Animals

Forty specific pathogen-free animals (SPF) male db/db mice (BKS-Leprem2Cd479/Gpt, strain number: T002407) aged 8 weeks and forty wt mice aged 8 weeks were obtained from the GemPharmatech Co., Ltd. (Jiangsu, China). Mice, housed 4–5 per cage, were allowed ad libitum access to normal food and water during the two-week acclimation period in a 12:12 light/dark cycle. For the first set animals, mice were randomized to subgroups as follows (1) wt-Con knockdown (KD), (2) wt-BMAL1 KD, (3) db/db-Con KD, (4) db/db-BMAL1 KD, (5) wt-Con overexpression (OE), (6) wt-BMAL1 OE, (7) db/db-Con OE, (8) db/db-BMAL1 OE, (n = 8 per group). After acclimation of two weeks, mice were injected with adeno-associated virus (AAV)-PHP. eB carrying either BMAL1 knockdown or overexpressing constructs through the tail vein (Fig. 1A). For the second set animals, mice were randomized to four groups: (1) db/db-Con OE + saline, (2) db/db-BMAL1 OE + saline, (3) db/db-Con OE + LY294002, (4) db/db-BMAL1 OE + LY294002 (n = 8 per group). Similarly, after acclimation of two weeks, mice were injected with AAV-PHP. eB carrying BMAL1 overexpressing constructs or control AAV through tail vein and intraperitoneally received PI3K inhibitor LY294002 (5 mg/kg) (purchased from MedChemExpress) or the same volume vehicle once a day. After 8 weeks, behavior tests were performed to explore the effect of BMAL1 on cognitive function in the eight groups before they were sacrificed. All animal experiments were approved by the Animal Care and Use Committee of Tongji Hospital (Approval number: TJH-202110034).

Fig. 1

BMAL1 downregulation exacerbated cognitive impairment. A The experimental timeline. B, C Western blots and quantitation analysis of the protein levels of BMAL1 (n = 3 per group). D Body weight (n = 8 per group). E Fasting blood glucose (n = 8 per group). F, G Blood glucose levels during GTT and ITT (n = 7 per group). H, I The total distance traveled and average velocity was assessed in the open field test (n = 8 per group). J, K The duration and frequency of exploring the novel object were assessed in the novel object recognition test (n = 8 per group). L, M Acquisition errors and probe errors of the Barnes maze test were assessed for spatial learning ability (n = 8 per group). Data were plotted as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. The data were analyzed by two-way ANOVA followed by post hoc Tukey's HSD test

AAV and tail vein injection

AAV-PHP.eB-CMV promoter-EGFP-MIR155(MCS)-SV40PolyA-Bmal1 and AAV-PHP.eB-CMV-betaGlobin-MCS-3Flag-SV40PolyA-Bmal1 vectors were purchased from GeneChem Company. Mice were anesthetized with isoflurane and injected intravenously with AAV-PHP.eB (3.0 × 1011 vg/mL per mouse) in a volume of 100 μl diluted by 0.9% NaCl after the tail was swabbed with alcohol.

Glucose tolerance tests (GTT) and insulin tolerance tests (ITT)

Mice were fasted for sixteen hours and then given an intraperitoneal injection of glucose (1.0 g/kg body weight, Hubei Kelun Pharmaceutical Co., Ltd.) in GTT. Six hours fasted mice were injected with human insulin (1 unit/kg Jiangsu Wanbang Biochemical Pharmaceutical Co., Ltd.) in ITT. Blood glucose concentrations were measured at 0, 15, 30, 60, and 120 min after injection by a blood glucose meter (ACCU-CHEK, Roche, Germany).

Behavioral tests

The behavioral tests were adapted according to our previous study [19]. On day 1, the open field test was conducted to assess general activities. On day 2, the novel object recognition test was used to detect hippocampal-related contextual learning ability. On day3-7, the Barnes maze test was performed to explore spatial learning and memory ability.

Open field test

For each trial, a mouse was placed in a 45 × 45 × 45 cm square open field box and allowed 10 min to freely explore. The mouse’s movements were recorded using an overhead video camera. The recorded data were then analyzed with the VisuTrack Animal Behavior Analysis System (XinRuan Co. Ltd., Shanghai) to determine the total distance traveled and the velocity of each mouse. To maintain cleanliness, the testing area was wiped with 75% ethanol between trials.

Novel object recognition test

In the initial trial, each mouse had 5 min to independently explore two identical objects within the open field. Afterward, the mouse was returned to its home cage and allowed a 30-min interval. In the subsequent trial, the mice were presented with two objects: one familiar and the other novel. They were given 5 min to explore both objects. The resulting data were expressed as the percentage of time spent investigating the novel object. This frequency was calculated as: [(frequency of investigating the novel object) / (total frequency of investigating both objects) * 100].

Barnes maze test

A Barnes maze consists of a circular platform (100 cm in diameter) elevated 90 cm above the floor. The platform, dark gray in color, featured 20 holes (5 cm in diameter), one of which led to an escape box. Spatial cues were placed around the platform to ensure visibility for the mice during the test. The Barnes maze protocol comprised two phases: “Spatial Acquisition” and “Probe Trials”. On the first day, the mice were given 5 min to familiarize themselves with the maze. After a 30-min interval, the first acquisition session commenced, lasting 5 min. If the mice failed to locate the escape box within the allotted time, they were gently guided to it and allowed to remain there for 2 min. Over the second and third days, the mice underwent two 5-min training trials each day, with a 30-min inter-trial interval. The fourth day served as a rest day. On the fifth day, a probe test was conducted: the escape box was removed, and the mice were given 5 min to explore the maze. The number of errors made by each mouse before locating the escape hole was recorded using a video camera and analyzed with VisuTrack Animal Behavior Analysis System (XinRuan Co. Ltd., Shanghai) to assess spatial memory retention.

Western blots

The total protein was extracted from hippocampus samples with RIPA extract buffer containing protease and phosphatase inhibitors (Boster, China). The collected supernatants were centrifuged at 12,000 × g for 15 min at 4 °C and then the protein concentration was measured via a bicinchoninic acid (BCA) kit (Boster, China). Proteins in the extracts (20 ug for each sample) were separated in 10% SDS-PAGE and transferred to a PVDF membrane (Millipore, China) at 300 mA. After blocking with the blocking buffer for one hour at room temperature, the membranes were incubated overnight with primary antibodies at 4 °C. After being washed with tris-buffered saline with tween 20 (TBST) buffer three times, the membranes were incubated with secondary antibodies for one and a half hours. Information of antibodies is presented in Table 1. Finally, protein bands were visualized with an enhanced chemiluminescence detection kit (Biosharp, China) and detected with a GelView 6000 Pro (Antpedia, China). Band density was analyzed using ImageJ software.

Table 1 Antibody informationEnzyme-linked immunoassay (ELISA)

ELISAs of Aβ1-40 and Aβ1-42 were performed as per the manufacturer’s instructions (Elabscience, Wuhan, China). Briefly, frozen hippocampal tissues were homogenized in RIPA lysis buffer containing protease and phosphatase inhibitors (Boster, Wuhan, China), followed by centrifugation at 20,000 g for 30 min at 4 °C. The supernatants were collected as soluble fractions. The levels of Aβ1-40 and Aβ1-42 were measured using ELISA kits (E-EL-M3009 and E-EL-M3010, Elabscience) according to the manufacturer’s instructions.

Immunohistochemical (IHC) analysis

Mouse brains were collected on ice after sacrifice, and the right brain was removed and fixed in 4% paraformaldehyde. Paraffin-embedded brain tissues were sectioned. After the procedure of dewaxing, hydration, and antigen retrieval, sections were incubated with 10% goat serum (Boster#AR1009) for 20 min at room temperature. Then, the sections were immunostained with anti-beta Amyloid 1–42 antibody (1:200, Abcam# ab201061) overnight at 4 °C followed by incubation of a secondary antibody (1:2000, Abcam#ab205718) for 45 min at 37 °C, and developed using diaminobenzidine (Maxim #DAB4033). Sections were counter-stained with hematoxylin, and IHC images were taken under a microscope (Olympus #CX31).

RNA extraction and Quantitative real time polymerase chain reaction (qRT-PCR)

We extracted total RNA from the hippocampus using Trizol reagent (Biosharp, Beijing, China). RNA concentration and quality were evaluated using a spectrophotometer (Denovix, USA). Subsequently, we synthesized cDNA using the Hifair II Reverse Transcription System and following the manufacturer’s protocol (Yeasen Biotech Co. Ltd, Shanghai, China). QRT-PCR was conducted using the SYBR Green qPCR Master Mix (Tolo Biotech Co. Ltd, Shanghai, China) and QuantStudioTM 1 system (Thermo Fisher Scientific biosystem). Gene expression was normalized using glyceraldehyde 3-phosphate dehydrogenase (Gapdh), and the relative gene expression was calculated using the Comparative CT method (2 − ΔCT).

RNA sequencing (RNA-seq) analysis

Total RNA of the hippocampus of db/db mice with or without BMAL1 knockdown was extracted by Trizol (Biosharp, Beijing, China). A minimum of 2 μg RNA was sent to BGI Tech (Wuhan, China) for quality control, library preparation, and next-generation sequencing. In brief, the RNA was segmented into 250–300 bp fragments and reverse transcribed to cDNA. Next, the cDNA was subjected to PCR for amplification. After the PCR product was denatured to a single chain, the cyclization reaction system was prepared to obtain the single-chain ring product. Transcriptome sequencing was performed with the Novaseq platform (Illumina, USA), which generated 150 bp paired-end reads. The read numbers were counted by featureCounts v1.5.0-p3, followed by the calculation of FPKM of each gene. Differential gene testing between the two groups was performed using DESeq2 (v1.4.5) with p-value ≤ 0.05. And we use kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis (https://www.kegg.jp/), with p value ≤ 0.05 as the threshold. Those meeting this condition are defined as significantly enriched in candidate genes.

Chromatin Immunoprecipitation (ChIP)

ChIP was conducted with Sonication ChIP Kit (Abclonal, RK20258). HT22 cells were crosslinked with 1% formaldehyde, followed by split and sonicated to shear the chromatin. Then it was incubated with ChIP grade anti-BMAL1 antibody (Cell Signaling TECHNOLOGY) or IgG for 4 h at 4 °C. After washing, the antibody-protein-DNA complexes were separated from the beads. Subsequently, the complexes were decrosslinked for 4 h at 65 °C, and DNA was purified.

Plasmid transfection and luciferase reporter assay

The pGL3-basic vector containing the Areg promotor (2000 bp) and HA-Bmal1 (purchased from GENECHEM) plasmid were constructed. Based on the pGL3-Areg promoter (2000 bp), we further constructed site-mutant plasmids in which the sequence CACGGG (-60 to -45 bp) or CAGGTG (-20 to -15 bp) was replaced by AGTTTT or AGTTGT. HT22 cells were seeded into a 12-well plate and cultured until 75% confluence was obtained. The firefly luciferase reporter plasmid (400 ng) and pRL-TK plasmids (20 ng) with or without HA-Bmal1 plasmid were co-transfected into the cells by Lipo3000™ Transfection Reagent (1.5 μL/well). After 48 h incubation, the cells were lysed with the lysis buffer, and the Firefly and Renilla luciferase were measured via the dual luciferase reporter assay system (Promega) according to standard protocols. Calculate the ratio of luminescence from the Firefly luciferase to the Renilla luciferase to assess the relative luciferase signal.

Statistical analysis

All values were presented as means ± SEM. Data were analyzed by two-way ANOVA with genotype and virus genotype as between-subject factors. For multiple comparisons, Tukey’s post hoc method or Dunn’s test was applied according to the result of the homogeneity of variance test. GraphPad Prism (version 8.0) was used to plot figures. The quantitative analysis of amyloid plaque in hippocampal sections was evaluated using ImageJ software (Version 1.8.0.112, NIH, Bethesda, Maryland, USA). P values are represented as follows: *P < 0.05, **P < 0.01, ***P < 0.001.