G1251

Heart mitochondrial function was assessed as previously described [49 (link)]. Briefly, cardiac samples were minced in isolation buffer (300 mM sucrose, 10 mM HEPES, 2 mM EGTA, pH 7.2, 4 ℃ ), containing type I protease (bovine pancreas; Sigma, P4630), centrifuged at 500× g to pellet cell debris, followed by centrifugation of supernatant at 9000× g, resulting in a mitochondrial-enriched pellet, which was resuspended in a minimal volume of isolation buffer. Mitochondrial O₂ consumption and H₂O₂ release were measured in 0.125 mg/mL of isolated mitochondria in experimental buffer (125 mM sucrose, 65 mM KCl, 10 mM HEPES, 2 mM inorganic phosphate, 2 mM MgCl₂, 100 μM EGTA, 0.01% BSA, pH 7.2), containing succinate 2 mM (Sigma, S3674), malate 2 mM (Sigma, M1000) and glutamate 2 mM (Sigma, G1251) substrates, with continuous stirring at 37 °C. ADP 1 mM (Amresco, 0160) was added to induce state 3 respiratory rate. Mitochondrial O₂ consumption was monitored using a computer-interfaced Clark-type electrode (OROBOROS Oxygraph−2k). Mitochondrial H₂O₂ release was measured using Amplex Red 25 μM (Molecular Probes A12222) horseradish peroxidase 0.5 U/mL (Sigma P8125) system, and detected using a fluorescence spectrophotometer (ʎex = 563/ʎem = 587 nm) (F-2500 Hitachi—Hitachi).

For induction of DNA damage, cells were incubated with etoposide (Sigma, E1383) and glutamate (Sigma, G1251) at 5 µM and 10 µM, respectively. For STING-activation experiments, 2′3′-cGAMP (Sigma, 5,318,890,001) was used at 20 μM in human iPSC-derived neurons, and DMXAA (Sigma, D5817) was used at 20 μg/ml in primary mouse cortical neurons. For STING-inhibition experiments, 1 μM H151 (Invitrogen, inh-h151) or 10 μM RU.521 (Sigma, SML2347) was used.

Hippocampal homogenate (100 µL) was added to a reaction medium (imidazole-HCl 100 mM, pH 7.2; glutamate 50 mM (Sigma, G1251); hydroxylamine-HCl 100 mM; MgCl₂ 20 mM; and 2-mercaptoethanol 25 mM (Sigma 63689)) at 37 °C. The reaction was started by adding 10 mM ATP, and after 30 min, the reaction was stopped by adding 1.0 mL FeCl₃ 370 mM/HCl 60 mM/TCA 3M [18 (link)]. The formation of gamma-glutamyl hydroxamate was spectrophotometrically determined at 540 nm. The activity of this enzyme was calculated from the molar extinction coefficient of gamma-glutamyl hydroxamate (0.7 × 10³ M/cm), and values were expressed in mM of gamma-glutamyl hydroxamate formed/min/mg protein.