Proteomic analysis links alterations of bioenergetics, mitochondria-ER interactions and proteostasis in hippocampal astrocytes from 3xTg-AD mice

Immortalized hippocampal astrocytes from 3xTg-AD mice

Generation of immortalized astrocytes from hippocampi of WT and 3xTg-AD mice (WT- and 3Tg-iAstro cells) was described elsewhere²⁷. iAstro lines were maintained in complete culture media containing Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, Cat. No. D5671) supplemented with 10% fetal bovine serum (Gibco, Cat. No. 10270), 2 mM L-glutamine (Sigma-Aldrich), and 1% penicillin/streptomycin solution (Sigma-Aldrich). Cells were passaged once a week and used for experiments between passages 12 and 20 from establishment²⁷.

Evaluation of iAstro energetic metabolism and ROS production

Mitochondrial respiration

Assessment of mitochondrial and glycolytic functionality was performed by Seahorse XFp Analyser (Agilent Technologies, Santa Clara, CA, USA) using Seahorse XFp Cell Mito Stress Test Kit (Agilent Technologies) and…