4E-BP2-dependent translation in parvalbumin neurons controls epileptic seizure threshold

doi:10.1073/pnas.2025522118

. 2021 Apr 13;118(15):e2025522118.

doi: 10.1073/pnas.2025522118.

4E-BP2-dependent translation in parvalbumin neurons controls epileptic seizure threshold

Vijendra Sharma^{1

2}, Rapita Sood^{3

2}, Danning Lou^{3

2}, Tzu-Yu Hung^{3

2}, Maxime Lévesque⁴, Yelin Han^{3

2}, Jeremy Y Levett^{3

2}, Peng Wang^{3

2}, Shravan Murthy^{3

2}, Shannon Tansley⁵, Siyan Wang⁴, Nadeem Siddiqui^{3

2}, Soroush Tahmasebi⁶, Kobi Rosenblum⁷, Massimo Avoli^{4

8}, Jean-Claude Lacaille⁹, Nahum Sonenberg^{1

2}, Arkady Khoutorsky¹⁰

Affiliations

¹ Department of Biochemistry, McGill University, Montreal, QC H3A 2B4, Canada; vijendra.sharma@mcgill.ca nahum.sonenberg@mcgill.ca arkady.khoutorsky@mcgill.ca.
² Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, QC H3A 2B4, Canada.
³ Department of Biochemistry, McGill University, Montreal, QC H3A 2B4, Canada.
⁴ Montreal Neurological Institute and Hospital, Department of Neurology & Neurosurgery, McGill University, Montreal, QC H3A 2B4, Canada.
⁵ Department of Anesthesia and Faculty of Dentistry, McGill University, Montreal, QC H3A 2B4, Canada.
⁶ Department of Pharmacology and Regenerative Medicine, University of Illinois at Chicago, Chicago, IL 60612.
⁷ Sagol Department of Neurobiology, University of Haifa, Mount Carmel, Haifa, 3498838, Israel.
⁸ Montreal Neurological Institute and Hospital, Department of Physiology, McGill University, Montreal, QC H3A 2B4, Canada.
⁹ Department of Neurosciences, Centre for Interdisciplinary Research on Brain and Learning, Research Group on the Central Nervous System, Université de Montréal, Montréal, QC H3C 3J7, Canada.
¹⁰ Department of Anesthesia and Faculty of Dentistry, McGill University, Montreal, QC H3A 2B4, Canada; vijendra.sharma@mcgill.ca nahum.sonenberg@mcgill.ca arkady.khoutorsky@mcgill.ca.

PMID: 33876772
PMCID: PMC8053921
DOI: 10.1073/pnas.2025522118

4E-BP2-dependent translation in parvalbumin neurons controls epileptic seizure threshold

Vijendra Sharma et al. Proc Natl Acad Sci U S A. 2021.

. 2021 Apr 13;118(15):e2025522118.

doi: 10.1073/pnas.2025522118.

Authors

Affiliations

¹ Department of Biochemistry, McGill University, Montreal, QC H3A 2B4, Canada; vijendra.sharma@mcgill.ca nahum.sonenberg@mcgill.ca arkady.khoutorsky@mcgill.ca.
² Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, QC H3A 2B4, Canada.
³ Department of Biochemistry, McGill University, Montreal, QC H3A 2B4, Canada.
⁴ Montreal Neurological Institute and Hospital, Department of Neurology & Neurosurgery, McGill University, Montreal, QC H3A 2B4, Canada.
⁵ Department of Anesthesia and Faculty of Dentistry, McGill University, Montreal, QC H3A 2B4, Canada.
⁶ Department of Pharmacology and Regenerative Medicine, University of Illinois at Chicago, Chicago, IL 60612.
⁷ Sagol Department of Neurobiology, University of Haifa, Mount Carmel, Haifa, 3498838, Israel.
⁸ Montreal Neurological Institute and Hospital, Department of Physiology, McGill University, Montreal, QC H3A 2B4, Canada.
⁹ Department of Neurosciences, Centre for Interdisciplinary Research on Brain and Learning, Research Group on the Central Nervous System, Université de Montréal, Montréal, QC H3C 3J7, Canada.
¹⁰ Department of Anesthesia and Faculty of Dentistry, McGill University, Montreal, QC H3A 2B4, Canada; vijendra.sharma@mcgill.ca nahum.sonenberg@mcgill.ca arkady.khoutorsky@mcgill.ca.

PMID: 33876772
PMCID: PMC8053921
DOI: 10.1073/pnas.2025522118

Abstract

The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) integrates multiple signals to regulate critical cellular processes such as mRNA translation, lipid biogenesis, and autophagy. Germline and somatic mutations in mTOR and genes upstream of mTORC1, such as PTEN, TSC1/2, AKT3, PIK3CA, and components of GATOR1 and KICSTOR complexes, are associated with various epileptic disorders. Increased mTORC1 activity is linked to the pathophysiology of epilepsy in both humans and animal models, and mTORC1 inhibition suppresses epileptogenesis in humans with tuberous sclerosis and animal models with elevated mTORC1 activity. However, the role of mTORC1-dependent translation and the neuronal cell types mediating the effect of enhanced mTORC1 activity in seizures remain unknown. The eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and 2 (4E-BP2) are translational repressors downstream of mTORC1. Here we show that the ablation of 4E-BP2, but not 4E-BP1, in mice increases the sensitivity to pentylenetetrazole (PTZ)- and kainic acid (KA)-induced seizures. We demonstrate that the deletion of 4E-BP2 in inhibitory, but not excitatory neurons, causes an increase in the susceptibility to PTZ-induced seizures. Moreover, mice lacking 4E-BP2 in parvalbumin, but not somatostatin or VIP inhibitory neurons exhibit a lowered threshold for seizure induction and reduced number of parvalbumin neurons. A mouse model harboring a human PIK3CA mutation that enhances the activity of the PI3K-AKT pathway (Pik3ca^H1047R-Pvalb ) selectively in parvalbumin neurons shows susceptibility to PTZ-induced seizures. Our data identify 4E-BP2 as a regulator of epileptogenesis and highlight the central role of increased mTORC1-dependent translation in parvalbumin neurons in the pathophysiology of epilepsy.

Keywords: epilepsy; mRNA translation; mTORC1.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interest.

Figures

**Fig. 1.**
Deletion of *Eif4ebp1/2/3* and *Eif4ebp2* alters susceptibility to PTZ-induced seizures. (A) Representative Western blot of hippocampal tissue from *Eif4ebp1/2/3* triple knockout (*Eif4ebp*^−/−/−) mice showing ablation of 4E-BP1 and 4E-BP2 (4E-BP3 was not detected in the brain). (B) Timeline of PTZ injection and videorecorded observations. Seizure incidence was measured by scoring seizure behaviors in mice. (C–E) *Eif4ebp*^−/−/− mice were injected with different concentrations of PTZ (50, 60, and 70 mg/kg s.c.), and the percentage of mice that survived or died 30 min post-PTZ injection was quantified. (F) Latency to the first PTZ-induced tonic-clonic seizure at different PTZ concentrations (mixed-effects model [REML]; F_1.55 = 6.426, P = 0.0141). The latency to the first seizure (onset) in response to 70 mg/kg of PTZ was significantly shorter in *Eif4ebp*^−/−/− mice than the wild-type control (Sidak's multiple comparisons test, P = 0.0006, n = 12, 12). (G) Cumulative percentage duration of seizures in *Eif4ebp*^−/−/− and wild-type mice after s.c. injection of different concentrations of PTZ (mixed-effects model [REML]; F_1.55 = 15.89, P = 0.0002). Seizures were recorded for 30 min after PTZ injection. Cumulative seizure percentage duration in response to 70 mg/kg PTZ injection is longer in *Eif4ebp*^−/−/− mice than in wild-type mice (Sidak's multiple comparisons test, P = 0.001, n = 12, 12). (H) Immunoblots of hippocampal protein lysate from *Eif4ebp1* knockout mice showing 4E-BP1 ablation. (I–K) *Eif4ebp1* knockout (*Eif4ebp1*^−/−) mice have similar mortality, seizure onset (t_16.69 = 1.186, P = 0.252, n = 10, 12) and percentage of seizures (t_19.35 = 1. 594, P = 0.127, n = 10, 12) for PTZ-induced seizure as control. (L) Immunoblot of hippocampal lysates from *Eif4ebp2* knockout (*Eif4ebp2*^−/−) mice shows the deletion of 4E-BP2. (M–O) The dose of 70 mg/kg of PTZ caused more mortality, reduced seizure onset (t_17.54 = 4.674, P = 2.02 × 10⁻⁴, n = 13, 18), and increased the percentage duration of seizures (t_24.37 = 3.915, P = 6 × 10⁻⁴, n = 13, 17) in *Eif4ebp2*^−/− mice. A two-tailed unpaired t test with Welch's correction was used to compare two groups. Data are mean ± SEM.

**Fig. 2.**
Global ablation of 4E-BP2 (*Eif4ebp2* KO) and inhibitory neuron-specific ablation of 4E-BP2 (cKO^Nkx2.1 and cKO^Gad2) increase seizure susceptibility. (A) Immunofluorescent labeling confirms the depletion of 4E-BP2 in *Eif4ebp2* knockout (*Eif4ebp2*^−/−) mice (t_9.985 = 12.24, P < 0.0001, n = 6, 6). (B–D) KA-induced dose-dependent induction of epileptiform activity was not lethal but ascribe early-onset (REML; F_1,16 = 8.866, P = 8.9 × 10⁻³; Sidak's multiple comparisons test, P = 0.034, n = 9, 9) and increased percentage duration of seizure events in *Eif4ebp2*^−/− mice (REML; F_1,44 = 6.098, P = 0.0175; Sidak's test, P = 0.046, n = 9, 9). (E) The genetic strategy for the cell type-specific deletion of 4E-BP2. (F–I) Selective ablation of 4E-BP2 in EMX1-expressing excitatory neurons (cKO^Emx1; t_6.239 = 9.023, P = 8.3 × 10⁻⁵, n = 5, 6) does not alter PTZ-induced mortality, the latency to the first seizure (t_29.65 = 0.275, P = 0.785, n = 17, 15), and duration of convulsive behavior (t_26.01 = 1.154, P = 0.259, n = 17, 15) as compared with control mice. (J–M) Deletion of 4E-BP2 in CAMK2α-expressing excitatory neurons (cKO^Camk2α; t_9.924 = 10.30, P = 1.3 × 10⁻⁶, n = 6, 6) does not change the PTZ-induced mortality, latency to the first seizure (t_23.77 = 0.623, P = 0.54, n = 17, 15), and total duration of seizures (t_19.22 = 0.716, P = 0.483, n = 17, 15). (N–Q) Ablation of 4E-BP2 in NKX2.1-expressing inhibitory neurons (cKO^Nkx2.1; t_14.2 = 12.27, P < 0.0001, n = 12, 14) increases PTZ-induced mortality, reduces latency to the first seizure (t_13.84 = 4.82, P = 2.82 × 10⁻⁴, n = 10, 11), and increases the duration of epileptic seizures (t_14.09 = 3.68, P = 2.48 × 10⁻³, n = 10, 11) as compared with control mice. (R–U) Deletion of 4E-BP2 in GAD2-expressing inhibitory neurons (cKO^Gad2; t_7.597 = 7.782, P = 7 × 10⁻⁵, n = 6, 5) enhances the susceptibility to PTZ-induced seizure onset (t_15.03 = 4.168, P = 8.2 × 10⁻⁴, n = 13, 14) and prolongs the duration of generalized tonic-clonic seizures (t_20.28 = 3.671, P = 1.5 × 10⁻³, n = 13, 14). (Scale bar, 20 μm.) The pyramidal cell layer (stratum pyramidale) of the hippocampal CA1 region was used for the immunofluorescence imaging and quantification of A, F, J, N, and R. A two-tailed unpaired t test with Welch’s correction was used to compare two groups. Data are mean ± SEM.

**Fig. 3.**
*Eif4ebp2*^Pvalb mice exhibit increased PTZ-induced epileptic behavior and electrographic seizures. (A–D) Immunofluorescent staining shows reduced 4E-BP2 in somatostatin-expressing neurons (t_9.532 = 5.011, P = 6.12 × 10⁻⁴, n = 6, 7). The threshold for PTZ (70 mg/kg)-induced behavioral seizure remains unaltered in *Eif4ebp2*^flx/flx;Sst-Cre⁺ (cKO^Sst) mice. Quantification of mortality, seizure onset (t_16.88 = 0.567, P = 0.578, n = 8, 11) and percentage of seizures (t_15.78 = 0.167, P = 0.87, n = 8, 11) shows similar results as compared with control mice (B; Dead [Black] and survived [White]). (E) The *Eif4ebp2*^flx/flx;VIP-Cre⁺ (cKO^VIP) mice hippocampus was injected with AAV9-EF1α-DIO-EYFP-WPRE-hGH to label Cre-positive VIP neurons in CA1 of dorsal hippocampus. Immunofluorescent staining shows reduced 4E-BP2 in eGFP-expressing VIP neurons (t_6.212 = 6.599, P = 5.04 × 10⁻⁴, n = 5, 5). (F–H) In cKO^VIP mice, the mortality rate, seizure onset (t_12.31 = 0.095, P = 0.926, n = 8, 10), and cumulative seizure duration in response to 70 mg/kg of PTZ are similar to control (t_15.99 = 0.191, P = 0.851, n = 8, 10). (I–L) In *Eif4ebp2*^flx/flx;Pvalb-Cre⁺ (cKO^Pvalb) mice, injection of 70 mg/kg of PTZ resulted in higher mortality and significantly reduced seizure threshold as seen in onset (t_14.64 = 3.99, P = 1.24 × 10⁻³, n = 10, 12) and percentage duration of seizures (t_17.02 = 3.74, P = 1.6 × 10⁻³, n = 10, 12). (M–P) In cKO^Pvalb mice, a lower dose of PTZ (50 mg/kg) induced more EEG seizure activity as measured by reduced onset (t_13.17 = 5.29, P = 1.4 × 10⁻⁴, n = 7, 9) and increased percentage duration of electrographic seizures (t_9.27 = 2.61, P = 0.0277, n = 7, 9). Representative EEG traces of seizure in cKO^Pvalb and control mice. (Scale bar, 20 μm.) The stratum oriens layer (A and E) and the pyramidal cell layer (I) of the hippocampal CA1 region were used for the immunofluorescence imaging and quantification. A two-tailed unpaired t test with Welch's correction was used to compare two groups. Data are mean ± SEM.

**Fig. 4.**
*Eif4ebp2*^Pvalb mice exhibit increased sensitivity to kainic acid and reduced number of parvalbumin-positive neurons and WFA/PNNs ratio in the hippocampus. (A–C) Administration of 30 mg/kg KA significantly reduces the behavioral seizure latency (t_7.224 = 2.413, P = 0.0455, n = 8, 9) and increases seizure events in cKO^Pvalb mice (t_12.14 = 2.67, P = 0.02, n = 8, 9). (D) Representative hippocampus sections of control and cKO^Pvalb mice. (Scale bar, 200 μm.) The cKO^Pvalb mice have a significantly lower percentage of PVALB⁺ neurons in the hippocampus than the control mice (t_12.93 = 2.261, P = 0.042, n = 7, 8). (E) The parvalbumin expression levels are identical in the control and cKO^Pvalb mice hippocampus (t_12.92 = 1.583, P = 0.138, n = 7, 8). (F) A reduced percentage of PVALB⁺ neurons also decreased the density of the PNNs surrounding PVALB⁺ neurons in the hippocampus of cKO^Pvalb mice (t_11.23 = 2.611, P = 0.0239, n = 7, 8). (Scale bar, 50 μm.) (G) However, the PNNs/PVALB⁺ ratio remains unchanged between the hippocampus of control and cKO^Pvalb mice (t_12.58 = 0.967, P = 0.352, n = 7, 8). (H) In cKO^Pvalb mice, PNNs show stronger WFA⁺ signal as compared with control mice (t_12.84 = 2.9, P = 0.0126, n = 7, 8). (Scale bar, 20 μm.) The pyramidal layer of the hippocampal CA1 region was used for the immunofluorescence imaging and quantification of E and H. A two-tailed unpaired t test with Welch's correction was used to compare two groups. Data are mean ± SEM.

**Fig. 5.**
An activating mutation of human *Pik3ca* in PVALB⁺ neurons is sufficient to lower the PTZ-induced seizure threshold in mice. (A) The genetic schematic of the conditional *Pik3ca*^H1047R mutation in PVALB⁺ neurons. (B–E) Immunofluorescent staining shows increased p-AKT Thr308 (t_10.64 = 2.653, P = 0.023, n = 6, 7) and p-S6K Thr389 (t_8.081 = 2.58, P = 0.032, n = 6, 7) as a result of a conditional *Pik3ca*^H1047R mutation in PVALB⁺ neurons. (F–H) The increased PI3K activity in *Pik3ca*^H1047R-Pvalb is sufficient to increase PTZ-induced seizures. The mortality was increased, the onset of seizures was significantly reduced (t_25.07 = 2.185, P = 0.0384, n = 13, 19), and percentage duration of seizure was significantly increased (t_28.80 = 2.726, P = 0.0108, n = 13, 19) in *Pik3ca*^H1047R-Pvalb as compared with WT^Pvalb control mice following the 70 mg/kg PTZ injection. (Scale bar, 20 μm.) The pyramidal layer of the hippocampal CA1 region was used for the immunofluorescence imaging and quantification of B and D. A two-tailed unpaired t test with Welch's correction was used to compare two groups. Data are mean ± SEM.

See this image and copyright information in PMC

Cited by

DEPDC5-dependent mTORC1 signaling mechanisms are critical for the anti-seizure effects of acute fasting.
Yuskaitis CJ, Modasia JB, Schrötter S, Rossitto LA, Groff KJ, Morici C, Mithal DS, Chakrabarty RP, Chandel NS, Manning BD, Sahin M. Yuskaitis CJ, et al. Cell Rep. 2022 Aug 30;40(9):111278. doi: 10.1016/j.celrep.2022.111278. Cell Rep. 2022. PMID: 36044864 Free PMC article.
mTORC1 function in hippocampal parvalbumin interneurons: regulation of firing and long-term potentiation of intrinsic excitability but not long-term contextual fear memory and context discrimination.
Khlaifia A, Honoré E, Artinian J, Laplante I, Lacaille JC. Khlaifia A, et al. Mol Brain. 2022 Jun 17;15(1):56. doi: 10.1186/s13041-022-00941-8. Mol Brain. 2022. PMID: 35715811 Free PMC article.
PARVing the Way to Cap Translation for Seizure Control.
Gross C. Gross C. Epilepsy Curr. 2021 Jun 27;21(5):360-362. doi: 10.1177/15357597211027010. eCollection 2021 Oct. Epilepsy Curr. 2021. PMID: 34924836 Free PMC article. No abstract available.
Parvalbumin Role in Epilepsy and Psychiatric Comorbidities: From Mechanism to Intervention.
Godoy LD, Prizon T, Rossignoli MT, Leite JP, Liberato JL. Godoy LD, et al. Front Integr Neurosci. 2022 Feb 17;16:765324. doi: 10.3389/fnint.2022.765324. eCollection 2022. Front Integr Neurosci. 2022. PMID: 35250498 Free PMC article. Review.
Expression of 4E-BP1 in juvenile mice alleviates mTOR-induced neuronal dysfunction and epilepsy.
Nguyen LH, Xu Y, Mahadeo T, Zhang L, Lin TV, Born HA, Anderson AE, Bordey A. Nguyen LH, et al. Brain. 2022 May 24;145(4):1310-1325. doi: 10.1093/brain/awab390. Brain. 2022. PMID: 34849602 Free PMC article.

See all "Cited by" articles

References

1. Theodore W. H., et al. ., Epilepsy in North America: A report prepared under the auspices of the global campaign against epilepsy, the International Bureau for epilepsy, the international league against epilepsy, and the World Health organization. Epilepsia 47, 1700–1722 (2006). - PubMed
1. Devinsky O., et al. ., Epilepsy. Nat. Rev. Dis. Primers 4, 18024 (2018). - PubMed
1. Mula M., Sander J. W., Psychosocial aspects of epilepsy: A wider approach. BJPsych Open 2, 270–274 (2016). - PMC - PubMed
1. Beghi E., Social functions and socioeconomic vulnerability in epilepsy. Epilepsy Behav. 100, 106363 (2019). - PubMed
1. Kim J. K., et al. ., Brain somatic mutations in MTOR reveal translational dysregulations underlying intractable focal epilepsy. J. Clin. Invest. 129, 4207–4223 (2019). - PMC - PubMed

Publication types

Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Substances

Actions
Actions
Actions
Actions
Actions
Actions

Grants and funding

LinkOut - more resources

Full Text Sources
Other Literature Sources
- scite Smart Citations
Medical
- MedlinePlus Consumer Health Information
- MedlinePlus Health Information
Molecular Biology Databases
- Mouse Genome Informatics (MGI)
Research Materials
- Addgene Non-profit plasmid repository
- NCI CPTC Antibody Characterization Program
Miscellaneous
- NCI CPTAC Assay Portal

[1] Theodore W. H., et al. ., Epilepsy in North America: A report prepared under the auspices of the global campaign against epilepsy, the International Bureau for epilepsy, the international league against epilepsy, and the World Health organization. Epilepsia 47, 1700–1722 (2006). - PubMed

[2] Theodore W. H., et al. ., Epilepsy in North America: A report prepared under the auspices of the global campaign against epilepsy, the International Bureau for epilepsy, the international league against epilepsy, and the World Health organization. Epilepsia 47, 1700–1722 (2006). - PubMed

[3] Devinsky O., et al. ., Epilepsy. Nat. Rev. Dis. Primers 4, 18024 (2018). - PubMed

[4] Devinsky O., et al. ., Epilepsy. Nat. Rev. Dis. Primers 4, 18024 (2018). - PubMed

[5] Mula M., Sander J. W., Psychosocial aspects of epilepsy: A wider approach. BJPsych Open 2, 270–274 (2016). - PMC - PubMed

[6] Mula M., Sander J. W., Psychosocial aspects of epilepsy: A wider approach. BJPsych Open 2, 270–274 (2016). - PMC - PubMed

[7] Beghi E., Social functions and socioeconomic vulnerability in epilepsy. Epilepsy Behav. 100, 106363 (2019). - PubMed

[8] Beghi E., Social functions and socioeconomic vulnerability in epilepsy. Epilepsy Behav. 100, 106363 (2019). - PubMed

[9] Kim J. K., et al. ., Brain somatic mutations in MTOR reveal translational dysregulations underlying intractable focal epilepsy. J. Clin. Invest. 129, 4207–4223 (2019). - PMC - PubMed

[10] Kim J. K., et al. ., Brain somatic mutations in MTOR reveal translational dysregulations underlying intractable focal epilepsy. J. Clin. Invest. 129, 4207–4223 (2019). - PMC - PubMed

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

4E-BP2-dependent translation in parvalbumin neurons controls epileptic seizure threshold

Affiliations

4E-BP2-dependent translation in parvalbumin neurons controls epileptic seizure threshold

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Medical

Molecular Biology Databases

Research Materials

Miscellaneous

Abstract

Conflict of interest statement

Figures

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Related information

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Medical

Molecular Biology Databases

Research Materials

Miscellaneous