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. 2015 Apr;56(4):636-46.
doi: 10.1111/epi.12946. Epub 2015 Mar 6.

mTOR inhibition suppresses established epilepsy in a mouse model of cortical dysplasia

Lena H Nguyen  1 , Amy L Brewster, Madeline E Clark, Angelique Regnier-Golanov, C Nicole Sunnen, Vinit V Patil, Gabriella D'Arcangelo, Anne E Anderson
Affiliations

mTOR inhibition suppresses established epilepsy in a mouse model of cortical dysplasia

Lena H Nguyen et al. Epilepsia. 2015 Apr.

Abstract

Objective: Hyperactivation of the mechanistic target of rapamycin (mTOR; also known as mammalian target of rapamycin) pathway has been demonstrated in human cortical dysplasia (CD) as well as in animal models of epilepsy. Although inhibition of mTOR signaling early in epileptogenesis suppressed epileptiform activity in the neuron subset-specific Pten knockout (NS-Pten KO) mouse model of CD, the effects of mTOR inhibition after epilepsy is fully established were not previously examined in this model. Here, we investigated whether mTOR inhibition suppresses epileptiform activity and other neuropathological correlates in adult NS-Pten KO mice with severe and well-established epilepsy.

Methods: The progression of epileptiform activity, mTOR pathway dysregulation, and associated neuropathology with age in NS-Pten KO mice were evaluated using video-electroencephalography (EEG) recordings, Western blotting, and immunohistochemistry. A cohort of NS-Pten KO mice was treated with the mTOR inhibitor rapamycin (10 mg/kg i.p., 5 days/week) starting at postnatal week 9 and video-EEG monitored for epileptiform activity. Western blotting and immunohistochemistry were performed to evaluate the effects of rapamycin on the associated pathology.

Results: Epileptiform activity worsened with age in NS-Pten KO mice, with parallel increases in the extent of hippocampal mTOR complex 1 and 2 (mTORC1 and mTORC2, respectively) dysregulation and progressive astrogliosis and microgliosis. Rapamycin treatment suppressed epileptiform activity, improved baseline EEG activity, and increased survival in severely epileptic NS-Pten KO mice. At the molecular level, rapamycin treatment was associated with a reduction in both mTORC1 and mTORC2 signaling and decreased astrogliosis and microgliosis.

Significance: These findings reveal a wide temporal window for successful therapeutic intervention with rapamycin in the NS-Pten KO mouse model, and they support mTOR inhibition as a candidate therapy for established, late-stage epilepsy associated with CD and genetic dysregulation of the mTOR pathway.

Keywords: Astrogliosis; Malformation of cortical development; Microgliosis; Rapamycin; Seizures.

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Conflict of interest statement

Disclosure

None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

Figures

Figure 1
Figure 1. Epilepsy progressively worsens with age in NS-Pten KO mice
(A) Representative EEG traces from 9 week-old WT and 4, 6, and 9 week-old NS-Pten KO mice are shown. NS-Pten KO mice exhibit progressively worsening epileptiform activity with age, while no abnormal EEG activity was observed in WT mice at any age. (B) Quantification of EEG activity in NS-Pten KO mice revealed a significant increase in the time animals spent in epileptiform activity at postnatal week 9 compared to postnatal week 4. n=12–14 mice/group; ***p<0.001 by Kruskal-Wallis ANOVA with Dunn’s post-hoc test; error bars = ± SEM. (C) Compressed EEG traces from a 90-min epoch of a recording session illustrating normal background EEG activity in WT mice and continuous subclinical polyspike and seizure activity in NS-Pten KO mice at late pathological stages are shown. (D) Representative EEG activity during a motor seizure that was associated with myoclonic jerks, tail and forelimb tonus-clonus, and loss of postural control is shown. Motor seizures were superimposed upon the continuous subclinical polyspike and seizure activity and were typically associated with a characteristic increase in spike frequency and amplitude that progressed into spike-and wave patterns, followed by post-ictal depression of EEG activity. (E) The frequency of motor seizures was significantly higher during postnatal weeks 8–11 compared to postnatal weeks 3–6. n=26–34 mice/group; *p=0.045 by Mann Whitney test; error bars = ± SEM.
Figure 2
Figure 2. Aberrant mTORC1 and mTORC2 signaling increases with age in NS-Pten KO mice
(A, B) Representative western blots and quantification of (A) p-S6 and (B) p-AKT protein levels in whole hippocampal tissue from 2, 4, 6, and 8–9 week-old NS-Pten KO and WT mice are shown. (A) p-S6 levels were significantly higher in NS-Pten KO compared to age-matched WT mice at postnatal weeks 6 and 8–9. Note that at postnatal week 2, both NS-Pten KO and WT mice displayed significantly higher levels of p-S6 compared to 8–9 week-old WT mice. (B) p-AKT levels were significantly higher in NS-Pten KO compared to age-matched WT mice at postnatal weeks 4, 6, and 8–9. (A, B) n= 6–15 mice/group; *p<0.05, ***p<0.001 (compared to age-matched WT), ‡p<0.05, ‡‡‡p<0.001 (compared to 8–9 week-old WT) by two-way ANOVA with Bonferroni post-hoc test; error bars = ± SEM. (C, D) Single confocal images of (C) p-S6- or (D) p-AKT-stained coronal sections of 6 week-old NS-Pten KO and WT hippocampi are shown. High magnifications of DG gcl showing co-labeling with NeuN are presented in the right panels. (C) p-S6 staining appeared more intense within DG gcl with weaker labeling in the CA3 area in NS-Pten KO compared to WT mice. No visible differences were found in the CA1 area between the two groups. (D) p-AKT staining appeared more intense within both DG gcl and DG ml with weaker labeling in the CA3 area in NS-Pten KO compared to WT mice. No visible differences were found in the CA1 area between the two groups. (C, D) n=3 mice/group. Abbreviations: DG, dentate gyrus; gcl, granule cell layer; ml, molecular layer. Dotted lines outline the hippocampal fissure.
Figure 3
Figure 3. Glial markers increase with age in NS-Pten KO mice
(A, B) Representative western blots and quantification of (A) GFAP and (B) IBA1 protein levels in whole hippocampal tissue from 2, 4, 6, and 8–9 week-old NS-Pten KO and WT mice are shown. (A) GFAP levels were significantly higher in NS-Pten KO compared to age-matched WT mice at postnatal weeks 6 and 8–9. (B) IBA1 levels were significantly higher in NS-Pten KO compared to age-matched WT mice at postnatal weeks 8–9. (A, B) n=6–15 mice/group; *p<0.05, ***p<0.001 by two-way ANOVA with Bonferroni post-hoc test; error bars = ± SEM. Black vertical line on the blot denotes gap in loading order; all bands are from the same gel and exposure time. (C, D) Photomicrographs of (C) GFAP- or (D) IBA1-stained coronal sections of 7–9 week-old NS-Pten KO and WT hippocampi are shown. Nissl-stained cell nuclei are shown in purple. High magnifications of the DG, CA1, and CA3 areas are shown in the right panels. Staining for (C) GFAP and (D) IBA1 was more intense in NS-Pten KO compared to WT mice. Several of the IBA1-stained microglia in NS-Pten KO hippocampi were hypertrophied and ameboid compared to normal appearing, ramified microglia observed in WT mice (insets). (C, D) n= 2–4 mice/group (2 WT, 3–4 KO).
Figure 4
Figure 4. Rapamycin treatment suppresses epileptiform activity and improves baseline EEG activity in severely epileptic NS-Pten KO mice
(A) Timeline for video-EEG monitoring and rapamycin treatment. Mice were monitored weekly with video-EEG recordings between postnatal weeks 8 and 13. Rapamycin was administered at postnatal week 9 until death. All EEG analyses were performed at postnatal week 12–13, after 3–4 weeks of treatment, except for motor seizure frequency, which was evaluated during the entire period between postnatal weeks 9–13. (B) Representative EEG traces illustrating normal activity in control and rapamycin-treated WT mice, epileptiform activity in control NS-Pten KO mice, and attenuated epileptiform activity in rapamycin-treated NS-Pten KO mice are shown. (C) Time spent in epileptiform activity was significantly reduced in rapamycin-treated NS-Pten KO mice compared to control NS-Pten KO mice. n=5–6 mice/group; **p=0.008 by Mann-Whitney test. (D) Within-subject comparisons between postnatal week 8, before treatment, and postnatal weeks 12–13, after 3–4 weeks of treatment, revealed a significant decrease in epileptiform activity following rapamycin treatment in NS-Pten KO mice. No changes were observed in control NS-Pten KO mice. Data are presented as percentages of postnatal week 8. n=5–6 mice/group; **p=0.0043 (compared to age-matched WT) by Mann-Whitney test, ‡‡p=0.005 (compared to 100% at postnatal week 8) by one sample t-test. (E) Spike frequency was significantly decreased in rapamycin-treated NS-Pten KO mice compared to control NS-Pten KO mice. n=5–6 mice/group; **p=0.0043 by Mann-Whitney test. (F) Motor seizure frequency was significantly decreased in rapamycin-treated NS-Pten KO mice compared to control NS-Pten KO mice. n=6–11 mice/group; *p=0.026 by Mann-Whitney test. (G) Total power was significantly increased in control NS-Pten KO compared to control WT mice. This aberrant increase was significantly suppressed in the rapamycin-treated NS-Pten KO mice. n= 4–6 mice/group; *p<0.05 by one-way ANOVA with Tukey’s post-hoc test. For all graphs, error bars = ± SEM.
Figure 5
Figure 5. Rapamycin treatment increases survival in severely epileptic NS-Pten KO mice
Kaplan-Meier survival curves for control and rapamycin-treated NS-Pten KO mice are shown. 86% of the rapamycin-treated NS-Pten KO mice (6 out of 7 mice) lived to postnatal week 16 compared to 33% of the control NS-Pten KO mice (4 out of 12 mice). Data from control and rapamycin-treated WT mice are plotted for comparison; all WT mice lived beyond postnatal week 16. n=3–12 mice per group; *p=0.034 by Kaplan-Meier log-rank test.
Figure 6
Figure 6. Rapamycin treatment suppresses mTORC1 and mTORC2 signaling and glial markers in severely epileptic NS-Pten KO mice
(A–D) Representative western blots and quantification of (A) p-S6, (B) p-AKT, (C) GFAP, and (D) IBA1 protein levels in whole hippocampal tissue from 11 week-old control and rapamycin-treated NS-Pten KO and WT mice are shown. (A) p-S6 levels were significantly higher in NS-Pten KO compared to WT mice in the control group. Rapamycin treatment suppressed p-S6 levels below control WT levels in both NS-Pten KO and WT mice. (B) p-AKT levels were significantly higher in NS-Pten KO compared to WT mice in the control group. Rapamycin treatment reduced p-AKT levels in NS-Pten KO mice to control WT levels but did not affect p-AKT levels in WT mice. (C) GFAP levels were significantly higher in NS-Pten KO compared to WT mice in the control group. Rapamycin treatment decreased GFAP levels in NS-Pten KO mice, however, the levels still remained higher than that of control WT mice. (D) IBA1 levels were significantly higher in NS-Pten KO compared to WT mice in the control group. Rapamycin treatment reduced IBA1 levels in NS-Pten KO mice to control WT levels. n=7–14 mice/group; *p<0.05, **p<0.01, ***p<0.001 (compared to control WT unless otherwise noted by connecting lines) by one-way ANOVA with Tukey’s post-hoc test; error bars = ± SEM.
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