Total tau

Homogenate samples of the left hippocampus and the cortex were prepared as described in a previous study [20 (link)]. The hippocampus or cortex was homogenized in tris-buffer solution (TBS) containing a protease inhibitor cocktail (BioVision, CA, USA) using a multi-beads shocker (Yasui Kikai, Osaka, Japan). The supernatant (first) was collected after centrifugation at 50,000×g for 20 min, and the pellet was homogenized again in TBS containing 1% Triton X-100 (Wako, Osaka Japan), and the supernatant (second) was collected after centrifugation at 50,000×g for 20 min. The total protein concentration of each supernatant was measured using the BCA Protein Assay Kit (Thermo-Scientific, Yokohama, Japan). The first supernatant was used to quantify soluble Aβ_1-42 (Wako), phosphorylated tau (pS199, ThermoFisher Scientific, Yokohama, Japan), total tau (ThermoFisher Scientific), synaptophysin (LSBio, Seattle, WA, USA), BDNF (Promega, Madison, WI, USA), and IGF-1 (R&D Systems, Minneapolis, MN, USA) by ELISA and cytokines and chemokines by a Bio-Plex assay system (Bio-Rad, Hercules, CA, USA). The second supernatant was used to quantify insoluble Aβ_1-42 (Wako) by ELISA.

ELISAs were performed according to the manufacturer’s instructions to obtain a relative quantity of Aβ₄₀, Aβ_42, p-tau181, p-tau396, and total tau (ThermoFisher). Data presented is % control after calculating either Aβ_42/40 ratio or normalizing p-tau data to total tau. Briefly, soluble protein or centrifuged culture media was diluted with kit diluent according to the table below: