N6-methyladenosine (m6A) dysregulation contributes to network excitability in temporal lobe epilepsy
Justine Mathoux, Marc-Michel Wilson, Sujithra Srinivas, Gabrielle Litovskich, Leticia Villalba Benito, Cindy Tran, Jaideep Kesavan, Aileen Harnett, Theresa Auer, Amaya Sanz-Rodriguez, Mohammad Kh. A.E. Alkhayyat, Mairéad Sullivan, Zining Liu, Yifan Huang, Austin Lacey, Norman Delanty, Jane Cryan, Francesca M. Brett, Michael A. Farrell, Donncha F. O’Brien, Pablo M. Casillas-Espinosa, Eva M. Jimenez-Mateos, Jeffrey C. Glennon, Mary Canavan, David C. Henshall, Gary P. Brennan
Justine Mathoux, Marc-Michel Wilson, Sujithra Srinivas, Gabrielle Litovskich, Leticia Villalba Benito, Cindy Tran, Jaideep Kesavan, Aileen Harnett, Theresa Auer, Amaya Sanz-Rodriguez, Mohammad Kh. A.E. Alkhayyat, Mairéad Sullivan, Zining Liu, Yifan Huang, Austin Lacey, Norman Delanty, Jane Cryan, Francesca M. Brett, Michael A. Farrell, Donncha F. O’Brien, Pablo M. Casillas-Espinosa, Eva M. Jimenez-Mateos, Jeffrey C. Glennon, Mary Canavan, David C. Henshall, Gary P. Brennan
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Research Article Cell biology Neuroscience

N6-methyladenosine (m6A) dysregulation contributes to network excitability in temporal lobe epilepsy

Abstract

Analogous to DNA methylation and protein phosphorylation, it is now well understood that RNA is also subject to extensive processing and modification. N6-methyladenosine (m6A) is the most abundant internal RNA modification and regulates RNA fate in several ways, including stability and translational efficiency. The role of m6A in both experimental and human epilepsy remains unknown. Here, we used transcriptome-wide m6A arrays to obtain a detailed analysis of the hippocampal m6A-ome from both mouse and human epilepsy samples. We combined this with human proteomic analyses and show that epileptic tissue displays disrupted metabolic and autophagic pathways that may be directly linked to m6A processing. Specifically, our results suggest that m6A levels inversely correlate with protein pathway activation. Finally, we show that elevated levels of m6A decrease seizure susceptibility and severity in mice. Together, our findings indicate that m6A represents an additional layer of gene regulation complexity in epilepsy and may contribute to the pathomechanisms that drive the development and maintenance of hyperexcitable brain networks.

Authors

Justine Mathoux, Marc-Michel Wilson, Sujithra Srinivas, Gabrielle Litovskich, Leticia Villalba Benito, Cindy Tran, Jaideep Kesavan, Aileen Harnett, Theresa Auer, Amaya Sanz-Rodriguez, Mohammad Kh. A.E. Alkhayyat, Mairéad Sullivan, Zining Liu, Yifan Huang, Austin Lacey, Norman Delanty, Jane Cryan, Francesca M. Brett, Michael A. Farrell, Donncha F. O’Brien, Pablo M. Casillas-Espinosa, Eva M. Jimenez-Mateos, Jeffrey C. Glennon, Mary Canavan, David C. Henshall, Gary P. Brennan

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Figure 9

m6A regulates neuronal structure and network activity.

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m6A regulates neuronal structure and network activity. (A) Schematic of ...
(A) Schematic of experimental approach using primary hippocampal neurons from E18 mouse pups to quantify dendritic spine density in cultures treated with either vehicle or METTL3 inhibitor. (B) Representative immunostaining (original magnification, ×10) of neuronal marker BIII_Tubulin and m6A levels following either control or METTL3 inhibitor treatment. (C) Colorimetric assay quantification of neuronal m6A levels over time following treatment with various concentrations of METTL3 inhibitor. Multiple unpaired 2-tailed t tests for each time point, 10 μM 24 hours t = 7.049, df = 8, P = 0.00032; 48 hours t = 1.7, df = 7, P = 0.25; 72 hours t = 0.55, df = 7, P = 0.59. (D) Representative confocal ICC images of primary hippocampal neurons stained for βIII-tubulin and with phalloidin to visualize dendritic spines (upper panels) and βIII-tubulin and PSD95 in the bottom panels as a postsynaptic density marker. Inset shows zoomed-in image with puncta staining. Scale bars: 20 μm. (E) Dendritic spine quantification characterized by distance from soma binned into 20-μm distances and according to branch order. Distance: 2-way repeated-measures (RM) ANOVA followed by Tukey’s post hoc multiple comparisons 100 μm t = 2.174, df = (62, 55), P = 0.0335; Branch order: 2-way ANOVA no significance. n > 32 dendrites from 22 neurons per group. (F) PSD95 puncta quantification according to distance from soma and by branch order. Distance: 2-way ANOVA 20 μm t = 4.367, df = (53, 91), P = < 0.0001; 100 μm t = 2.269, df = (61, 21), P = 0.027. Branch order: 2-way ANOVA followed by multiple comparison t tests Order 1 t = 2.747, df = (63, 00), P = 0.03; Order 4 t = 3.283, df = (13, 00), P = 0.03. n > 32 dendrites from 22 neurons per group. (G) Experimental design to test whether elevated m6A levels alter seizure threshold in mice. (H) Kaplan-Meier curve depicting survival rates among vehicle- and FB23-2–treated animals following high-dose PTZ seizure challenge. Kaplan-Meier χ2 = 3.4, df = 1, P = 0.063. (I and J) Quantification of the maximum seizure stage reached (I) and total seizure numbers (J) in vehicle- or FB23-2–treated mice following PTZ administration over a 25-minute recording period, as measured using an adapted Racine scale. Seizure stage/number χ2 df = (79.37, 5), P < 0.0001. *P < 0.05; ***P < 0.01; ***P < 0.001; ****P < 0.0001.