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. 2023 Aug 23:17:1257056.
doi: 10.3389/fnins.2023.1257056. eCollection 2023.

4E-BP1 expression in embryonic postmitotic neurons mitigates mTORC1-induced cortical malformations and behavioral seizure severity but does not prevent epilepsy in mice

Lena H Nguyen  1   2 , Manas Sharma  2 , Angelique Bordey  2
Affiliations

4E-BP1 expression in embryonic postmitotic neurons mitigates mTORC1-induced cortical malformations and behavioral seizure severity but does not prevent epilepsy in mice

Lena H Nguyen et al. Front Neurosci. .

Abstract

Hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway during neurodevelopment leads to focal cortical malformations associated with intractable seizures. Recent evidence suggests that dysregulated cap-dependent translation downstream of mTORC1 contributes to cytoarchitectural abnormalities and seizure activity. Here, we examined whether reducing cap-dependent translation by expressing a constitutively active form of the translational repressor, 4E-BP1, downstream of mTORC1 would prevent the development of cortical malformations and seizures. 4E-BP1CA was expressed embryonically either in radial glia (neural progenitor cells) that generate cortical layer 2/3 pyramidal neurons or in migrating neurons destined to layer 2/3 using a conditional expression system. In both conditions, 4E-BP1CA expression reduced mTORC1-induced neuronal hypertrophy and alleviated cortical mislamination, but a subset of ectopic neurons persisted in the deep layers and the white matter. Despite the above improvements, 4E-BP1CA expression in radial glia had no effects on seizure frequency and further exacerbated behavioral seizure severity associated with mTORC1 hyperactivation. In contrast, conditional 4E-BP1CA expression in migratory neurons mitigated the severity of behavioral seizures but the seizure frequency remained unchanged. These findings advise against targeting 4E-BPs by 4E-BP1CA expression during embryonic development for seizure prevention and suggest the presence of a development-dependent role for 4E-BPs in mTORC1-induced epilepsy.

Keywords: 4E-BP; cortical development; corticogenesis; epilepsy; mTOR; malformation of cortical development; seizures.

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Conflict of interest statement

LN and AB are co-inventors on a patent application, PCT/US2020/054007 entitled “Targeting Cap-Dependent Translation to Reduce Seizures in mTOR disorders”. 2021-04-08: Publication of WO2021067752A1. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
4E-BP1CA expression (by embryonic targeting of radial glia) decreases neuron hypertrophy, improves cortical lamination, and exacerbates behavioral seizure severity in RhebCA mice. (A) Schematic diagram of plasmids used for IUE and experimental timeline. (B) Representative images showing tdTomato+ cells in electroporated cortices in 12-week-old RhebCA + GFP and RhebCA + 4E-BP1CA mice. Scale bar = 25 μm. (C) Quantification of tdTomato+ cell soma size. Each data point represents averaged values from 50 cells/animal. Data were analyzed using unpaired t-test. Gray dotted line shows previously reported levels for adult control mice electroporated with GPF for visual comparison (Nguyen et al., 2019). (D) Representative images showing tdTomato+ cell placement in electroporated cortices in 12-week-old RhebCA + GFP and RhebCA + 4E-BP1CA mice. A defined region of the cortex (purple rectangle, enlarged on the bottom) spanning from the WM border to pia was divided into 10 equal bins, and the % of cells in each bin was quantified to assess cell distribution. Scale bars = 100 μm. (E) Quantification of tdTomato+ cell placement in the cortex. Each data point represents averaged values from 2 brain sections/animal. Data were analyzed using two-way repeated measures ANOVA with Šídák’s post-hoc test. (F) Representative EEG trace showing a typical electrographic seizure in an 11-week-old RhebCA + GFP mouse. Expanded traces from the region indicated by the blue lines are shown at the bottom. (G) Quantification of seizure frequency. Each data point represents mean seizures/day from 7 days per animal. Data were analyzed using Mann–Whitney U test. (H) Quantification of seizure incidence. Data were analyzed using Fisher’s Exact test. (I) Quantification of behavioral seizure severity based on a modified Racine scale. Data were analyzed using Pearson’s Chi-Squared test with accompanying z-tests to compare proportions. (C,E,G–I) n = 6 RhebCA + GFP, 7 RhebCA + 4E-BP1CA mice. *p < 0.05, ***p < 0.001, ****p < 0.0001. Error bars are ± SEM. IUE, in utero electroporation; CTX, cortex; WM, white matter; cb, cingulum bundle; cc, corpus callosum.
Figure 2
Figure 2
c4E-BP1CA expression (by selective embryonic targeting of postmitotic migratory neurons) decreases neuron hypertrophy, improves cortical lamination, and mitigates behavioral seizure severity in RhebCA mice. (A) Schematic diagram of plasmids used for IUE and experimental timeline. (B) Representative images showing GFP+ cells in electroporated cortices in 10-week-old RhebCA + cDsRed and RhebCA + c4E-BP1CA mice. Scale bar = 25 μm. (C) Quantification of GFP+ cell soma size. Each data point represents averaged values from 20 cells/animal. Data were analyzed using unpaired t-test. Gray dotted line shows previously reported levels for adult control mice electroporated with GPF for visual comaprison (Nguyen et al., 2019). (D) Representative images showing GFP+ cell placement in electroporated cortices in 10-week-old RhebCA + cDsRed and RhebCA + c4E-BP1CA mice. A defined region of the cortex (purple rectangle, enlarged on the bottom) spanning from the WM border to pia was divided into 10 equal bins, and the % of cells in each bin was quantified to assess cell distribution. Scale bars = 100 μm. (E) Quantification of GFP+ cell placement in the cortex. Each data point represents averaged values from 2 brain sections/animal. Data were analyzed using two-way repeated measures ANOVA with Šídák’s post-hoc test. (F) Representative EEG trace showing a typical electrographic seizure in a 9-week-old RhebCA + cDsRed mouse. Expanded traces from the region indicated by the blue lines are shown at the bottom. (G) Quantification of seizure frequency. Each data point represents mean seizures/day from 7 days per animal. Data were analyzed using Mann–Whitney U test. (H) Quantification of seizure incidence. Data were analyzed using Fisher’s Exact test. (I) Quantification of behavioral seizure severity based on a modified Racine scale. Data were analyzed using Pearson’s Chi-Squared test with accompanying z-tests to compare proportions. (C,E,G–I) n = 8 RhebCA + cDsRed, 12–13 RhebCA + c4E-BP1CA mice, *p < 0.05, **p < 0.01, ****p < 0.0001. Error bars are ± SEM. IUE, in utero electroporation; CTX, cortex; WM, white matter; cb, cingulum bundle; cc, corpus callosum.
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