Human subjects

Human post-mortem cortical tissue sections were obtained from Guizhou Medical University, consisting of both patients diagnosed with AD and healthy control subjects. The additional details were presented in Supplementary Table s1. Subjects’ consent was acquired following the Declaration of Helsinki and authorized by the Tongji Medical School Ethics Committee.

Animals

Male mice with homozygous IDH3β knockout exhibited infertility and an inability to produce mature spermatozoa, leading to an extended breeding cycle. Consequently, IDH3β knockdown heterozygotes were generated in C57BL/6 mice for this investigation. Shulaibao Biotechnology in Wuhan, China, supplied the male mice with either the wild-type (WT) or 5xFAD, which express the Florida (APPI716V) APP, London (APPV717I), and Swedish (APPK670N/M671L) mutations, in addition to the PS1L286V and PS1M146L mutations. The mice were either 5 or 11 months old. The Animal Care and Use Committee at Huazhong University of Science and Technology approved all procedures involving animals. These procedures were conducted in adherence to the regulations set forth by the Administration of Laboratory Animals in China. The mice were kept in a controlled laboratory environment with a temperature of 23 °C, unlimited access to food and water, and a 12-h cycle of light and dark. In every experiment, male mice weighing between 20 and 30 g were utilized. For immunostaining and Golgi staining, brain slices from 6-, 9-, and 12-month-old 5xFAD or age-matched WT mice were utilized; for Western blotting or metabolite assessments, brain tissue homogenate was utilized.

Stereotactic injection

Mice were anesthetized with isoflurane, and recombinant adeno-associated virus (rAAV) rAAV-CMV-3xFLAG-P2A-mNeonGreen-tWPA (empty vector) and rAAV-CMV-IDH3β-3xFLAG-P2A-mNeonGreen-tWPA (IDH3β), as well as AAV-U6-shRNA (PAX6)-CMV-mScarlet-WPRE and AAV-U6-shRNA (NC2)-CMV-mScarlet-WPRE, were injected into the bilateral HP between the CA1 and DG subsets. From OBIO Technology (Shanghai, China), these viral vectors were obtained. The coordinates for the injection were as follows: −1.94 mm anterior-posterior, ±1.30 mm medial-lateral, and −1.75 mm dorsoventral from the bregma and dura assuming a flat cranium. The specific short hairpin RNA (shRNA) sequences targeting PAX6 were 5′-GAGTTTGAGAGGACCCATTAT-3′. Behavior assessments were performed on the mice 1 month later. After the behavioral evaluations, the mice were sacrificed for further biochemical analysis.

Behavioral tests

The novel object recognition (NOR) test was conducted following previously established procedures.58 Mice underwent 5 min of handling per day for three consecutive days before the test. Placed in a box (50 × 50 × 50 cm) containing two objects positioned in the corner on the same side, mice were allowed 5 min of free exploration. After 24 hs, one of the objects was replaced, and mice were again given free exploration. Using a video tracking system (Sans, Jiangsu, China), the mice’s real-time location was monitored and their preference for the object within 3 cm of exploration was documented. The system, equipped with a top-mounted camera, recorded distance, time, and position. Before each test, both the box and objects underwent cleaning with 75% ethanol.

The Morris water maze (MWM) test was performed as described in a previous study.59 Mice underwent training in a circular pool, partitioned into four quadrants. The 120 cm in diameter and 60 cm high walls of the pool were outfitted with a camera video monitoring system (Taimeng, China) above the center to keep an eye on the mice’s whereabouts and speed. A week before the experiment, the experimental environment was provided for the mice to acclimate. The mice were allowed to swim freely after being introduced to the pool gently for 5 days. The latency was measured in seconds and the mouse was guided to remain on the platform for an additional 30 s if it could be detected within 60 s. A mouse was led to the platform and guided to remain there for 30 s if it was unable to locate it in 60 s. Mice were placed in various quadrants of the pool, excluding the quadrant containing the platform, and underwent three training sessions per day for 5 days. The interval between two consecutive training sessions for the same mice was at least 30 min.

On day 7, the platform was removed to assess spatial memory. The latency was quantified as the duration required for the mice to reach to the position on the platform. Within 60 s, the target platform crossing number was the number of times the mice traversed the platform area. The duration spent in the target quadrant constituted the time in target quadrant.

The contextual fear condition test (CFT) was performed as previously described.60 Mice were situated in a soundproof enclosure measuring 17 cm × 17 cm × 25 cm, featuring a floor capable of administering electric shocks. Time, distance, and motion trajectories were captured by a camera positioned above the center of the enclosure. The mice were allowed unrestricted movement within the enclosure for 3 min, following which three electric shocks of 2 s each (1 mA, with 1-min intervals) were administered. After a 24-h interval, the mice underwent testing for freezing time, defined as the duration when their displacement from the original position was less than 10%. The freezing time of the mice for 3 min was recorded using a video tracking system (Sans, China). Following each mouse’s assessment, the enclosure was cleaned with 75% alcohol.

Immunohistochemistry and immunofluorescence

Immunohistochemistry (IHC) and immunofluorescence (IF) experiments were conducted following established protocols.61 Mice were anesthetized with isoflurane, fixed in an abdominal upward position, and intracardially perfused with 0.9% saline. Subsequently, brains were extracted and fixed in a 4% paraformaldehyde solution (PFA) in 0.01 M PBS at pH 7.4 for 24 h, followed by dehydration with 30% sucrose-PBS for a minimum of 48 h.

For IHC, brains were sectioned into 5 µm slices and paraffin-embedded. The dewaxing process included two 30-min treatments with xylene, followed by rehydration with ethanol at concentrations of 70%, 90%, and 95%. Antigen retrieval was performed by immersing the samples in a citric acid and sodium citrate solution (0.4 grams citric acid monohydrate, 2.64 grams sodium citrate dihydrate in one liter of ddH2O), boiled for 15 min. Subsequently, samples were treated with hydrogen peroxide for 30 min to eliminate endogenous hydrogen peroxidase. Following a 30-min blocking step with 5% bovine serum albumin/0.5% Triton X-100/PBS, the sections were subjected to an overnight incubation at 4 °C with primary antibodies. Subsequently, the sections were treated with secondary and third antibodies, respectively, for 1 h at 37 °C. Finally, samples were prepared utilizing a DAB peroxidase substrate kit (ZSGB-BIO), dehydrated through a series of alcohol concentrations (70%, 90%, and 95%), followed by two rounds of 10-min immersion in xylene, and sealed with neutral resin. Imaging was performed using a virtual slide microscope (Olympus SV120, Japan).

Brain sections were obtained for IF using a cryostat microtome (CM1900, Leica, Germany), with a dimension of 30 µm coronal sections. Following blocking for 30 min with 5% bovine serum albumin/0.5% Triton X-100/PBS, the sections were subjected to overnight incubation at 4 °C in the presence of primary antibodies, followed by incubation with secondary antibodies for 1 h at 37 °C and Hoechst for 10 min, then sealed with a mixture of 50% glycerol and 50% PBS. Slices were scanned using a confocal microscope (Carl Zeiss LSM800, Germany) for image acquisition. The steps for IHC on brains sliced with a cryostat microtome were the same as paraffin sectioning, except for the absence of antigen retrieval. The antibodies that were utilized are detailed in Supplementary Table s2.

Western blotting

Western blotting was performed using an established in our laboratory.4,62 In summary, hippocampal tissue was homogenized in a buffer at a 1:10 ratio. Depending on the sample concentration, SDS-polyacrylamide gel electrophoresis (10% or 15% gel) was employed to separate proteins based on their molecular weight. Subsequently, the proteins of different molecular weights were transferred from the gel to nitrocellulose (NC) membranes using the wet transfer method. The primary antibodies were incubated at 4 °C for a whole night after the membranes were blocked with 5% skim milk. The next day, the membranes were cleaned and treated with Odyssey secondary antibodies. The images were captured using Odyssey (LI-COR Biosciences, USA) and quantified using ImageJ software. Supplementary Table s2 contains a list of the antibodies that were employed.

Golgi staining

Utilizing the FD Rapid GolgiStain Kit (PK401, FD Neurotechnology) following the manufacturer’s guidelines, Golgi staining was performed. After anesthetizing the mice, their brains were immersed in an AB mixture (1:1) for one month. Throughout this period, the AB solution was refreshed on the first day and the mixture was agitated every 2 days, with the brains being shielded from light. Following that, the brains were immersed in solution C for 3–7 days, sectioned, and subjected to staining at a 100 µm thickness.

The staining procedure comprised the subsequent stages: a double wash in ddH2O for 4 min each, followed by a 10-min immersion in a solution mixture consisting of solution D, solution E, and ddH2O (in a 1:1:2 ratio), and then another double wash in ddH2O for the same duration. The samples were then dehydrated in 50%, 75%, and 95% alcohol gradients for 4 min each, with final dehydration in anhydrous ethanol for 4 min, and then in xylene for 3 rounds of 4 min each. After a waiting period of several hours, the samples were sealed, imaged using a light microscope (Nikon, Japan), and analyzed as previously described.35,63

Cell culture and transfection

As previously described,6 90% DMEM/Basic Glucose medium with 10% FBS was utilized to culture both mouse neuroblastoma N2a cells (N2a, RRID: CVCL_0470, Cat#: GDC0162, China Centre for Type Culture Collection) and human embryonic kidney 293 cells (HEK293, RRID: CVCL_0045, Cat#: GDC0067, China Centre for Type Culture Collection). The incubator was kept at 37 °C in a humidified atmosphere with 5% CO2. Tests for Mycoplasma on the validated N2a and HEK293 cell lines have yielded negative results. The Lipo-3000 RNA and DNA transfection kit (Cat#: L3000015, Thermo Fisher) was utilized to transfect cells with plasmids (pEGFP-MCS-2A-GFP, pEGFP-IDH3β-2A-GFP, pEGFP-PAX6-2A-GFP), or siRNAs, as directed by the manufacturer.

Primary hippocampal neurons were cultured as described previously.64 Neurons were obtained from Sprague Dawley rats (17 days embryos). The HP was removed, trypsinized at 37 °C for 10 min, dispersed equally with repeated blowing, filtered via a 0.45 µm filter, and cultured in 6- or 12-well plates with F-12 media containing 10% FBS. After 4 h, the F-12 medium was substituted with a 2 mL Neurobasal medium containing 2% B27 and 1% GlutaMAX. At 2 days in vitro (div), the neurons were infected with lentivirus (pcSLenti-CMV-IDH3β-3xFLAG-P2A-EGFP-WPRE and pcSLenti-CMV-3xFLAG-P2A-EGFP-WPRE), and 50% maintenance medium was renewed every 3 days. Cells were exposed to oligomeric Aβ after 6 days for 24 h. Cells that have been treated were analyzed using Western blotting analysis or IF.

Reverse transcription and real-time quantitative PCR

In our laboratory, reverse transcription and real-time quantitative PCR were carried out following the established protocols.3 Total RNA was extracted using RNAisO PIus (Cat#: 9109, Takara), and then a reverse transcription reagent kit (Cat#: RR037A, Takara; Cat#: BL699A, Biosharp) was utilized to obtain cDNA. RT-PCR was carried out using a StepOnePlus Real-Time PCR Detection System (AB Applied Biosystems, Singapore). The PCR system is as follows: a blend of 1 µL cDNA, 1 µL forward and reverse primers, 7 µL RNase-free H2O, and 10 µL SYBR Green PCR master mixes (Cat#: Q711-02, Vazyme, China). GAPDH mRNA served as a reference for the mRNA levels of the relevant genes. Every experiment was conducted three times. The primer information was listed in Supplementary Table s3.

Chromatin immunoprecipitation (ChIP) assay

The ChIP assay was performed using the ChIP kit (P2078, Beyotime) following the manufacturer’s instructions, following published methods.64 Using the DNA Purification Kit (D0033, Beyotime), purified DNA was produced. The quantification of DNA fragments in immunoprecipitated samples was carried out through quantitative real-time qPCR. Every experiment was conducted for a minimum of three times.

Luciferase activity assay

The binding sequences of PAX6 to IDH3β gene promoter region, as well as their mutations, were synthesized and inserted into pGL3 luciferase reporter vectors. The Dual-Lumi Luciferase Assay Kit (RG088S, Beyotime) manufacturer’s protocol was followed throughout the experimental process. Utilizing the multifunctional enzyme marker Synergy 2 (BioTek, USA), relative luciferase activity was measured. At least three replications of each experiment were conducted. The binding sequences are listed in Supplementary Table s4.

Metabolite assays

IDH3 activity was measured using the Isocitrate Dehydrogenase Assay Kit (Colorimetric) (ab102528, Abcam), while L-lactate levels were assessed with L-lactate Assay Kit (Colorimetric/Fluorometric) (ab65330, Abcam). The quantification of NAD+ and NADH was conducted using the NAD+/NADH Assay Kit (Colorimetric) (ab65348, Abcam). Alpha Ketoglutarate levels were determined with an Alpha-Ketoglutarate (alpha KG) Assay Kit (ab83431, Abcam). Additionally, ATP levels were measured utilizing the ATP Assay Kit (S0027, Beyotime). The Deproteinizing Sample Preparation Kit—TCA (ab204708, Abcam) was employed to remove proteins from the samples. Details of the reagents and resources utilized are provided in Supplementary Table s5.

Statistical analyses

GraphPad Prism V9.0 (San Diego, CA, USA) was utilized for statistical analysis. These results are expressed as means ± SEM. The significance level was established at P < 0.05. Student’s t-test or ANOVA multiple comparisons test with Turkey or Bonferroni’s post hoc testing determined group differences.