Animals

SAMP8 and SAMR1 mice (11 weeks old) were purchased from Beijing Weixuan Technology Co., Ltd. (Beijing, China), and acclimated for 1 week before experimental interventions.

For the Aβ1–42 oligomer-induced AD model, Aβ1–42 peptides (Abcam, UK) were prepared into oligomeric forms as described previously [30]. The peptide films were dissolved in DMSO (5 mM), diluted with sterile PBS to 100 μM, and incubated at 4 °C for 24 h. C57BL/6 J mice (Yisi Experimental Animal Technology Co., Ltd., Changchun, China) were anesthetized with 1% pentobarbital sodium and placed in a stereotaxic apparatus. Aβ1–42 oligomers (5 μL, 100 μM) were bilaterally injected into the hippocampus (AP: − 2.0 mm, ML: ± 1.5 mm, DV: − 2.0 mm) at a rate of 0.5 μL/min. The needle was retained for 5 min to prevent reflux Sham mice received an equal volume of PBS. Mice were allowed 1 week of recovery before subsequent interventions.

5xFAD transgenic mice (12 weeks old) were purchased from Cavens Laboratory Animal Co., Ltd. (Changzhou, China), and age-matched wild-type littermates were used as controls. All mice were acclimated for 1 week prior to experimentation.

For all experiments, only male mice were used to minimize sex-related variability. Each experimental group included 10–15 mice, depending on the specific behavioral, molecular, or histological analyses performed. The mice were housed in a room with suitable temperature and maintained on a 12 h day/night cycle. All animal procedures were approved by the Ethics Committee of Jilin University [SYXK(JI)2024–0023] and conducted in accordance with the ARRIVE guidelines.

Exercise training protocol

Treadmill exercise intervention was conducted using a motorized rodent treadmill (Taimeng, Chengdu, China). Mice first underwent a 3-day adaptation period with gradually increasing speeds (5 m/min, 10 m/min, and 15 m/min, respectively). Formal training commenced on day 4, consisting of running at a 5° incline, 15 m/min for 40 min per session, 5 days per week for 8 consecutive weeks. The running speed was increased by 1 m/min each week to progressively enhance exercise intensity. Throughout the intervention, mice were closely monitored for behavioral status, and body weight was recorded regularly.

For 5xFAD mice in the 4-CIN group, a physiological saline solution of 4-CIN (200 mg/kg, Selleck Chemicals, Houston, USA) was administered intraperitoneally 1 h prior to each exercise session throughout the intervention period.

NaLA administration

In the Aβ1–42 injection model, NaLA (Sigma-Aldrich, St. Louis, USA) was administered via intraperitoneal injection at a dose of 2 g/kg, 5 days per week for 8 consecutive weeks. Throughout the intervention, mice were closely monitored for general health status and behavioral activity, and body weight was recorded regularly.

Lactate measurement

Blood lactate levels were measured using a portable lactate meter (VivaChek, Hangzhou, China). Tail vein blood was collected 15 min after treadmill exercise or intraperitoneal NaLA administration.

Y-maze test

After the final intervention, animals were transferred to the behavioral testing room and maintained under standard housing conditions for 24 h to acclimate to the new environment. Behavioral testing was initiated 24 h later.

The Y-maze apparatus consisted of three arms (30 cm long, 6 cm wide, and 15 cm high) arranged at 120° angles, designated as the start arm, novel arm, and other arm. During the training phase, the novel arm was blocked, and each mouse was placed at the end of the start arm and allowed to freely explore the other two arms for 8 min. After a 4-h retention interval, the novel arm was opened, and the mouse was reintroduced into the start arm for the test phase. Behavioral activity was recorded and analyzed using Smart 3.0 software (PanLab, Barcelona, Spain).

Morris water maze test

The spatial learning and memory ability of mice was assessed using the Morris water maze (MWM), following our previously established protocol [31]. The test was conducted in a circular pool (diameter: 150 cm; water depth: 16 cm; water temperature: 26 °C). To obscure the submerged platform, the water was made opaque by adding non-toxic white paint. The pool was virtually divided into four quadrants, with a hidden platform placed in one of them.

The test consisted of 5 days of training trials and a probe trial on the sixth day. During each training session, mice were released from various starting positions and given 60 s to locate the hidden platform. If a mouse failed to find the platform within the allotted time, it was gently guided to the platform and allowed to remain there for 15 s to aid spatial learning. On the sixth day, the platform was removed, and mice were allowed to swim freely for 60 s. The percentage of time spent in the target quadrant and swimming trajectories were recorded to assess memory retention performance. Animal behavior was recorded and analyzed using Smart 3.0 software (PanLab, Barcelona, Spain).

Novel object recognition test

The novel object recognition (NOR) test was used to evaluate recognition memory based on the innate tendency of rodents to explore novel stimuli. The apparatus consisted of a square open-field arena (40 cm × 40 cm × 40 cm). On the day prior to the formal test, each mouse was individually placed in the empty arena and allowed to freely explore for 10 min to habituate to the environment. The experiment consisted of two phases: familiarization and test. During the familiarization phase, two identical objects (Object A and Object B) were placed in opposite corners of the arena, and each mouse was allowed to explore freely for 10 min. After a 4-h interval, one of the familiar objects was replaced with a novel object, and the mice were returned to the arena for a 5-min test session. All behaviors were recorded and analyzed using Smart 3.0 software (PanLab, Barcelona, Spain). Exploration was defined as the mouse directing its nose toward or sniffing the object within a distance of ≤ 2 cm.

The recognition index was calculated as:

$$\mathrm\;\mathrm\;(\%)\;=\;\lbrack\mathrm\;(\mathrm\;\mathrm)\;/\;(\mathrm\;(\mathrm)\;+\;\mathrm\;(\mathrm))\rbrack\;\times\;100\%.$$

Animal euthanasia and tissue collection

Animals were euthanized 24 h after the final behavioral test to ensure the stabilization of physiological responses. Euthanasia was conducted via intraperitoneal injection of pentobarbital sodium (50 mg/kg), followed by transcardial perfusion with ice-cold saline. Brain tissues were subsequently either fixed in 4% paraformaldehyde or snap-frozen in liquid nitrogen, depending on the requirements of downstream analyses.

HE staining and Nissl staining

To assess neuronal damage, HE staining and Nissl staining (Solarbio, Beijing, China) were performed following the manufacturer’s protocols. After staining, sections were examined and imaged using a digital imaging system (OPLENIC, Japan). Neuronal density and morphology were analyzed, and the number of neurons was quantified using ImageJ software.

Immunohistochemistry

Paraffin-embedded hippocampal sections were deparaffinized and rehydrated, followed by immunostaining procedures performed according to the protocols of the DAB and IHC kits (MXB Biotechnology, Fuzhou, China). Primary antibodies used included anti-Syn (1:100, Proteintech, Wuhan, China) and anti-PSD95 (1:100, Proteintech, Wuhan, China). Positive staining signals were visualized under a light microscope, and the immunoreactive area was quantified using ImageJ software.

Cell culture

N2a cells were cultured in DMEM/F12 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Oricell, USA) and maintained in a humidified incubator at 37 °C with 5% CO₂.

Primary hippocampal neurons were isolated from neonatal C57BL/6 J mice within 24 h after birth. Briefly, hippocampal tissues were dissected in pre-cooled DMEM/F12 medium under sterile conditions and minced into small fragments. The tissue was digested with 0.05% trypsin containing DNase I at 37 °C for 15 min. Digestion was terminated by adding medium containing FBS, followed by centrifugation and filtration through a 0.45-μm cell strainer. The resulting cell pellet was resuspended in complete culture medium. Cells were plated at a density of 3 × 105 cells/mL for immunofluorescence assays and 8 × 105 cells/mL for Western blot analysis. After 3 h of initial attachment, the medium was replaced with Neurobasal medium (Gibco, USA) supplemented with 2% B27 (Gibco, USA), 1% GlutaMAX, and 1% penicillin–streptomycin (Solarbio, Beijing, China) to support neuronal survival and maturation.

Immunofluorescence staining

At DIV2 and DIV4, neurons cultured on poly-D-lysine–coated coverslips were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.3% Triton X-100 in PBS for 10 min. After blocking with 5% BSA for 1 h, cells were incubated overnight at 4 °C with anti-MAP2 antibody (1:1000, Abcam, UK). After washing, cells were incubated with Alexa Fluor–conjugated secondary antibody (1:500, Abcam, USA) for 1 h at room temperature in the dark. Coverslips were mounted with antifade mounting medium and imaged using an FV3000 laser scanning confocal microscope (Olympus, Japan). Axonal length was quantified using ImageJ software.

Real-time quantitative polymerase chain reaction (qPCR)

Total RNA was extracted from collected cells using TRIzol Reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. Reverse transcription was performed using the TransScript® First-Strand cDNA synthesis SuperMix (TransGen Biotech, Beijing, China) to synthesize complementary DNA (cDNA). Quantitative PCR was carried out using SYBR Green Master Mix (Roche, Basel, Switzerland) on a real-time PCR system. Gene expression levels were analyzed using the 2−ΔΔCt method, with β-actin as the internal reference gene. Primer sequences are listed in Additional file 1: Table S1.

Western blot

N2a cells were treated with varying concentrations of sodium NaLA, dichloroacetate (DCA), or oxamate for 24 h, or co-treated with 15 mM NaLA and 10 μM Aβ1–42 for 24 h. For primary hippocampal neurons, 5 mM NaLA was added at DIV1, and cells were harvested at DIV2 and DIV4. Western blot was performed as previously described [32]. Following treatment, cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (Beyotime, Shanghai, China), and centrifuged at 12,000 × g for 15 min at 4 °C. Protein concentrations were determined using a BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). After blocking with 5% non-fat milk, membranes were incubated overnight at 4 °C with primary antibodies including anti-Syn (1:1000, Proteintech, Wuhan, China), anti-PSD95 (1:1000, Proteintech, Wuhan, China), and anti-α-Tubulin (1:1000, Proteintech, Wuhan, China). After incubation with HRP-conjugated secondary antibodies, protein bands were visualized using enhanced chemiluminescence (ECL, Thermo Fisher Scientific, USA) and quantified using ImageJ software.

Transcriptome sequencing (RNA-seq)

Total RNA was extracted from hippocampal tissues using TRIzol reagent (Invitrogen, USA). RNA quality was assessed by NanoDrop and Agilent 2100 Bioanalyzer. High-quality RNA (RIN ≥ 7.0) was used for library preparation and sequencing, which were performed by Novogene (Beijing, China). Libraries were constructed using the NEBNext® Ultra™ RNA Library Prep Kit and sequenced on an Illumina NovaSeq 6000 platform (150 bp paired-end reads). Clean reads were aligned to the mouse reference genome (GRCm38/mm10) using HISAT2. Gene expression was quantified using featureCounts and normalized as FPKM. Differentially expressed genes (DEGs) were identified with DESeq2 (|log2 FC|> 0, p < 0.05). FDR-adjusted p-values were also calculated using the Benjamini–Hochberg method, but were not used as the primary threshold due to the limited number of DEGs under FDR < 0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to explore biological functions and pathways associated with DEGs. The full list of DEGs is provided in Additional file 2: Table S2.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8.0.2 software. Data are presented as mean ± SEM. Group differences were analyzed using one-way or two-way ANOVA, followed by Tukey’s multiple comparisons test. A p-value < 0.05 was considered statistically significant.