Mitochondrial Function Assessment in Brain

Mitochondrial function was assessed ex vivo in homogenates of the right frontal cortex. Brain homogenates (1 mg/mL) were prepared fresh as previously described and a respirometric Substrate–Uncoupler–Inhibitor Titration (SUIT) protocol was applied for in-depth characterization of the OXPHOS system and electron transport systems (ETS)^{30 (link)}. Chemical reagents and concentrations used in the SUIT protocol are provided in Supplementary Table S1. In brief, specific mitochondrially targeted CI-, CII- and CIV-linked substrates were used to assess functionality of the ETS system, the OXPHOS system, and to evaluate the integrity of the inner mitochondrial membrane. For more details on the SUIT protocol see Jang, et al.^{31 (link)} After completion of the respirometry protocol, the chamber contents were stored at − 80 °C and citrate synthase (CS) activity was measured to correct for any difference in mitochondrial content between samples. CS activity was measured using a commercially available kit according to the manufacturer’s instructions (CS0720; Sigma-Aldrich, St. Louis, MO).