubated with corresponding secondary antibody (detailed in Table 1) for 1 h at RT. After three additional TBS-T washes of 5 min, immunoreactive bands were visualized using Immobilon® Western Chemiluminescent HRP Substrate (#WBKLS0500, Merck Millipore, Darmstadt, Germany) and imaged using an ImageQuant LAS 500 system (GE Healthcare, Chicago, IL, USA). The optical density of the obtained bands was quantified with Image Lab Software (Bio-Rad, Hercules, California, United States).

Immunohistochemistry

For immunohistochemical analyses, animals (n = 5 per group) were anaesthetized with 0,33 mg/g of pentobarbital and intracardially perfused with a 4% (v/v) paraformaldehyde (PFA) solution diluted in 0.1 M phosphate buffer (PB). Brains were dehydrated in a 30%/w/v) sucrose + 2% (v/v) sodium azide solution diluted in 0.1 M phosphate-buffered saline (PBS) for at least 3 days and coronal sections of 20 μm of thickness were obtained with a cryostat (Leica Microsystems, Wetzlar, Germany) and stored at −20ºC in cryoprotectant solution (10% (v/v) PB 0,1 M, 30% (v/v) ethylene glycol and 30% (v/v) glycerol diluted in water) until use.

Free-floating sections were processed as previously described by or group [43]. The corresponding antibodies used are detailed in Table 1.

20 × Images were obtained with a Leica Thunder Microscope (Leica Thunder Imager; Leica Microsystems) and quantified by ImageJ. The resulting integrated density of glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (IBA1) was quantified in the hippocampal DG using ImageJ [44]. Ki67-immunoreactive positive cells were quantified in the subgranular zone (SGZ) of the hippocampal DG, differentiating suprapyramidal (SP) and infrapyramidal (IP) layers.

Thioflavin-S staining

Free-floating sections (n = 5 per group) were rinsed 5 min in 0.1 M PBS (pH 7.35) three times, then 5 min in PBS-T for five times. Subsequently, sections were submerged in blocking solution for 2 h at RT and washed five times for 5 min with PBS-T. Afterwards, slices were incubated with 0.0033% Thioflavin-S diluted in 0.1 M PBS for 8 min, followed by two washes with 50% (v/v) ethanol for 1 min. Then, sections were washed 3 times with 0.1 M PBS for 5 min.

Finally, slices were mounted in Superfrost™ microscope slides (J1800AMNZ, Epredia Netherlands B.V., Essedonk, Netherlands) using Fluoromount-G medium (00–4958-02, Thermo Fisher Scientific). Twenty × Images were obtained with a Leica Thunder Microscope (Leica Thunder Imager; Leica Microsystems) and quantified by ImageJ.

PAS Staining

For histological staining studies, after intracardiac PFA-perfusion livers were isolated and stored into a 50% (v/v) ethanol solution until paraffin-embedding.

Glycogen storage was assessed using Periodic acid–Schiff (PAS) staining on 4-μm liver sections (n = 3 per group) obtained from PFA-fixed paraffin-embedded samples.

Hepatic glycogen quantification

Liver samples (n = 6 per group) were homogenized in 30% (w/v) KOH and incubated at 100ºC for 30 min. Samples were then centrifugated at 5000 rpm for 10 min at RT. The supernatant was collected and spotted on 31ET paper. The papers were washed once with cold 66% (v/v) ethanol for 15 min, followed by two times with 66% (v/v) ethanol at RT for 30 min each. Subsequently, the papers were washed with acetone and allowed to dry at RT.

Each paper was then incubated in a 50% (w/v) α-amiloglucosidase (#A7420, Sigma Aldrich, Germany) solution prepared in 0.4 M sodium acetate buffer (pH 4.8) for 120 min at 37 ºC. The resulting supernatant was collected and centrifuged at 2000 rpm for 15 min at 4ºC. Glycogen was then quantified as glucose equivalent in the supernatant using the Glucose (GO) Assay Kit (#GAGO20, Sigma Aldrich, Germany) following the manufacturer’s instructions.

RNA isolation

Hippocampi (n = 6 per group) were homogenized using TRItidy G™ reagent (A4051; Panreac Química S.L.U., Barcelona, Spain) and centrifuged at 12,000 g for 5 min at 4 °C. The supernatant was collected, mixed with chloroform, and centrifuged again under the same conditions. The upper phase was mixed with isopropanol and incubated on ice for 10 min, followed by centrifugation at 14,000 g for 10 min at 4 °C. The resulting RNA pellet was washed with 70% (v/v) ethanol and centrifuged at 7,500 g for 5 min at 4 °C. Finally, it was air-dried at room temperature and resuspended in 20 μL DEPC-treated water.

RNA concentration and integrity were assessed using a NanoDrop™ One/OneC Microvolume UV–Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For cDNA synthesis, 2.000 ng of RNA was reverse-transcribed using a High-Capacity Reverse Transcription Kit (4,368,813; Applied Biosystems, Foster City, CA, USA). The resulting cDNA was diluted in DEPC-treated water to 2 ng/μL.

Real-time polymerase chain reaction

Real Time Polymerase Chain Reaction (RT-PCR) was performed in duplicate using six cDNA samples per group. Reactions contained equal volumes of cDNA and Maxima SYBR Green qPCR Master Mix with ROX (K0253, Thermo Scientific) along with specific primers (detailed in Table 2). Amplification was performed using a QuantStudio™ 3 real-time PCR system (Thermo Fisher Scientific). Gene expression was normalised to Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) and expressed relative to the Saline group.

Table 2 Primers for RT-PCRAmyloid β1–42 quantification

Cortical tissue samples (n = 10 per group) were collected after cervical dislocation and stored at −80 °C until analysis. Aβ₄₂ levels were measured using a Human Aβ₄₂ ELISA Kit (KHB344, ThermoFisher Scientific) following manufacturer’s instructions. Absorbance was measured using a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific). Results were normalized to total protein content and expressed as pg Aβ₄₂/μg protein.

β-secretase activity

β-Secretase (BACE) activity was analyzed in −80ºC-stored cortex samples (n = 7 per group) using a fluorometric β-Secretase activity assay kit (ab65357, Abcam). following manufacturer's instructions. Fluorescence was measured using a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific). Results were expressed as relative fluorescent units (RFU) 335/495 nm ratio.

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 8.3.0 (San Diego, CA, United States, www.graphpad.com). When comparing two groups, an unpaired Student t-test was performed. Meanwhile, when comparing the four groups a two-way ANOVA followed by Tukey’s post-test for multiple comparisons was applied. In cases where data did not follow a normal distribution, the Kruskal–Wallis non-parametric test followed by Dunn’s post-test was applied. Results are expressed as mean ± SEM. Differences were considered statistically significant when *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Genotype- and treatment-dependent significant differences were depicted with & and $, respectively.