Second-hit mosaic mutation in mTORC1 repressor DEPDC5 causes focal cortical dysplasia-associated epilepsy

Abstract

DEP domain-containing 5 protein (DEPDC5) is a repressor of the recently recognized amino acid-sensing branch of the mTORC1 pathway. So far, its function in the brain remains largely unknown. Germline loss-of-function mutations in DEPDC5 have emerged as a major cause of familial refractory focal epilepsies, with case reports of sudden unexpected death in epilepsy (SUDEP). Remarkably, a fraction of patients also develop focal cortical dysplasia (FCD), a neurodevelopmental cortical malformation. We therefore hypothesized that a somatic second-hit mutation arising during brain development may support the focal nature of the dysplasia. Here, using postoperative human tissue, we provide the proof of concept that a biallelic 2-hit - brain somatic and germline - mutational mechanism in DEPDC5 causes focal epilepsy with FCD. We discovered a mutation gradient with a higher rate of mosaicism in the seizure-onset zone than in the surrounding epileptogenic zone. Furthermore, we demonstrate the causality of a Depdc5 brain mosaic inactivation using CRISPR-Cas9 editing and in utero electroporation in a mouse model recapitulating focal epilepsy with FCD and SUDEP-like events. We further unveil a key role of Depdc5 in shaping dendrite and spine morphology of excitatory neurons. This study reveals promising therapeutic avenues for treating drug-resistant focal epilepsies with mTORC1-targeting molecules.

Keywords: Epilepsy; Genetics; Molecular genetics; Neurodevelopment; Neuroscience.

Figure 1. DEPDC5 biallelic mutations in a patient.

(A) Family pedigree. The black square indicates the patient with FCD-associated epilepsy. Sanger sequence chromatograms show the germline nonsense variant (c.856C>T/p.Arg286*, black arrowheads) in the mother and child, and the somatic nonsense variant (c.865C>T/p.Gln289*, red arrowhead) that was only detected in the DNA extracted from the seizure-onset zone (SOZ), and not from the surrounding epileptogenic zone (EZ). (B) Integrative genomics viewer displays mutated bases in red in aligned reads: c.856C>T in blood (51%, 828 of 1,618 reads) and SOZ cortex DNA (48%, 748/1551 reads) and c.865C>T in SOZ cortex DNA only (10%, 148 of 1,505 reads). The 2 variants were systematically observed on different reads, indicating that they are in trans configuration. (C) Immunostaining against pS6 protein (red) and the neuronal marker NeuN (green) of a paraffin-embedded brain section from the patient. DAPI is shown in blue. White arrowheads indicate enlarged pS6⁺ neurons. Scale bar: 50 μm.

Figure 2. Delayed migration and increased mTORC1 activity in brain cortex of Depdc5^fKO mice.

(A) Schema of IUE (in utero electroporation) procedure. (B) Representative coronal sections of control (n = 5), Depdc5^fKO-gRNA1 (n = 6), and Depdc5^fKO-gRNA1 plus rapamycin (n = 6) E18.5 embryo neocortices. DAPI, blue; GFP, green. Bottom: Histogram shows the distribution of electroporated GFP⁺ cells in neocortex. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. ***P < 0.0001, by 2-way ANOVA. Scale bars: 100 μm. (C) Representative coronal sections of control (n = 5), Depdc5^fKO-gRNA1 (n = 5), and Depdc5^fKO-gRNA2 (n = 4) adult brain cortices. DAPI, blue; GFP, green. White arrowheads indicate ectopic neurons in the deeper cortical layers. CC, corpus callosum. Histogram shows the percentage of GFP⁺ cells in cortical layers II/III and IV. ***P = 0.0001, by 2-way-ANOVA. Scale bars: 100 μm. (D) Representative coronal sections of control (n = 5), Depdc5^fKO-gRNA1 (n = 5), and Depdc5^fKO-gRNA2 (n = 4) adult brain over the cortical layer III/IV boundary. GFP, green; pS6, red. Box and whisker plot shows fold changes in pS6 levels and soma size between ipsilateral GFP⁺ cells and contralateral GFP^– cells in control, Depdc5^fKO-gRNA1, and Depdc5^fKO-gRNA2 adult brain cortices. ***P < 0.0001, by 1-way ANOVA (mean fold change in pS6: control = 1.05, Depdc5^fKO-gRNA1 = 2.69, Depdc5^fKO-gRNA2 = 3.82; mean fold change in soma size: control = 1.05, Depdc5^fKO-gRNA1 = 1.8, Depdc5^fKO-gRNA2 = 2.13; n = 30–70 cells per slice). Scale bars: 70 μm. (E) H&E staining of coronal brain sections from control (n = 3), Depdc5^fKO-gRNA1 (n = 5), and Depdc5^fKO-gRNA2 (n = 4) adult mice over cortical layers III/IV. Red arrowheads indicate balloon-like cells. Scale bars: 20 μm.

Figure 3. Epileptic activity of adult Depdc5^fKO mice.

(A) Schematic representation of implanted electrode locations. (B) EEG recordings of a cluster of seizures in a mouse aged P54. Hp, hippocampal; M1, primary motor cortex (R, right; L, left); LPtA, lateral parietal-temporal–associated cortex. Boxes below show different phases of EEG activity or suppression. A flat EEG after the last seizure was associated with the death of the animal.

Figure 4. Morphological and functional changes in Depdc5^fKO neurons.

(A) Neuronal reconstruction of representative control (n = 5) and Depdc5^fKO-gRNA1 (n = 5) recorded neurons. Scale bars: 100 μm. (B) Quantification of branching complexity by Sholl analysis. ***P < 0.0001, by 2-way-ANOVA for group effects. Nb, number. (C) Apical tuft width of control (n = 5) and Depdc5^fKO-gRNA1 (n = 5) neurons. ***P = 0.0079, by Mann-Whitney U test. (D) Representative images of level-2 basal dendrite. Scale bars: 5 μm. (E) Dot plot showing the distribution of spine head widths (μm). **P = 0.0072, by unpaired t test (n = 4 neurons/group; n = 200 spines/neuron). (F) Capacitance (C_m) and (G) input resistance (R_in) for control (n = 11) and Depdc5^fKO (n = 12) neurons. ***P = 0.002, by Mann-Whitney U test (mean C_m: controls = 130.5 pF, Depdc5^fKO-gRNA1 = 238.1 pF; mean R_in: controls = 95.6 MOhm, Depdc5^fKO-gRNA1 = 67.6 MOhm). (H) Left: Firing pattern of control Depdc5^fKO-gRNA1 cells for 250 pA and 300 pA current input, respectively. Right: Mean frequency-current curve. ***P < 0.0001, by 2-way-ANOVA for group effects. (I) Left: Representative traces of sEPSC events over a 1-minute period. Right: Cumulative frequency distribution and dot plot of sEPSC mean amplitude. Mean amplitude: controls (n = 10) = 11.07 ± 0.12 pA; Depdc5^fKO-gRNA1 (n = 8) = 12.1 ± 0.52 pA. *P = 0.0219, by Mann-Whitney U test.