Aβ1 42 peptide

Wistar rat pups (newborn P0–1) were obtained from the Valladolid University animal facility. All animals were handled according to the ethical standard of Valladolid University under protocols approved by the animal housing facility and in accordance with the European Convention 123/Council of Europe and Directive 86/609/EEC. Fura-2/AM and Rhod-5N are from Invitrogen (Barcelona, Spain). Fetal bovine serum (FBS) is from Lonza (Barcelona, Spain). Horse serum, neurobasal medium, HBSS medium, B27, L-glutamine and gentamycin are from Gibco (Barcelona, Spain). Papain solution is from Worthington (Lakewood, NJ, USA). The poly-D-lysine and annexin V are from BD (Madrid, Spain). DNase I and antibody against the MCU are from Sigma (Madrid, Spain). IP₃R1 and IP₃R2 primary antibodies are from Santa Cruz Biotechnology (Dallas, TX, USA). IP₃R3 primary antibody is from BD Transduction Laboratories (Madrid, Spain). ER tracker, mitotracker, tetramethylrhodamine, methyl ester (TMRM) and CM-H2DCFDA are from ThermoFisher Scientific. Aβ_1–42 peptide is from Bachem AG (Bubenforf, Switzerland). Other reagents and chemicals were obtained either from Sigma or Merck.

Prior to establishing an acute AD model through stereotactic injection of Aβ, aggregation of Aβ was evaluated by a Thioflavin T (ThT) assay (Figure S5). Aβ_1–42 peptide (Bachem, Bubendorf, Switzerland) was solubilized in 0.1 M aqueous ammonia solution to prepare a 27.5-μM Aβ peptide solution. ThT (Tokyo Chemical Industry, Tokyo, Japan) was dissolved in 50 mM glycine buffer to prepare a 15-μM ThT solution. A final 100 μL reaction solution was made, consisting of 67.5 μL 27.5-μM Aβ peptide solution, 25 μL 15-μM ThT solution, and 7.5 μL of distilled water. As a positive control to inhibit Aβ oligomerization, 2 mM Morin (Sigma-Aldrich, St. Louis, MO, USA), an inhibitor of Aβ oligomerization [74 (link)], was used instead of triple distilled water. The ThT reaction solution was incubated in a 96-well black plate (SPL life Sciences, Pocheon-si, Republic of Korea) for 1, 2, 3, and 4 h at 37 °C, after which the fluorescence intensity of ThT was measured at Ex/Em = 440 nm/484 nm using a SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA).

Cortical neurons and microglia were incubated with Aβ solutions after 11 days of culture. The Aβ_1–42 preparation was done following the procedure described by^{24 (link)}. Briefly, Aβ_1–42 peptide (Bachem, Weil-am-Rhein, Germany) was dissolved in the defined culture medium mentioned above, at an initial concentration of 40 μmol/L. This solution was gently agitated for 3 days at 37 °C in the dark and immediately used after being properly diluted in control medium to the concentrations used (5 μM of Aβ_1–42 preparation containing 0.5 μM of Aβ oligomers (AβO) measured by WB). The control medium consisted in the defined culture medium (as described above).
AZP2006 and anti-PGRN (5 μg/mL, MAB2557, R&D Systems, Noyal Châtillon sur Seiche, France) were dissolved in culture medium and incubated with Aβ_1–42 preparation on primary cortical neurons for 72 h.