Activation of the NR4A1–AMPK pathway reduced dendritic spine number in vitro. A, Time-lapse imaging of TMRM fluorescence in primary cortical neurons (DIV14 with 10 d of doxycycline treatment). Yellow box is one example of an ROI. B, ΔF (F − F0/F0 × 100) traces are the mean ± SEM of N = 10 GFP, N = 10 NR4A1, and N = 7 ΔAF1 neurons subjected to all treatments subsequently. Statistical comparison between GFP and NR4A1 groups at indicated epochs (last 3 min of each treatment) by unpaired t test: during 100 μm glutamate, *p = 0.009 (N = 16 GFP, N = 27 NR4A1 cells); during washout, op = 0.0039 (N = 16 GFP, N = 27 NR4A1 cells); and during 1 μm oligomycin administration, §p = 0.035 (N = 10 GFP, N = 10 NR4A1 cells). C, Dissipation of ATP upon stimulation of primary cortical neurons (DIV14 with 10 d of doxycycline treatment) with 100 μm glutamate compared with 1 μm oligomycin for 1 h. Mean ± SEM of N = 8 GFP, N = 10 NR4A1, and N = 5 ΔAF1 independent samples; unpaired t test comparing GFP and NR4A1, *p = 0.024. D, Phosphorylation of AMPK and its substrate ACC in primary cortical neurons (DIV14 with 10 d of doxycycline treatment) after stimulation with 25 μm glutamate for 3 h (N = 18 GFP, N = 15 NR4A1, N = 5 ΔAF1 samples/group). Pearson correlation between levels of p-ACC and p-AMPK normalized to GFP as controls. Group comparison by unpaired t test for p-ACC: NR4A1 vs GFP, p < 0.0001; and ΔAF1 vs GFP, p = 0.009; for p-AMPK: NR4A1 vs GFP, p < 0.0001; and ΔAF1 vs GFP, p = 0.004. E, Modulation of AMPK by 10 μm STO609 and 1 μm A769662 for 24 h in primary cortical neurons (DIV14). Pearson correlation between levels of p-ACC and p-AMPK normalized to vehicle controls (N = 6 samples/group). Comparison by unpaired t test for p-ACC: STO609 vs vehicle, p = 0.0061; and A769662 vs vehicle, p = 0.0007; for p-AMPK: STO609 vs vehicle, p = 0.2; and A769662 vs vehicle; p = 0.0013. F, Effect of STO609 (S) and A769662 (A) compared with vehicle (V) on dendritic spine density in primary cortical neurons expressing GFP, NR4A1, or ΔAF1 (DIV21 with 10 d of doxycycline treatment). Mean ± SEM of N = 99 GFP, N = 192 NR4A1, and N = 160 ΔAF1 dendrites; ANOVA post hoc Tukey's test for comparing the following: NR4A1 and ΔAF1, **p < 0.0001; GFP and NR4A1, †p = 0.0019; GFP and ΔAF1, ¶p = 0.002; STO609 and vehicle on NR4A1 cells, #p < 0.0001; A769662 and vehicle on ΔAF1 cells, §p = 0.023; interaction of NR4A1 and A769662, *p < 0.045. See extended data Figure 6-1.
