Bioinformatic analysis

The GSE206478 dataset, downloaded from the GEO database, examines gene expression through RNA sequencing in microglia to elucidate the functional characteristics of genetic variants in AD. Differentially expressed genes (DEGs) between the two groups were analyzed using the online tool Geo2R provided by the GEO database. Genes were screened based on criteria of P < 0.05 and ∣log2FC∣≥1.5.

Animals

The amyloid precursor protein/presenilin 1 (APP/PS1) transgenic AD mouse model used in this study is derived from a C57BL/6J background and harbors the human APP mutation site (K595N/M596L) and the human PS1 mutation site (deletion of the ninth intron). These model mice exhibit learning and memory deficits at 3 months of age, begin to develop age spots at 5 months, and show a significant number of age spots by 12 months, presenting a pathological phenotype similar to that of AD [10]. For this experiment, 7-month-old male APP/PS1 transgenic mice (30–40 g) and their non-transgenic littermates were purchased from the Nanjing University-Nanjing Institute of Biomedicine. Mice were maintained at a room temperature of 22 ± 1 °C under a 12-hour light/dark cycle. All animal procedures were approved by the Animal Experimentation Ethics Committee of Tongren Hospital, Shanghai Jiao Tong University School of Medicine.

Adeno-associated viruses (AAVs) carrying short hairpin RNA (shRNA) targeting NEK7 (AAV-sh-NEK7) and a negative control (AAV-sh-NC) were obtained from GenePharma. Each mouse received an intravenous injection of AAV at a dose of 5 × 10^10 plaque-forming units (PFU) in 20 µL of phosphate-buffered saline (PBS) via the tail vein. The mice were divided into four groups:

Normal group (n = 5): Non-transgenic C57BL/6J mice served as the normal control group.

AD group (n = 5): APP/PS1 transgenic AD mice.

AD + AAV-sh-NC group (n = 65): AD model mice injected with AAV-sh-NC.

AD + AAV-sh-NEK7 group (n = 5): AD model mice injected with AAV-sh-NEK7.

Morris water maze test

The Morris water maze is a circular pool with a diameter of 120 cm and a height of 60 cm, artificially divided into four quadrants. Different colored reference objects are placed on the walls of the pool in each quadrant. The water maze is equipped with an image acquisition and processing system that automatically records the swimming trajectories of the mice. Parameters such as the time required for the mice to locate the submerged platform, their swimming speed, and other relevant metrics were recorded.

Three days prior to the start of the experiment, the mice were transferred to the behavioral testing room to acclimate to the new environment. Daily handling helped reduce their stress and anxiety. Prior to the commencement of the experiment, the pool was filled to a predetermined depth of approximately 35 cm with water, and food-grade titanium dioxide was added to ensure the water was opaque. A circular platform (approximately 9 cm in diameter) was positioned in the center of the first quadrant. The platform was adjusted to be approximately 1.0 cm above the water surface. The operator then placed the mice on the platform for 60 s to allow them to acclimate to the environment. Subsequently, the mice were released into the water sequentially from the first, second, third, and fourth quadrants (with the mouse’s head directed towards the pool wall) to search for 60 s. Using a long stick to guide them, the mice were allowed to rest on the platform for 20 s after locating it. Adaptive training commenced on Day 1 and continued for a total of three days. Each mouse was given a rest period of 1 h between consecutive water trials. Following the adaptive phase, the water level was raised to submerge the circular platform, positioning it approximately 1.0 cm below the surface. Each mouse was then placed into the water from each of the four quadrants (first, second, third, and fourth) with its head directed towards the pool wall. The recording started when the mouse entered the water and ended when it located the submerged platform and remained there for 5 s. The escape latency was defined as the time taken to find the platform. Additionally, the number of times the mouse crossed the platform position, the swimming distance, and the time spent swimming were used to assess learning and memory abilities.

Immunofluorescence staining

The mice were anesthetized with sodium pentobarbital (P276000, 100 mg/kg, i.p., AmyJet). Following anesthesia, the brains were collected, and the mice were euthanized by cervical dislocation. The left hemispheres were fixed in 4% paraformaldehyde in PBS at 4 °C overnight and subsequently processed for paraffin embedding. For immunostaining, 5 μm coronal serial sections were deparaffinized and subjected to antigen retrieval using citrate buffer (0.01 M, pH 6.0) at 95 °C for 20 min. The sections were then incubated with 3% hydrogen peroxide (H₂O₂) and washed three times with PBS. Next, the sections were permeabilized and blocked with 10% normal goat serum in 0.3% Triton X-100-PBST for 1 h at room temperature. Following blocking, the sections were incubated with primary antibodies: anti-IBA1 (#17198, Cell Signaling Technology) or anti-NEK7 (MA5-47007, Thermo Fisher). After overnight incubation at 4 °C, the sections were washed with PBS and incubated with the appropriate HRP-labeled secondary antibodies. The immunoreactivity was visualized using diaminobenzidine (DAB). All images were acquired using an Olympus IX73 inverted microscope equipped with a DP80 camera or a Leica TCS SP8 confocal microscope. For each animal, three to seven coronal sections spanning the cortex and hippocampus at different depths were analyzed. Six images were captured from matching areas per section.

Cell culture and treatment

BV2 microglial cells (American Type Culture Collection) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Hyclone) and penicillin/streptomycin (Beyotime Biotechnology) in a humidified atmosphere with 5% CO₂ at 37 °C. To establish an AD model, BV2 cells cultured in four-well chambers were treated with Aβ1-42 (5 µM, Sigma-Aldrich) for 24 h [11, 12]. Small interfering RNAs (siRNAs) targeting NEK7 (si-NEK7), LDHA (si-LDHA), and a control siRNA (si-NC) were obtained from Invitrogen. To knock down NEK7 or LDHA, BV2 cells were transfected with si-NEK7 or si-LDHA using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer’s instructions.

Western blotting

Western blotting was performed to evaluate protein levels, following established protocols [12, 13]. The antibodies utilized in the Western blotting study included anti-Aβ (sc-28365) from Santa Cruz Biotechnology, anti-NEK7 (ab133514) and anti-caspase-1 (ab138483) from Abcam, anti-GSDMD-N (#39754) from Cell Signaling Technology, anti-L-Lactyl Lysine Rabbit pAb (PTM-1401), anti-L-Lactyl-Histone H4 (Lys12) Rabbit mAb (PTM-1411RM), and Anti-Histone H4 Mouse mAb (PTM-1009) from PTM Biolabs.

CCK-8 assay

The viability of BV-2 cells in each group was assessed using the Counting Kit-8 (CCK-8) assays (Dojindo) according to the manufacturer’s instructions. Following transfection and different treatments, BV-2 cells were seeded into 96-well plates and incubated with 10 µL of CCK-8 solution. After incubation at 37 °C in a 5% CO₂ atmosphere for 2 h, the absorbance was measured at 450 nm using a microplate reader (BMG Labtech).

Flow cytometry

Activation of caspase-1 and positive PI staining indicate pyroptosis [14], which was detected using the FAM-FLICA Caspase-1 Assay Kit (ImmunoChemistry Technologies) in this study. Briefly, BV-2 cells (3 × 105) were resuspended in 300 µL of PBS and incubated with 10 µL of FLICA staining solution in the dark for 1 h at 37 °C. The cells were then stained with PI at a concentration of 0.5% (v/v). Detection was performed using a Beckman CytoFLEX flow cytometer, and the results were analyzed with FlowJo software.

ELISA

The protein levels of IL-1β and IL-18 in the cell supernatants were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits from Sigma-Aldrich, following the manufacturer’s instructions.

qRT-PCR analysis

Total RNA was extracted using Trizol reagent (Invitrogen). Subsequently, cDNA was synthesized using the Reverse Transcriptase kit (Promega). Gene expression was quantified by real-time PCR using an ABI 7500 Real-Time PCR detection system (Applied Biosystems) with SYBR Green Master Mix (Roche). The cycling conditions consisted of an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. The specificity of each amplified product was confirmed by analyzing the melting curve of each sample. The relative mRNA expression levels of each gene were normalized to the expression of the housekeeping gene GAPDH. The primer sequences are provided below:

5ʹ-GCTGTCTGCTATATGAGATGGC-3ʹ (NEK7-F) and 5ʹ-CCGAATAGTGATCTGACGGGAG-3ʹ (NEK7-R); 5ʹ-CAAAGACTACTGTGTAACTGCGA-3ʹ (LDHA-F) and 5ʹ-TGGACTGTACTTGACAATGTTGG-3ʹ (LDHA-R); 5′-GTCGCCAGCCGAGCC-3′ (GAPDH-F) and 5′-TGAAGGGGTCATTGATGGCA-3′ (GAPDH-R).

Chromatin immunoprecipitation PCR (ChIP-PCR)

BV-2 cells were cross-linked with 1% formaldehyde, and the genomic DNA was sheared to an average fragment size of 400 bp. Immunoprecipitation was performed using antibodies specific to H4K12la. The ChIP-PCR primers were designed to amplify the promoter regions containing potential H4K12la binding sites within the NEK7 gene. A positive control antibody (RNA polymerase II) and a negative control non-immune IgG were used to validate the efficacy of the kit reagents (P-2025-48, Epigentek Group). The immunoprecipitated DNA was then purified, released, and eluted. The eluted DNA was used for subsequent PCR analysis.

NEK7 transcriptional activity determination

Mutated NEK7 luciferase reporter vectors were transfected into BV-2 cells with or without LDHA knockdown, followed by lactate treatment (25 µM, Sigma-Aldrich) [15]. The cells were then lysed, and luciferase activity was measured using a Promega luciferase assay kit (Promega) and a microplate reader (Bio-Rad).

Statistical analysis

Normally distributed data were compared using Student’s t-test or one-way analysis of variance (one-way ANOVA). Results are presented as the mean ± SD. Statistical significance was set at p < 0.05p < 0.05. All statistical analyses were conducted using IBM SPSS 22.0 software.