Figure 2.

Effects of βOHB on mitochondrial respiration and neuroprotection of aralar-KO primary neuronal cultures. Effects of 30-min preincubation with 5 mm βOHB on WT or aralar-KO neurons maintained in 25 mm glucose NB until DIV9 (A–D), on basal respiration (A, OCR, nmol O₂/min/mg protein) or on glutamate 50 μm (Glu50)-stimulated respiration (B–D, as % of basal values). E–H, Effects of 48-h βOHB treatment on WT and aralar-KO neurons maintained in 5 mm glucose NB-A from DIV7 to DIV9 on basal respiration (E, F) or Glu50-stimulated respiration (G, H). Mean ± SEM from three to six embryos per condition measured in three to nine replicates; ***p ≤ 0.001, *p ≤ 0.05 (two-way ANOVA followed by post hoc Bonferroni-corrected t test). I, J, Effects of 30-min treatment with 5 mm βOHB or 5 mm AcAc on glutamate 50 μm (Glu50)-stimulated respiration on aralar-KO neurons. Mean ± SEM (three to six wells/condition); ***p ≤ 0.001, *p ≤ 0.05 (one-way ANOVA followed by post hoc Bonferroni-corrected t test). Mitochondrial function was determined at DIV9 in 2.5 mm glucose and 2 mm Ca²⁺ DMEM. Sequential addition of glutamate (Glu50), 6 μm oligomycin (Oli), 0.5 mm dinitrophenol (DNP), and 1 μm rotenone/1 μm antimycin (R/A) was performed where indicated by the dashed lines in B. K, Percentage of dead cells maintained in 25 mm glucose NB in basal conditions of after treatment with glutamate (Glu), with or without 5 mm βOHB during 30 min or 96 h. Data calculated as an increase in % of dead cells versus basal. Mean ± SEM from four to six replicates coming from four to five different embryos per condition; ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 (two-way ANOVA followed by post hoc Bonferroni-corrected t test). L, Percentage of MTT reduction in WT and aralar-KO neurons under basal conditions or preincubated 96 h with 5 mm βOHB. Data expressed as % of WT basal (mean ± SEM from 8 to 12 different embryos per condition measured in triplicate); *p ≤ 0.05 (two-way ANOVA followed by post hoc Bonferroni-corrected t test).