Animals and ethics statement
We obtained APP/PS1 double transgenic mice and B6C3-Tg (APPswe, PSEN1dE9) 85Dbo/J (original species No. 004462) from Jax Laboratory (Bar Harbor, ME, USA). Our lab maintained transgenic mice on a standard 12-h light/dark cycle under constant temperature with free access to water and food. Animal ethics committee in Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine approved this study. All protocols followed “Guide for the Care and Use of Laboratory Animals” from National Institutes of Health (Bethesda, MD, USA), and we made all efforts to minimize the number of animals utilized and any discomfort experiments.
ADSCs isolation, culture, and identification
Our lab washed adipose tissue harvested from healthy people or normal mice with phosphate-buffered saline (PBS) and minced it before digestion with 0.2% collagenase I (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37°C with intermittent shaking. Technician washed tissue that digested with Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich) including fetal bovine serum (FBS, Gibco BRL, Frederick, MD, USA) of 15%, and centrifuged it at 1000 rpm for 10 min to eliminate mature adipocytes. Our lab resuspended pellet in DMEM supplied with FBS of 15%, 100 U/mL penicillin, and 100 μg/mL streptomycin and cultured it at 37°C with CO2 of 5%. When the ADSCs had reached 80%~90% confluency, our lab detached them with 0.02% ethylenediaminetetraacetic acid (EDTA)/0.25% trypsin (Sigma-Aldrich) for 5 min at room temperature and then replated them. For phenotypic analyses, we leveraged fluorescein isothiocyanate (FITC)-conjugated CD29, CD44, CD90, CD105, and vWF antibodies. An IgG-matched isotype was employed as internal control for every antibody. Normoxic ADSC cultures were in 95% air (20% O2) and CO2 of 5%. For hypoxia induction, our lab cultured ADSCs in 94% N2, 1% O2, and CO2 of 5%.
ADSCs multilineage differentiation
To validate ADSCs multilineage differentiation, third-passage mouse ADSCs were cultured in adipogenic differentiation medium (Sigma-Aldrich), which were stained with oil red O after 2 w, or cultured in osteogenic differentiation medium (Sigma-Aldrich) and stained with alizarin red after 3 w.
ADSC-derived exosome isolation and identification
After reaching 80%~90% confluency, our lab rinsed ADSCs with PBS and cultured them in FBS-free endothelial cell growth medium (EGM)-2MV supplemented with 1× serum replacement solution (PeproTech, Cranbury, NJ, USA) for another 2 d. We erased conditioned culture medium and centrifuged it at 300 × g for 10 min and then at 2000 × g for 10 min to erase apoptotic cells and cellular debris. Briefly, we removed cell debris and large membrane vesicles by sequential centrifugation at 300 × g for 10 min, 2,000 × g for 10 min, and 10,000 × g for 0.5 hr, followed by filtration through 0.22-μm syringe filters. Afterwards, our lab transferred supernatant that cleared to fresh tube and spun it at 100,000 × g for 70 min. Then, we completely removed supernatant, and washed the pellet with PBS to collect exosomes. We characterized exosomes via transmission electron microscopy and western blotting, and the size was determined by dynamic light scattering using nanoparticle tracking analysis (NTA; NanoSight, Malvern, Worcestershire, UK).
We labelled exosomes using lipophilic dye DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; Thermo Fisher Scientific, Waltham, MA, USA) for biodistribution detection.
Strand-specific high-throughput RNA-Seq library construction
Our team extracted total RNA from ADSCs exosomes (Exo) and hypoxia-pretreated ADSC exosomes (HExo) with or without hyperglycemia pretreatment through TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). About 3 μg total RNA from every sample was subjected to VAHTS Total RNA-seq (H/M/R) Library Prep Kit from Illumina (Vazyme Biotech Co., Ltd, Nanjing, China). In this way, we removed ribosomal RNA and retained other types of RNA including ncRNA and mRNA. Our lab treated purified RNA with 40 U RNase R (Epicenter, Madison, WI, USA) at 37°C for 3 h, followed by TRIzol purification. Our lab made RNA-seq libraries via KAPA Stranded RNA-Seq Library Prep Kit (Roche, Basel, Switzerland), which were subjected to deep sequencing with an Illumina HiSeq 4000 at Aksomics, Inc. (Shanghai, China).
We intravenously treated two-month-old APP/PS1 mice with PBS (AD), exosomes derived from ADSCs (Exo), hypoxia-pretreated ADSCs (HExo), or circ-Epc1-expressing ADSCs (circ-Epc1-Exo) monthly for 2 months (n=10 mice per group). Of all groups, the injection volume was 100 μL. Exosomes from 1×109 ADSCs were dissolved in 100 μL PBS. There was no drop-out during normal breeding along with injection period. To detect exosome presence in brain, we euthanized three mice from each group at 5 h post-injection to examine brain slices by confocal microscopy after counterstaining with 4′,6-diamidino-2-phenylindole (DAPI).
Morris water maze (MWM) test
Our lab tested memory and learning function with MWM . Operator blinded to treatment groups performed all examinations. The apparatus consisted of a round steel pool (diameter: 122 cm; height: 60 cm) that was filled with water to 1 cm higher than the platform (diameter: 10 cm; depth: 30 cm) top. Blue curtain with cues surrounded the pool, which was put in an isolated room (20°C, 60% humidity). We maintained water at 21°C and opacified it via inserting titanium dioxide.
Our team performed testing for 5 d. The first 4 d (P40–P43) comprised a place navigation (reference memory) test including 16 training trials (4 trials per day for 4 d, with 30–40 min inter-trial interval). At the start of every trial, we put mice in the water facing the wall in various starting locations (south, north, west or east), which were allowed 1 min to discover and 15 s to stay on top of the hidden platform. If the mouse could not locate the platform within 1 min, it was guided to and allowed to stay on the platform for 15 s. We employed video tracking system to track swimming activity regarding every mouse. Escape latency, say, timing from placement into the water to staying on the platform, was tracked. We performed spatial probe test where the platform was moved out of the pool. We placed the animal in opposite quadrant and allowed it to swim freely for 2 min. We tracked platform crossing numbers. Our team analyzed data via motion-detection software for MWM test (Shanghai Mobile Datum Information Technology Co., Shanghai, China).
BV2 cell culture and transfection
Our lab maintained BV2 cells (Wuhan Biofavor Biotechnology Service Co., Ltd., Wuhan, China) in DMEM (Invitrogen) supplemented with FBS (Invitrogen) of 10% under atmosphere containing 5% CO2/95% air. For phenotypic analyses, we transfected BV2 cells with a small interfering (si)RNA against TREM2, a circ-Epc1 overexpression plasmid, or a miR-770-3p overexpression plasmid (mimic) (GeneCopoeia, Shanghai, China) through Lipofectamine 2000 (Invitrogen) following standard procedures. We used cells for further experimentation after 2 d and exposed them to LPS (1 μg/mL) for 1 d before phenotypic analysis.
RNA and miRNA extraction with real-time (RT)-PCR
Our lab isolated total RNA from serum, cells, or brain tissues via TRIzol reagent. We synthesized first strand cDNA through PrimeScript RT Master Mix (Perfect Real Time) Kit (RR036A, Shiga, Japan), which was leveraged for RT-PCR, along with reverse and forward primers and Power SYBR Green PCR Master Mix (Life Technologies, Carlsbad, CA, USA). U6 and GAPDH were utilized as internal controls. We analyzed data via 2−ΔΔCt method.
Luciferase reporter assays
We cloned putative miR-770-3p binding site in 3′-UTR of target gene TREM2 and circ-Epc1 (Wt or Mut) into psi-CHECK vector (Promega, Madison, WI, USA) downstream of firefly luciferase 3'-UTR or circ-Epc1 as primary luciferase signal with Renilla luciferase as normalization signal, these vectors were termed TREM2-Wt/circ-Epc1-Wt and TREM2-Mut/circ-Epc1-Mut, respectively. psi-CHECK vector provided Renilla luciferase signal as normalization to compensate the differences between transfection and harvesting efficiencies. We performed transfection into HEK293 cells through Lipofectamine 2000 (Invitrogen). Our lab detected Renilla and firefly luciferase activities 1 d after transfection with Dual-Luciferase Reporter Assay System (Promega) through luminometer (Molecular Devices, San Jose, CA, USA). Our lab detected relative Renilla luciferase activities following the manufacturer (Promega) instructions.
Immunohistochemistry (IHC) and immunofluorescence (IF) analyses
Our team fixed brain tissue samples in 10% formalin solution, embedded them in paraffin, which were sectioned at 5 μm. Our lab stained tissue sections with TUNEL detection kit (Zeiss, Oberkochen, Germany) for apoptosis evaluation. Our lab performed IF staining for Iba-I, CD11b, and CD206 to validate microglial polarization. We analyzed results through Axiophot light microscope (Zeiss) or a fluorescence microscope (Nikon, Tokyo, Japan), which were photographed with digital camera.
Enzyme-linked immunosorbent assay (ELISA)
We collected cell culture medium after the treatments that mentioned above. We utilized ELISA kits (R&D Systems, Minneapolis, MN) to obtain the interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α levels following the instructions.
Continuous variations are denoted as mean ± standard deviation (SD). We used one-way analysis of variance for multiple comparisons via GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA). A P-value ≤0.05 inferred statistically significance.