Cell culture, animals and reagents

N1E-115 cells (ECACC #88112303), which are derived from a mouse neuroblastoma C1300 tumor, were originally obtained from DS Pharma Biomedical Co., Ltd. (Osaka, Japan). Cells were grown in Dulbecco’s minimum essential medium containing 10% heat-inactivated fetal calf serum (FCS) (Biological Industries, Beit Haemek, Israel), 2 mM glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin, and were plated in wells, dishes, or cell culture inserts of laminin [derived from mouse Engelbreth-Holm-Swarm (EHS) sarcoma]-coated plates at a density of 4.0 × 10⁵ cell/ml. Medium from cells cultured for 48 h was collected and used as conditioned medium. To elicit neurite elongation, after confirming cell adhesion, the medium was changed to conditioned medium containing 1% dimethyl sulfoxide (DMSO) solution. After 48 h, cells were used in experiments (Fig. 1A).

Morphology of N1E-115 cells before and after elongation of neurites, and induction of neurite degeneration in 10 µM hydrogen peroxide-treated N1E-115 cells (A). Scale bar is 50 µm. Hydrogen peroxide induces cell death in a concentration-dependent manner in N1E-115 cells (B). After treatment with various concentrations of hydrogen peroxide, dead cells were counted by trypan blue dye exclusion. Survival on untreated group was set to 100%. Data were analyzed by Dunnet’s test; * indicates p<0.05, ** indicates p<0.01. Each result represents at least four independent experiments.

All animal experiments were performed with the approval of the Animal Protection and Ethics Committee of the Shibaura Institute of Technology. Three-month-old control animals (C57BL/6, male) were purchased from Japan SLC, Inc. (Hamamatsu, Japan). Twenty-four-month-old (C57BL/6, male) mice were obtained from the Tokyo Metropolitan Institute of Gerontology (Tokyo, Japan). AD transgenic mice [#008730, MMRRC034848, B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax, Alias/5XFAD] were purchased from the Jackson Laboratory (Bar Harbor, ME). All mice were maintained under controlled conditions of temperature (22 ± 2°C), 12 h light/dark cycle, and had free access to food and water. The aged-mice were acclimated to their new environment for one week before each experiment. Normal diet pellets (Labo MR Stock) were purchased from Nosan Corp (Kanagawa, Japan). After dissection, the cortex regions were homogenized and used as brain samples.

All other chemical agents were obtained from either FUJIFILM Wako Pure Chemical Co. (Osaka, Japan) or Sigma-Aldrich Co. (St. Louis, MO). All tissue culture plates and dishes were purchased from Becton Dickinson and Company (Franklin Lakes, NJ).