Prevention of premature death and seizures in a Depdc5 mouse epilepsy model through inhibition of mTORC1

Abstract

Mutations in DEP domain containing 5 (DEPDC5) are increasingly appreciated as one of the most common causes of inherited focal epilepsy. Epilepsies due to DEPDC5 mutations are often associated with brain malformations, tend to be drug-resistant, and have been linked to an increased risk of sudden unexplained death in epilepsy (SUDEP). Generation of epilepsy models to define mechanisms of epileptogenesis remains vital for future therapies. Here, we describe a novel mouse model of Depdc5 deficiency with a severe epilepsy phenotype, generated by conditional deletion of Depdc5 in dorsal telencephalic neuroprogenitor cells. In contrast to control and heterozygous mice, Depdc5-Emx1-Cre conditional knockout (CKO) mice demonstrated macrocephaly, spontaneous seizures and premature death. Consistent with increased mTORC1 activation, targeted neurons were enlarged and both neurons and astrocytes demonstrated increased S6 phosphorylation. Electrophysiologic characterization of miniature inhibitory post-synaptic currents in excitatory neurons was consistent with impaired post-synaptic response to GABAergic input, suggesting a potential mechanism for neuronal hyperexcitability. mTORC1 inhibition with rapamycin significantly improved survival of CKO animals and prevented observed seizures, including for up to 40 days following rapamycin withdrawal. These data not only support a primary role for mTORC1 hyperactivation in epilepsy following homozygous loss of Depdc5, but also suggest a developmental window for treatment which may have a durable benefit for some time even after withdrawal.

Figure 1

Depdc5^Emx1 CKO mice display changes in gross brain/body weight and demonstrate both seizures and reduced survival. (A) Schematic demonstrating targeting of the Depdc5 allele. Genotyping primers are indicated by red arrows. (B) Representative genotyping of tail DNA as well as DNA from primary neuronal cultures. Wt mice show a 701 bp band, while conditional mice (F) show a larger 981 bp band due to inclusion of the FRT and LoxP sites. Mice with a conditional allele show a 381 bp band representing excisional loss of exon 5 and a LoxP site. Cre + mice are identified using primers targeting Cre. Top image, genotyping from mouse tail DNA. Bottom image, genotyping from cultured primary cortical neurons. (C) Depdc5-Emx1 mice have increased brain weights, are smaller than littermate controls, and cease to gain body weight by P20. CKO mice thus have an increased brain/body weight ratio in contrast littermate controls and heterozygotes. *P < 0.05, two-way ANOVA with Bonferroni’s multiple comparisons. n = 4–7 controls, n = 2–5 heterozygotes, n = 3–9 CKOs. (D) Survival was significantly shortened in CKO animals, with only a single CKO animal surviving past P26 (P < 0.0001 using Log-rank Mantel–Cox test). CKO male: n = 17, median survival = 22 days; CKO female, n = 18, median survival = 23 days; in contrast control (n = 37) and heterozygous (n = 28). Tick marks indicate animals that were censored due to sacrifice for biochemical experiments. (E) CKO animals (n = 9) showed decreased flurothyl-induced seizure threshold in contrast littermate controls (n = 10) and heterozygotes (n = 2). *P < 0.05, **P < 0.01, one-way ANOVA.

Figure 2

Neurohistologic abnormalities in CKO animals. (A) CKO animals display grossly thickened cortices and hippocampi by P5; hematoxylin and eosin (H&E) stain. Scale bar, 1000 μm. (B) CKO animals show diffusely positive pS6 (s240/244) immunofluorescence signal intensity (red) in the cortex and hippocampus. Image location indicated by the black box in panel A. Scale bar, 1000 μm. (C) Quantification of cortical width shows increased cortical thickness in CKO animals. Four separate sites were measured and averaged in at least two sections from n = 3 animals per genotype. *P < 0.05, Student’s t-test. (D and E) CKO cortical neurons (NeuN, green) are larger with more intense pS6 (s240/244) signal (red). ***P < 0.001, Student’s t-test, n ≥ 50 neurons per animal from 7 CKOs and 4 littermate controls. Image location indicated by the green box in (A). Scale bar, 200 μm. (F–G) Representative immunoblot and quantitative analysis of cortical lysates from P17–18 CKO and littermate controls (n = 5 mice per genotype). Expression levels were normalized to respective total protein (for pS6 and pAkt) or total protein by Coomassie (for NPRL2 and GFAP), then shown as percent of the average littermate control expression. β-actin is also shown as a loading control. Groups were compared using unpaired t-tests: pS6 s235/235, P = 0.0006; pS6 s240/244, P = 0.0004; pAkt, P = 0.009; NPRL2, P = 0.0015; GFAP, P = 0.5659. Bars represent mean ± SEM.

Figure 3

Astrocyte cultures from CKO mice display increased mTORC1 activation. (A and B) Semi-quantitative reverse transcriptase PCR of mRNA extracts from primary astrocyte cultures. Data represent mean ± SEM. Groups were compared using Student’s t-test, independent cultures from n = 4 control and n = 3 CKO mice. ***P < 0.001. (C–H) Primary astrocyte cultures from the CKO cortex demonstrate increased pS6 expression relative to littermate controls. Scale bar, 200 μm. (I–M) Protein immunoblots from primary astrocyte cultures isolated from WT and CKO cortex demonstrate increased expression of mTORC1 signaling targets at baseline and when subjected to amino acid deprivation (−AA). Blots were cropped to show relevant bands. Bars represent mean ± SEM. Groups were compared using two-way ANOVA with Tukey’s multiple comparisons, n = 3 independent cultures per genotype. *P < 0.05, **P < 0.01.

Figure 4

Brain slice cultures from CKO mice demonstrate increased capacitance and changes in inhibitory neurocircuitry. (A) Representative electrophysiologic tracing demonstrating mIPSCs in cortical excitatory neurons. (B) Capacitance was increased in CKO neurons. (C) mIPSC amplitude was decreased in CKO neurons. (D) Frequency of mIPSCs tended to be higher in CKO mice but did not reach statistical significance. (E and F) Both the rise time and rise time constant for mIPSCs were increased in CKO cortical excitatory neurons. n = 8–10 neurons from N = 3 mice per group. Groups were compared with the Student’s t-test, *P < 0.05.

Figure 5

Rapamycin treatment prolongs survival even after withdrawal. (A) Survival after rapamycin (P < 0.0001 using Log-rank [Mantel–Cox] test). (B) Without treatment, CKO cortical lysates demonstrated increased pS6 (s240/244) (P18) in contrast littermate controls. This was attenuated during rapamycin treatment (P29) and remained attenuated 1 week after rapamycin withdrawal (P37). (C) Quantification of pS6 s240/244 expression. n = 4 animals per genotype. *P < 0.05, ***P < 0.001 by two-way ANOVA with Bonferroni’s multiple comparison test. (D–F) Brain weight (D), body weight (E) and brain-to-body ratio (F) immediately after the rapamycin treatment window (P30), 7 days after treatment withdrawal (P37) and 40 days after treatment withdrawal (P70). n = 3–6 CKOs and n = 4–20 littermate controls. *P < 0.05 by 2-way ANOVA with Bonferroni’s multiple comparison test.