Mice and Human
APN KO mice (B6;129-Adipoqtm1Chan/J) were purchased from the Jackson Laboratory (Maine, USA). The WT mice (C57BL/6J) were purchased from Weitong Lihua Limited Company (Beijing, China). All mice were housed in a pathogen-free facility with a 12 h light-dark cycle (lights on at 6:00 am, lights off at 6:00 pm) and ad libitum access to food and water. D-Galactose (200 mg/kg/d) was used to induce the animal model of accelerated aging. The animal experiments were approved by the Animal Care and Use Committee of the Experimental Animal Center at Shenzhen Center for Disease Control and Prevention.
Informed consent for all human samples was obtained following the Ethical Committee of Shenzhen Center for Disease Control and Prevention. Young individuals (n=17) and BMI-matched older individuals (n=15) have additional characteristics shown in Table S1. As shown by %HbA1c, all individuals were normoglycemic. The exclusion criteria included smoking, antibiotics usage, auto-immune disease, diabetes, hypertension, and pregnancy.
SA-β-gal for frozen sections
Firstly, 15.5 month-old WT and APN KO mice were deeply anesthetized with 4% chloral hydrate and perfused with PBS to remove intravascular blood. According to the mid-sagittal plane, the brains were rapidly removed from the skull and divided into two halves. The left hemi-brains were made of paraffin-embedded sections and used for immunofluorescence staining. The right hemi-brains were made frozen sections. For SA-β-gal staining, the right hemi-brains were fixed with 4% paraformaldehyde (PFA) for 2 days and dehydrated through a sucrose gradient (10% sucrose, 1 day; 20% sucrose, 1 day; 30% sucrose, 2 days). After that, 20-µm frozen sections were cut and rehydrated 3 times with PBS in a 6-well plate. SA-β-gal staining was performed using a kit (BestBio, China). Briefly, sections were immersed in a fixation solution for 15 min and subsequently washed with PBS 3 times. Then 2 ml per well of working solution of β-galactosidase with X-gal was placed, and the plate was maintained at 37℃ for 48 h. SA-β-gal positive areas were quantified by counting stained and unstained areas and expressed as the percent of SA-β-gal positive areas over the total area.
The paraffin-embedded sections were deparaffinized by dimethyl benzene and rehydrated by graded alcohol, followed by a citric acid antigen repair buffer to unmask the epitope. After washing 3 times with PBS, the sections were blocked with blocking buffer (0.3% Triton X-100 + 3% bovine serum albumin in PBS) for 60 min, followed by primary antibodies including mouse monoclonal anti-GFAP and rabbit polyclonal anti-Iba1 overnight at 4℃. After primary antibodies incubation, the sections were labeled with fluorescent secondary antibodies as follows: Alexa Fluor 488 goat anti-mouse IgG (H+L) and Alexa Fluor 568 goat anti-rabbit IgG (H+L). DAPI (4, 6-diamidino-2-phenylindole) was used to counterstain the nuclei. The images were acquired using a confocal microscope and analyzed using ImageJ software.
Total RNA was extracted using TRIZOL reagent (Invitrogen, Germany) from brain tissues of 15.5 month-old WT and APN KO mice. RNA integrity and concentration were verified using Nanodrop2000 (Thermo, USA). PrimeScript RT-PCR Kit (TAKARA, Japan) was used to perform reverse transcription to synthesize cDNA. Gene expression was quantified following the instructions provided with SYBR PremixEx TaqTM (TAKARA, Japan). The β-actin gene was selected as a housekeeping reference. The following primers were used for quantification: EHMT1 (F) GGC ACC TTT GTC TGC GAA TAC and (R) AGA ACC GAG CGT CAA TGC AG; Baz2b (F) GCT CTA GAC GTC AGG CTT GTT and (R) TTC ACA CCG CTG GTC TTG TT; β-actin (F) TCC GGC TCA GAA CTA CAG TGT AAT and (R) TGC GGC GTT TTC ATG GT. The RT-PCR procedure was performed as the following amplification conditions: pre-denaturation at 95℃ for 2 min; 40 cycles including denaturation at 95℃ for 5 s, annealing at 54℃ for 30 s and extension at 70℃ for 34 s. Finally, the 2-ΔΔCt method was used to analyze gene expression.
Human plasma cytokine assays
Human plasma cytokine levels of IL-1β, IL-2, IL-6, IL-8, IFN-γ, TNF-α, IL-4, IL-10, IL-12p70, and IL-13 were measured by the Meso Scale Discovery (MSD) according to the manufacturer’s protocol. Briefly, plasma samples were added to the plate and incubated at room temperature. After 2 h incubation, the plate was washed 3 times before the addition of detection antibodies and read using a MESO QuickPlex SQ120. The data were analyzed using the MSD Discovery Workbench software v.4.0.
The levels of human APN, mouse APN, inflammatory cytokines including IL-1β, IL-6, TNF-α, MCP-1, IFN-γ, IL-18, IL-4, IL-10, IL-13, and TGF-β2 were measured using ELISA kits from Elascience according to the manufacture's protocol. Dopamine signaling including dopamine (DA) and serotonin (5-HT) was detected using ELISA kits from Nanjing Jiancheng Bioengineering Institute.
Mitochondrial function and oxidative damage
Mitochondrial function was assessed by measuring ATP production using commercially available kit (Beyotime, China) according to the manufacture's protocol. Oxidative damage was assessed by measuring lipid peroxidation and GSH content. The lipid peroxidation was determined using an MDA assay kit (Beyotime, China), and the GSH level was determined using the GSH assay kit (Nanjing Jiancheng Bioengineering Institute, China).
The mouse microglia cell line (BV2) was purchased from the Cell Bank of the Chinese Academy of Sciences (Beijing, China). The BV2 cells were cultured in 6-well plates with DMEM/F12 supplemented with 10% FBS and incubated at 37℃ in an atmosphere of 5% CO2. For senescence induction, BV2 cells were treated with 100 nM rotenone (Rot) or 10 nM antimycin A (Anti A) for 5 days. Aged BV2 cells were treated with 5μM AdipoRon.
Primary microglia culture was performed according to a method previously developed . Briefly, brain tissues from postnatal (day 1-3) APN KO mice and WT mice were isolated, cut into tiny pieces, and followed by digestion with trypsin for 15 min. Dissociated tissues mixed with glia were then plated into T-25 culture flasks containing DMEM/F12 with 10% fetal bovine serum, GlutaMAX (Invitrogen), and 1% penicillin/streptomycin. After culture in a 5% CO2/37℃ incubator for 14 days, the flasks were shaken at 220 rpm for 4 h at 37℃ to harvest the primary microglia. After that, the microglia were plated in 6-well plates at a density of 5 x 105 cells per well for inducing senescence and 10μM Cpd-60 treatment.
Flow cytometry was used to measure the intracellular ROS level. In brief, the aged BV2 cells with or without AdipoRon treatment were incubated using 10μM 2', 7’-dichlorofluorescein diacetate (DCFH-DA, Sigma, USA) for 30 min at 37℃. Then cells were washed 3 times with PBS and collected for analysis through BD Accuri C6 Plus (BD Bioscience, USA).
Protein samples from animals or cells were extracted with RIPA lysis solution containing protease and phosphatase inhibitor (Thermo, USA) and then separated by SDS-PAGE and transferred to PVDF membrane. The membrane was blocked with 5% nonfat milk for 1 h at room temperature and subsequently incubated with primary antibody and secondary antibody. The protein level was measured using the PierceTM ECL Western Blotting Substrate kit (Thermo, USA) and quantified using ImageJ software.
Mouse Behavioural Assays
Open field and elevated plus-maze tests were used to assess experimental mice's anxiety-like behavior. A fear conditioning test was used to assess cognitive impairment.
Open field test
The open field consisted of a plexiglas box (50 x 50 x 40 cm). The bottom of this apparatus was divided into 16 equal squares. The inner 25 x 25 cm area was defined as central; the remaining regions were defined as peripheral areas. Mice were allowed to freely explore the device for 5 min. The device was cleaned with 75% ethanol and allowed to dry completely between each trial. Total distance traveled and the time spent in the center were recorded by Xeye software (Biowill, Shanghai, China).
Elevated-plus maze test
The elevated plus-maze was comprised of two open and two closed arms extended out from a central platform. Each mouse was individually placed in the center area and allowed to explore the device for 5 min. The time and movement of experiment mice were recorded by Xeye software (Biowill, Shanghai, China). The time spent in the open arms was calculated to assess anxiety-like behavior.
Fearing condition test
Fear conditioning was performed according to the previously described . The apparatus consisted of an acrylic chamber (25 × 25 × 25 cm) equipped with a stainless-steel grid floor. On the training day, mice were first allowed to freely explore the chamber for 6 min in the absence of any other behaviorally relevant stimulus. After that, mice received 3 paired presentations of a 30 s, 4 kHz, 80 dB auditory cue (CS) co-terminating with a 2 s, 0.5 mA scrambled footshock (US), followed by the addition of 2 min of free exploration without tone or shock stimuli. Each inter-trial interval was 2 min. The chamber was cleaned with 75% ethanol between sessions to avoid residue from the previous session. On the second day, mice were placed into the original chamber to assess contextual memory. The experimental animals were allowed to explore the chamber for 8 minutes without tone or shock stimuli. On the third day, the cued memory was tested in a novel context in which different odorants and shapes of the chamber. After a brief baseline period with no tone, the 80 dB tone sounded for 30 s at trial time points 120 s, 270 s, and 420 s. Then mice received the addition of 2 min freely exploration without any tone. The movement of mice was recorded and analyzed using the video tracking system (Biowill, Shanghai, China)
The data was presented as the mean±SEM and analyzed using GraphPad Prism 8.0 statistical software (GraphPad Software, Inc., La Jolla, CA, USA). A two-tailed unpaired Student's test was applied to compare two groups statistically. Simultaneously, One-way analysis of variance (ANOVA) was employed to determine the statistical significance of differences among groups and follow Dunnett's multiple comparison test. A probability value of p* < 0.05, p** < 0.01, p*** < 0.001 and p**** < 0.0001 was considered statistically significant.