A mouse model of DEPDC5-related epilepsy: Neuronal loss of Depdc5 causes dysplastic and ectopic neurons, increased mTOR signaling, and seizure susceptibility

Abstract

DEPDC5 is a newly identified epilepsy-related gene implicated in focal epilepsy, brain malformations, and Sudden Unexplained Death in Epilepsy (SUDEP). In vitro, DEPDC5 negatively regulates amino acid sensing by the mTOR complex 1 (mTORC1) pathway, but the role of DEPDC5 in neurodevelopment and epilepsy has not been described. No animal model of DEPDC5-related epilepsy has recapitulated the neurological phenotypes seen in patients, and germline knockout rodent models are embryonic lethal. Here, we establish a neuron-specific Depdc5 conditional knockout mouse by cre-recombination under the Synapsin1 promotor. Depdc5^flox/flox-Syn1^Cre (Depdc5cc+) mice survive to adulthood with a progressive neurologic phenotype that includes motor abnormalities (i.e., hind limb clasping) and reduced survival compared to littermate control mice. Depdc5cc+ mice have larger brains with increased cortical neuron size and dysplastic neurons throughout the cortex, comparable to the abnormal neurons seen in human focal cortical dysplasia specimens. Depdc5 results in constitutive mTORC1 hyperactivation exclusively in neurons as measured by the increased phosphorylation of the downstream ribosomal protein S6. Despite a lack of increased mTORC1 signaling within astrocytes, Depdc5cc+ brains show reactive astrogliosis. We observed two Depdc5cc+ mice to have spontaneous seizures, including a terminal seizure. We demonstrate that as a group Depdc5cc+ mice have lowered seizure thresholds, as evidenced by decreased latency to seizures after chemoconvulsant injection and increased mortality from pentylenetetrazole-induced seizures. In summary, our neuron-specific Depdc5 knockout mouse model recapitulates clinical, pathological, and biochemical features of human DEPDC5-related epilepsy and brain malformations. We thereby present an important model in which to study targeted therapeutic strategies for DEPDC5-related conditions.

Keywords: Conditional knockout; DEPDC5; Familial focal epilepsy; Focal cortical dysplasia; Megalencephaly; Seizures; mTOR.

Figure 1. Conditional neuron-specific Depdc5 knockout (Depdc5cc+) mice have minimal weight loss but evidence of a progressive neurologic phenotype and reduced survival

(A) Depdc5 PCR product is truncated (arrow) in brain tissue from Depdc5^f/f mice with a copy of the Syn-Cre allele (Depdc5cc+), but not from tail or control mice samples. (B) Left: Quantitative analysis of RT-PCR of Depdc5 transcript is reduced in Depdc5cc+ cortical tissue (0.33 ± 0.04; p = 0.002) but not liver tissue (0.90 ± 0.13) compared to controls (cortex 1± 0.18; liver 1± 0.05). Right: Gel from Depdc5 RT-PCR product with a truncated product (arrow) only in Depdc5cc+ cortical tissue homogenate. (C) Compared to littermate controls, Depdc5cc+ male mice had reduced body weight in weanlings, P21–30, (1 ± 1.3 gram reduction; p = 0.04) and aged mice (3.1 ± 1.2 gram reduction; p = 0.03), whereas Depdc5cw+ males showed a significant weight reduction only in aged mice (3.2 ± 1.3 gram reduction; p = 0.046). No differences in female mice or between Depdc5cc+ and Depdc5cw+ were seen, n > 6 per sex within each genotype. (D) As a test of neurologic dysfunction, aged, >90 days old, Depdc5cc+ mice had evidence of hind limb strain (n=13; score = 1.8 ± 0.2) unlike control (n=17; score = 1.18 ± 0.1) and Depdc5cw+ (n=9; score = 1.22 ± 0.15) mice. (E) Monitoring survival of littermate mice revealed a markedly shortened lifespan of Depdc5cc+ (n=29; median survival = 115 days) compared to Depdc5cw+ (n=25) and control (n=30) mice. Error bars represent mean ± SEM. *p < 0.05 (2-way ANOVA and Tukey’s multiple comparison test).

Figure 2. Neuropathological defects in conditional neuron-specific Depdc5 knockout (Depdc5cc+) mice

(A) Left: Representative images of adult dissected brains show increased brain size of Depdc5cc+ compared to littermate controls. Right: Brain weight is increased in Depdc5cc+ (n=10) mice compared to littermate control (n=13) adult >60 day old mice. (B) Increased cortical thickness with less distinct layers in Depdc5cc+ adult cortex compared to littermate controls. Top: Representative NeuN stained coronal sections, scale bars: 100μm. Bottom: Cortical thickness measurements at 6 paired sites in at least 3 sections per brain. (C) Neuronal density was unchanged in layers I–III and decreased in layers IV–V in Depdc5cc+ mice compared to matched controls; measured from on average 100 NeuN+ neurons per layer in each animal. (D) Neuron soma size is larger in Depdc5cc+ layers IV–V of M1 region of cortex compared to controls, measured from at least 175 NeuN-positive neurons per genotype. (E) Dysplastic neurons within Depdc5cc+ brains are evident by SMI 311 staining. Top panels in control mouse cortex with linear SMI 311 staining and minimal p-S6 staining. Bottom panels in Depdc5cc+ cortex with strong, disorganized SMI 311 staining colocalizes with p-S6 staining. Scale bars: 50μm. Sections from n=4 Depdc5cc+ and n=3 control brains. Graph error bars represent mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test).

Figure 3. Widespread architectural abnormalities and evidence of mTORC1 hyperactivation in adult conditional neuron-specific Depdc5 knockout (Depdc5cc+) mice

A–F: Increased pS6 expression in NeuN+ neurons throughout the RSG region of the motor cortex of Depdc5cc+ brains (D–F) compared to controls (A–C). G–L: Occasional large pS6+ neurons within the CA1 pyramidal cell layer of the hippocampus of Depdc5cc+ mice (J–L) and also ectopically in the stratum oriens (L; arrowheads). M–R: In the CA3 region of the hippocampus, nearly all neurons are pS6+ including those outside the pyramidal cell layer (arrowheads) in Depdc5cc+ mice (P–R). S–X: In the dentate gyrus, robust pS6 positive neurons in the granule cell layer and hilus of Depdc5cc+ mice (V–X) with a few ectopic neurons (arrowheads). Sections from Depdc5cc+ (n=4) and control (n=3) brains. Scale bars: 100μm.

Figure 4. mTORC1-signaling is increased in conditional neuron-specific Depdc5 knockout (Depdc5cc+) cortical lysates

(A) Immunoblots and (B) quantitative analysis of cortical brain lysates from adult (92–122 day old) Depdc5cc+ and littermate control mice demonstrate altered regulation of mTOR1C by increased p-S6(S240/244) and decreased p–AKT(S473) in Depdc5cc+ brains, which is independent of TSC1 and TSC2. Analysis from ≥3 samples per genotype. Expression of levels were normalized to alpha-tubulin, and p-S6(S240/244) and p–AKT(S473) were normalized to total levels of S6 and AKT, respectively. All ratios for the control samples are normalized to 1 with mean ± SEM. *p < 0.05; **p < 0.01 (Student’s t-test).

Figure 5. Evidence of reactive astrogliosis in conditional neuron-specific Depdc5 knockout (Depdc5cc+) mice

Astrogliosis was absent in control cortical sections (n=3 mice) (Fig. 5A–C) and present in adult Depdc5cc+ cortical sections (n=4 mice) (Fig. 5D–F). Scale bars: 50 μm. (F) Inset demonstrates lack of GFAP and pS6 colocalization, scale bar = 10 μm. (G) Immunoblots and quantitative analysis of cortical brain lysates for GFAP from adult Depdc5cc+ and littermate control mice (n=8 per genotype). Expression of levels were normalized to alpha-tubulin and control samples are normalized to 1 with mean ± SEM. *p < 0.05; **p < 0.01 (Student’s t-test).

Figure 6. Electrophysiologic defects are evident in conditional neuron-specific Depdc5 knockout (Depdc5cc+) mice

(A) Representative baseline EEG data from control and Depdc5cc+ mice demonstrates 2-second spike trains (horizontal bars; a normal rodent EEG finding) intermixed with a normal background EEG pattern. (B) Representative EEG tracings after PTZ injection demonstrate a shorter latency to GTC seizures in Depdc5cc+ mice. Clinical seizure onset corresponds to left box margin. (C) Depdc5cc+ mice (n=8) have a shorter latency to GTC seizures after 40mg/kg PTZ administration compared to controls (n=11). p<0.05, one-tailed Mann-Whitney test. (D) Increased mortality of Depdc5cc+ mice (n=8) compared to controls after 40mg/kg PTZ, p<0.05 by Chi-Square analysis.