Research ArticleEpilepsy

Filamin A inhibition reduces seizure activity in a mouse model of focal cortical malformations

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Science Translational Medicine  19 Feb 2020:
Vol. 12, Issue 531, eaay0289
DOI: 10.1126/scitranslmed.aay0289
  • Fig. 1 FLNA expression is increased in cortices of patients with FCDII.

    (A) MRI scans of 2- and 5-year-old patients with FCDII with seizures. The red arrow points to the FCM. (B and C) Images of phospho-S6 (B) and FLNA (C) staining in hematoxylin-stained sections from the FCM in the brain of patients whose scans are shown in (A). Black and green arrows point to FLNA-positive balloon cells and dysmorphic neurons, respectively. (D and E) Coimmunostaining for FLNA and a marker of cytomegalic neurons, SMI-311, and 4′,6-diamidino-2-phenylindole (DAPI) counterstain in human FCDII tissue. (F) Magnification of SMI-311 or FLNA staining with DAPI from images in the white square in (D) and (E). (G) Quantification of FLNA staining in SMI-311–positive neurons relative to surrounding SMI-311–negative cells. Only SMI-311–negative cells with visible FLNA staining (to outline the cell body) were used for quantification. (H and I) Immunoblots of FLNA and tubulin from resected FCDII tissue (H) and quantification from 12 patients with FCDII (I). The numbers correspond to those of the patients listed in tables S1 and S2. FLNA and tubulin size were 280 and 55 kDa, respectively. Unpaired Student’s t test. Data are means ± SEM. n numbers are listed in table S3. Scale bars, 100 μm.

  • Fig. 2 FLNA expression is increased in mouse cortices containing RhebCA-induced FCM.

    (A) FLNA (280 kDa), phospho-S6 (pS6) (32 kDa), S6 (32 kDa), and glyceraldehyde phosphate dehydrogenase (GAPDH) (37 kDa) immunoblots in cultured neurons expressing plasmids encoding GFP, mTOR mutants, or RhebCA. (B and C) Quantification of the blots shown in (A). (D) Diagram of the experimental procedure for generating FCM in mice followed by video-EEG recordings and immunoblotting or immunochemistry. (E and F) FLNA immunostaining in sections from mouse FCM (E) and quantification in GFP+ RhebCA-expressing cells relative to surrounding GFP cells (F). Scale bars, 100 μm. (G) FLNA and GAPDH immunoblots. Ipsi, ipsilateral; Contra, contralateral. (H) FLNA/GAPDH quantification in FCM-containing cortices relative to contralateral cortices in mice electroporated with RhebCA. One-way ANOVA (B and C) and unpaired and paired Student’s t test (F and H, respectively). Data are means ± SEM. n numbers are listed in table S3.

  • Fig. 3 Normalizing the amount of FLNA in dysmorphic neurons of mTOR-driven FCM partially prevents cytoarchitectural abnormalities and attenuates seizure activity.

    (A) Images of control or RhebCA-expressing neurons coexpressing luciferase or Flna shRNA in littermate mice, and quantification of neuronal placement in the different conditions. Scale bar, 250 μm. (B) Images of GFP+ electroporated neurons in different conditions and quantification of soma size. Scale bars, 50 μm. (C) Basal dendrite reconstruction and corresponding Sholl analysis. Scale bars, 50 μm. (D) Bar graphs of the basal total dendritic length (TDL). (E) Diagram of the experimental procedure. (F) Representative EEG traces in a control shRNA seizing mouse. Scales: 200 μV/5 s and 1 s (inset). (G) Frequency of seizures in the luciferase (Luc) or Flna shRNA condition. (H) Phospho-S6 immunostaining and quantification in GFP+ RhebCA-expressing neurons (normalized to surrounding GFP neurons) in EEG-recorded mice. Scale bars, 50 μm. Mann-Whitney tests (G), one-way ANOVA (A, B, and D) and two-way repeated-measures ANOVA (C), and Student’s t test (H). Data are means ± SEM. n numbers are listed in table S3.

  • Fig. 4 Treatment with a small molecule modulator of FLNA, PTI-125, before seizure onset partially prevents cytoarchitectural abnormalities and reduces seizure activity.

    (A) Diagram of experimental paradigm. (B) Images of neuronal soma after treatment with vehicle or different PTI-125 doses and quantification. Scale bars, 50 μm. (C and D) Reconstructions of basal dendrites (C), Sholl analyses, and basal total dendritic length (D) under different treatment conditions. (E) Immunoblots of phospho-S6, S6, and GAPDH from the cortices of mice treated with vehicle (saline) or PTI-125, and quantification. (F) Diagram of experimental paradigm. (G) Seizure frequency (6-day-long recordings) after vehicle or PTI-125 treatment at 12 mg/kg. (H) Plots of the weight gain during vehicle or PTI-125 treatment from P8 to P60. Inset, mean body weight between P52 and P60. (I) Quantification of cell misplacement in mice treated with vehicle or PTI-125. (J) Images of phospho-S6 immunostaining and GFP fluorescence in coronal sections containing RhebCA-expressing cells (GFP+) and corresponding phospho-S6 quantification normalized to the vehicle-treated condition. Scale bars, 50 μm. Student’s t test (E, H, I, and J), two-way repeated-measures ANOVA (D), Mann-Whitney test (G), and one-way ANOVA (B and D). Data are means ± SEM. n numbers are listed in table S3.

  • Fig. 5 Treatment with a small molecule modulator of FLNA, PTI-125, after seizure onset alleviates neuronal dysmorphogenesis and seizure activity.

    (A) Diagram of experimental paradigm. (B and C) Images of control GFP+ neurons and GFP+ RhebCA-expressing neurons in mice treated with vehicle (saline) or PTI-125 (B) and quantification of soma sizes (C). Scale bar, 120 μm. (D and E) Sholl analyses and basal total dendritic length. (F) Heat map of seizure activity over time (per day) per mouse treated with either vehicle (saline) or PTI-125. (G) Seizure frequency after and during vehicle or PTI-125 treatment from P29 to P54. (H) Diagram of experimental paradigm. (I) Heat map of seizure activity per individual mouse over time. In the upper heat map, mice received vehicle injections and were recorded from P61 to P92. In the bottom heat map, mice received vehicle from P61 to P72 and then PTI-125 until P106. For mouse #5, the wires were unplugged for 2 days, leading to loss of recordings that are colored white on the heat map. (J) Bar graph of the seizure frequency at days (D) 1 to 5 and D27 to D31 of recordings (corresponding to P61 to P65 and P88 to P92) under continuous vehicle treatment or vehicle–to–PTI-125 treatment. (K) Plots of the seizure frequency before and after PTI-125 treatments. One-way ANOVA (C, E, and J), two-way repeated-measures ANOVA (D), Mann-Whitney tests (G), and Wilcoxon matched-pairs test (K). Data are means ± SEM. n numbers are listed in table S3.

  • Fig. 6 PTI-125 decreases the percentage of seizing mice and seizure frequency.

    (A) Percentage (%) of seizure-free mice in both conditions. Two mice in vehicle-treated condition died of seizures during EEG recordings and thus were not included in (B). (B and C) Heat map of seizure frequency for all conditions (B) and corresponding scatter plot (C). Fisher’s exact test (A) and Mann-Whitney test (C). Data are means ± SEM.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/531/eaay0289/DC1

    Fig. S1. FLNA expression is increased in cortices of patients with FCDII.

    Fig. S2. FLNA expression is increased in cytomegalic cortical neurons of patients with FCDII.

    Fig. S3. Original immunoblots for FLNA in human FCDII samples.

    Fig. S4. The quantity of FLNA is not correlated to the type of FCDII or age of seizure onset or epilepsy duration.

    Fig. S5. Knocking down FLNA does not affect the degree of S6 phosphorylation.

    Fig. S6. Delineation of layer 2/3 and layer 5 neurons using ER81 immunostaining.

    Fig. S7. Single-cell labeling for dendrite (Sholl) analysis.

    Fig. S8. Original immunoblots.

    Table S1. Information for patients with FCDII.

    Table S2. Information for control patients.

    Table S3. Summary of statistical tests.

    Table S4. Plasmids.

    Table S5. Primary and secondary antibodies.

    Movie S1. A Racine grade 4 to 5 seizure in a RhebCA-expressing mouse.

    Data file S1. Individual-level data for Figs. 1 to 6 and figs. S1, S4, and S5 (provided as separate Excel file).

  • The PDF file includes:

    • Fig. S1. FLNA expression is increased in cortices of patients with FCDII.
    • Fig. S2. FLNA expression is increased in cytomegalic cortical neurons of patients with FCDII.
    • Fig. S3. Original immunoblots for FLNA in human FCDII samples.
    • Fig. S4. The quantity of FLNA is not correlated to the type of FCDII or age of seizure onset or epilepsy duration.
    • Fig. S5. Knocking down FLNA does not affect the degree of S6 phosphorylation.
    • Fig. S6. Delineation of layer 2/3 and layer 5 neurons using ER81 immunostaining.
    • Fig. S7. Single-cell labeling for dendrite (Sholl) analysis.
    • Fig. S8. Original immunoblots.
    • Table S1. Information for patients with FCDII.
    • Table S2. Information for control patients.
    • Table S3. Summary of statistical tests.
    • Table S4. Plasmids.
    • Table S5. Primary and secondary antibodies.
    • Legend for movie S1
    • Legend for data file S1

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). A Racine grade 4 to 5 seizure in a RhebCA-expressing mouse.
    • Data file S1. Individual-level data for Figs. 1 to 6 and figs. S1, S4, and S5 (provided as separate Excel file).

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