Tg2576 mice

For DNA microarray analysis, brain samples were obtained from the mice previously reported^{9 (link)}. In brief, 5-month-old female Tg2576 mice (Taconic Farms, Germantown, NY) were used. They were divided into a control group fed a normal diet, and the RA group was fed an RA diet for 10 months. It was confirmed immunohistochemically for these mice that RA significantly decreased Aβ deposition in the brain. All animal studies were approved by the Institutional Animal Experiment Committee of Kanazawa University. For all other experiment, 7–10-week-old female C57BL/6 J mice (CLEA Japan Inc., Tokyo, Japan) were used. The mice were divided into two groups, a control (C) group fed with AIN-93G diet made in our laboratory from the necessary ingredients (Oriental Yeast Co., Shiga, Japan) as a normal diet, and an RA group with 0.5% RA added to the normal diet. All mice were housed individually in a room with a 12-h light/dark cycle with food and water ad libitum. All animal experiments were approved by the animal experiment committee of the University of Tokyo and performed in accordance with the relevant guidelines and regulations (approval number: P15-010 and P15–100).

All animal procedures were carried out following the National Institutes of Health Guidelines for the Humane Treatment of Animals, with approval from the Institutional Animal Care and Use Committee of Seoul National University (IACUC No. SNU-121012-03). Tg2576 mice were obtained from Taconic Farms (Germantown, NY, USA) and were bred by mating male mice with C57B16/SJL F1 female mice, as recommended by the suppliers and as described by others.^{52 (link)} Comparisons were performed between heterozygous transgenic (Tg2576) mice and age-matched transgene-negative littermates (WT).

Tg2576 mice were purchased from Taconic Biosciences (strain: B6;SJL-Tg(APPSWE)2576Kha; cod. 1349-M). A colony of Tg2576 was obtained by crossing heterozygous transgenic male mice with C57BL/6J x SJL hybrid female mice, as previously described [13 (link)]. For colony genotyping, tail tissues were sampled from in order to extract genomic DNA, according to standard procedures. DNA yield and purity were assessed by evaluating 260/280 nm and 260/230 nm absorbance ratios using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Milan, Italy). An amount of 10 ng of DNA per sample was amplified by means of polymerase chain reaction (PCR): all assays were carried out using specific primers (FW 5′-CTG ACC ACT CGA CCA GGT TCT GGG T-3′, REV 5′-GTG GAT AAC CCC TCC CCC AGC CTA GAC CA-3′, Sigma-Aldrich (St. Louis, MO, USA)). The thermal profile was a 1′ initial denaturation step at 95 °C followed by 35 cycles of amplification (denaturation: 15″ at 95 °C; annealing: 15″ at 55 °C; and extension: 10″ at 72 °C). Genotyping was later performed by running amplified DNA samples on a 2% agarose gel (Sigma-Aldrich) in presence of 0.01% of GelRedTM nucleic acid staining (Biotium, Hayward, CA, USA). Tg2576 mice were identified by the presence of a ≈500 bp band.

The rabbit polyclonal antibodies used in this project include APP (Cell Signaling, Danvers, MA, USA), ferriportin (Thermo, Inc., Rockford, IL, USA), NFATC1 (BD biosciences, San Jose, CA, USA), phospho-Erk1/2 (pErk1/2) and phospho-Akt (pAkt)(Cell Signaling, Danvers, MA, USA). Murine macrophage colony-stimulating factor (M-CSF) was obtained from R&D Systems (Minneapolis, MN, USA), and the recombinant GST-RANKL proteins were provided by Dr. Xu Feng (University of Alabama at Birmingham), which were generated and purified as previously described [7 (link), 17 (link), 18 (link)].
As described previously[19 (link)], the Tg2576 mice, purchased from Taconic, Inc. (Hudson, NY, USA), express human APP695 with double mutations at KM670/671NL (Swedish mutations)(called APPswe) under the control of hamster prion promoter. Tg2576 has been crossed into C57BL/6 genetic background for more than 6 generations. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the Georgia Regents University, in accordance US National Institutes of Health guidelines.

Wild-type C57BL/6J mice (CLEA Japan Inc, Kawasaki, Japan) and Tg2576 mice (Taconic Biosciences Inc., Hudson, NY, USA) at 10- and 17-months-old were used for RNA extraction from brain cortex tissue. For the isolation of primary murine neuronal cells, pregnant C57BL/6J mice (embryonic-day 17) were euthanized and the embryos were collected. For behavioral testing, male C57BL/6J mice (13–14-weeks-old) were housed in plastic cages and kept in a regulated environment with a 12-h light/dark cycle. Food and tap water were available ad libitum.
All animal studies were performed according to the protocols approved by the Ethics Committees of the National Center for Geriatrics and Gerontology, Japan. The procedures involving mice and their care were conducted in line with international guidelines, specifically the Principles of Laboratory Animal Care (National Institutes of Health Publication 85–23, revised 1985).

Triple transgenic (3XTg-AD) mice harboring APP_SWE, PSEN1 (PS1/M146V) and tau (P301L) mutations (3XTg-AD, The Jackson Laboratory, Bar Harbor, ME, USA), Tg2576 mice harboring APP_SWE (Taconic, Hudson, NY, USA), and wild-type B6129SF2/J mice (the Jackson Laboratory) were housed under standardized 12 h-light/12-h dark cycle at ambient temperature and humidity with diet and water available ad libitum at the USF vivarium. The mice were allowed to acclimate for a period of one week before any treatment. All experiments were conducted in accordance with USF Institutional Animal Care and Use Committee approved protocols and guidelines of the National Institutes of Health.