Fig 2: NMDAR‐dependent decrease in APT1 expression is insensitive to Akt inhibition or Ago2 phosphorylation at S387 (related to Fig 7) Endogenous APT1 protein levels are rapidly reduced in response to NMDAR stimulation. Cortical neuronal cultures were exposed to NMDA or vehicle for 3 min, and lysates were prepared 10, 20 or 40 min after NMDA washout and analysed by Western blotting. Graphs show quantification of APT1 expression normalised to vehicle control; n = 6. **P < 0.01; ***P < 0.001 one‐way ANOVA, Bonferroni post hoc test. Error bars are SEM.NMDAR‐dependent decrease in APT1 is Akt‐independent. Cortical neuronal cultures were treated with Akti‐1/2 20 min before NMDA or vehicle application, and lysates were prepared 40 min after NMDA washout and analysed by Western blotting. Graphs show quantification of APT1 expression normalised to vehicle control; n = 6. *P < 0.05; two‐way ANOVA, Bonferroni post hoc test. Error bars are SEM.NMDAR‐dependent decrease in dendritic APT1 expression is unaffected by Ago2 phosphorylation at S387. Cortical neurons were transfected with molecular replacement constructs expressing Ago2 shRNA plus shRNA‐resistant GFP‐Ago2 (WT, S387A or S387D), fixed 40 min after NMDA washout, permeabilised and stained with APT1 antibodies (red channel). GFP signal was maximised at acquisition so that dendrites could be effectively visualised. Graph shows APT1 staining intensity in dendrites normalised to vehicle control. Scale bar = 20 μm; n = 10 cells from five independent experiments. ***P < 0.001 two‐way ANOVA, Bonferroni post hoc test. Error bars are SEM. Source data are available online for this figure.

Fig 3: NMDA‐induced dendritic spine shrinkage requires Akt activation, Ago2 phosphorylation at S387 and miRNA‐mediated reduction in LIMK1 expression S387 phosphorylation is required for NMDA‐induced spine shrinkage. Cortical neurons were co‐transfected with mRUBY as a morphological marker, and molecular replacement constructs expressing Ago2 shRNA plus shRNA‐resistant GFP‐Ago2 (WT, S387A or S387D). Forty minutes after NMDA or vehicle application, cells were fixed, permeabilised and stained with anti‐mCherry antibody to amplify the mRUBY signal, from which spine size and density were measured. Graph shows quantification of spine size and spine density; n = 4 independent experiments (nine cells per condition). *P < 0.05; **P < 0.01; ***P < 0.001; two‐way ANOVA, Bonferroni post hoc test. Scale bar = 10 μm. Mean ± SEM.Akt activation is required for NMDA‐induced spine shrinkage. Cortical neurons were transfected with GFP as a morphological marker, and Akti‐1/2 was applied 20 min before NMDA or vehicle application. Forty minutes after NMDA washout, cells were fixed, and spine size and density were measured. Graph shows quantification of spine size and spine density; n = 4 independent experiments (10 cells per condition). *P < 0.05; two‐way ANOVA, Bonferroni post hoc test. Scale bar = 10 μm. Mean ± SEM.Loss of LIMK1 is required for NMDA‐induced spine shrinkage. Cortical neurons were co‐transfected with mRUBY as a morphological marker, and mycLIMK1 or empty vector. Forty minutes after NMDA or vehicle application, cells were fixed, permeabilised and stained with anti‐myc antibody (green) and anti‐mCherry antibody (magenta) to amplify the mRUBY signal, from which spine size and density were measured. Graphs show quantification of spine size (left), spine density (middle) and mycLIMK1 expression (right); n = 5 independent experiments (10 cells per condition). *P < 0.05; two‐way ANOVA, Bonferroni post hoc test. Scale bar = 10 μm. Mean ± SEM. Source data are available online for this figure.

Fig 4: Experimental design and small RNA sequencing. (A) Schematic showing the full study design. Step 1: Three rodent models of epilepsy were generated: IAKA (intraamygdala kainic acid-induced status epilepticus in C57BL/6 mice), PILO (pilocarpine-induced status epilepticus in NMRI mice), and PPS (perforant pathway stimulation-induced hippocampal lesioning in Sprague-Dawley rats). Step 2: Hippocampi were extracted at six (mice) or seven (rat) time points and processed for Ago2 immunoprecipitation and small RNA sequencing (Ago2-seq). Step 3: miRNAs with consistent up-regulation in all three models were selected for an antagomir-based screen for antiseizure phenotypes and neuroprotection. Step 4: Pathway modeling and biophysical analyses were used to investigate the function of the miRNAs. (B) The read mapping distribution for the three rodent models. Note, the majority of small RNA reads mapped to miRNAs. (C) Expression of the top 50 miRNAs between the three models showing highly similar expression levels.

Fig 5: Mutation of the SUMOylation consensus site in Ago2 abolishes SUMOylation. A) Myc-Ago2 can be modified at K402 by both SUMO1 and SUMO2. N2a cells were transfected as indicated, and SUMOylated proteins enriched on GFP-trap beads followed by western blotting. For one set, after the pulldown and extensive washing, the beads were treated with the recombinant catalytic domain of the SUMO-specific protease SENP1 to further confirm the identity of the higher molecular weight species of Ago2 as Ago2-SUMO. B) Ablation of the SUMOylation consensus site surrounding K402 reduces the residual SUMOylation observed upon mutation of K402 to alanine. Cells were transfected as indicated, followed by enrichment of YFP-SUMOylated proteins on GFP-trap beads and western blotting.