Elsevier

Neurobiology of Disease

Volume 111, March 2018, Pages 91-101
Neurobiology of Disease

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

https://doi.org/10.1016/j.nbd.2017.12.010Get rights and content

Highlights

  • Conditional neuron-specific Depdc5 knockout mice (Depdc5cc+) have dysplastic cortical neurons.

  • Similar to other mTORopathy models, Depdc5cc+ mice have larger brains and increased neuron size.

  • Neuronal Depdc5 loss results in neuronal mTORC1 hyperactivation and evidence of gliosis.

  • Depdc5cc+ mice demonstrate a decreased seizure threshold, and some display seizure.

  • Depdc5cc+ mice have increased mortality, likely due in part to terminal seizures.

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. Depdc5flox/flox-Syn1Cre (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.

Introduction

DEPDC5 is a gene that has been recently associated with familial focal epilepsy as well as sporadic epilepsy (Dibbens et al., 2013, Epi4K and Epilepsy Phenome/Genome Project, 2017, Ishida et al., 2013, Lal et al., 2014, Picard et al., 2014). The list of epilepsy syndromes associated with pathogenic variants in DEPDC5 is expanding, with the severe early onset epilepsy syndrome known as infantile spasms recently included (Carvill et al., 2015). DEPDC5 variants may also confer an increased risk for Sudden Unexplained Death in Epilepsy (SUDEP) (Bagnall et al., 2016, Nascimento et al., 2015). In addition to a role in non-lesional focal epilepsy, DEPDC5 has also been associated with epileptogenic structural brain malformations, from focal cortical dysplasia (FCD) (Baulac et al., 2015, D'Gama et al., 2015a, Scheffer et al., 2014) to large cortical malformations, such as hemimegalencephaly (D'Gama et al., 2015b, Ricos et al., 2016, Scerri et al., 2015). Pathological examination of resected human brain tissue from patients with pathogenic variants in DEPDC5 reveals dysplastic neurons and evidence of mTOR pathway disruption (Scerri et al., 2015).

Altered mTOR complex 1 (mTORC1) signaling is implicated in many neurologic conditions including epilepsy, brain malformations, and autism (Lipton and Sahin, 2014). At the cellular level, the mTORC1 pathway is activated by pro-growth factors such as neurotrophins, growth factors, and amino acids, and inhibited by metabolic stress such as nutrient starvation and endoplasmic reticulum stress. The product of DEPDC5, pleckstrin (DEP) domain-containing protein 5 (DEPDC5) is a key member of the amino acid sensing machinery and negatively regulates the mTORC1 pathway (Bar-Peled et al., 2013). DEPDC5 is ubiquitously expressed in the developing and adult brain, with high levels of expression in neurons (Dibbens et al., 2013).

DEPDC5 is a component of the GATOR1 complex along with NPRL2 and NPRL3. Collectively, the GATOR1 complex inhibits RagA/B- and RagC/D-mediated mTORC1 recruitment to lysosomal membranes and inhibits downstream mTORC1-mediated phosphorylation of S6 kinase and its substrate S6 (Saxton and Sabatini, 2017, Wolfson et al., 2017). The GATOR1 complex is a key regulator of cellular amino acid and nutrient detection in non-neuronal cell lines. These signaling pathways are unique to the GATOR complex and function independently of TSC1/TSC2 signaling (Shimobayashi and Hall, 2016). Given the relatively recent recognition of their role in epilepsy and brain development, the role of DEPDC5 and GATOR1 signaling in neuronal function remains largely unexplored.

Recently, Baulac and colleagues demonstrated a post-zygotic, somatic mutation of DEPDC5 in the brain lesion of a patient with a germline DEPDC5 mutation (Baulac et al., 2015). This provides evidence that a “two-hit” functional knockout of DEPDC5 may underlie brain malformations in some patients, particularly those with MRI-evident brain lesions (Scheffer et al., 2014). No animal model of DEPDC5-related epilepsy exists, and many animal models of GATOR1 complex genes are embryonic lethal, including a Depdc5−/− mouse (Dickinson et al., 2016). A Depdc5−/− rat model is embryonic lethal, and the Depdc5+/− rats do not display spontaneous seizures and seizure thresholds were not evaluated (Marsan et al., 2016). Animal models of other mTOR pathway regulators are lethal in the embryonic period, including Tsc1 and Tsc2 (Han and Sahin, 2011) and Pten (Di Cristofano et al., 1998). The synapsin promoter driving Cre-recombinase expression starting at embryonic day 12–13 has been successfully utilized to generate neuron-specific inactivation models of other mTORopathies (Meikle et al., 2007, Yuan et al., 2012, Zhu et al., 2001).

Here, we report the generation and characterization of a neuron-specific Depdc5 mouse model, Depdc5flox/flox-Syn1Cre (Depdc5cc+), which displays a larger brain size, early mortality, lowered seizure threshold, evidence of mTOR hyperactivation, ectopic neurons in the hippocampus, and dysplastic neurons in the cortex. Taken together, we present the first mammalian model that recapitulates many features of the human conditions associated with pathogenic variants in DEPDC5.

Section snippets

Mouse alleles, breeding strategy, and phenotyping

Mouse experiments were performed in a mixed-strain background using equal numbers of male and female mice. All mice were housed in a 12-h light-dark cycle, climate controlled room, with access to food and water ad lib. Depdc5tm1c(EUCOMM)Hmgu conditional mice (referred to as Depdc5c/c) contain loxP sites flanking exon 5 of the Depdc5 gene. Germline loss of Depdc5 exon 5 results in embryonic lethality (Dickinson et al., 2016). To generate the neuron-specific Depdc5 conditional knockout mice, the

Conditional neuron-specific Depdc5 knockout mice survive to adulthood

We generated a conditional neuron-specific Depdc5 knockout mouse model to evaluate DEPDC5 function in the brain in vivo. Depdc5c/c mice contain loxP sites flanking exon 5 of the Depdc5 gene and are embryonic lethal when Cre is constitutively expressed (Dickinson et al., 2016). We crossed Depdc5c/c mice with a line containing the SynapsinI promoter-driven cre recombinase allele (SynICre) that leads to recombination of loxP sites in neurons beginning at E12.5 (Zhu et al., 2001). We generated

Discussion

We present the first evidence of an animal model of DEPDC5-related epilepsy that recapitulates many features of the human conditions associated with this important epilepsy- and malformation-related gene. Humans with heterozygous loss-of-function DEPDC5 variants exhibit epilepsy with or without developmental cortical malformations. Histopathology from resected tissue from an individual with a pathogenic DEPDC5 variant has been reported to show increased mTORC1 activity by p-S6 staining in

Conclusions

We demonstrate in a neuron-specific conditional knockout mouse model that Depdc5 loss of function plays a critical role in epileptogenesis and brain development. Depdc5cc+ mice display a propensity to proconvulsant-induced and rare spontaneous behavioral seizures, sometimes associated with increased mortality. We thus report the first animal model of DEPDC5-related epilepsy and present a model that may be studied in future to elucidate the currently elusive mechanisms of SUDEP. Thus, our Depdc5

Acknowledgments

We would like to thank the IDDRC Cellular Imaging Core at Boston Children's Hospital and Dr. Anthony Hill for imaging assistance, Samantha Murphy for aid in generating mice, Samantha Schaeffer for animal breeding, Dr. Alessia Di Nardo for antibodies, Allison Mazzella for technical assistance, and the Experimental Neurophysiology Core at Boston Children's Hospital.

Funding

This work was supported by the NIH 2R25NS070682-07 (CJY) and U54HD090255 (MS), and the Translational Research Program at Boston Children's Hospital (AP).

References (47)

  • J.M. Han et al.

    TSC1/TSC2 signaling in the CNS

    FEBS Lett.

    (2011)
  • J.O. Lipton et al.

    The neurology of mTOR

    Neuron

    (2014)
  • E. Marsan

    Depdc5 knockout rat: a novel model of mTORopathy

    Neurobiol. Dis.

    (2016)
  • R.A. Saxton et al.

    mTOR signaling in growth, metabolism, and disease

    Cell

    (2017)
  • K. Switon

    Molecular neurobiology of mTOR

    Neuroscience

    (2017)
  • S. Amin

    Causes of mortality in individuals with tuberous sclerosis complex

    Dev. Med. Child Neurol.

    (2017)
  • S. Anderl

    Therapeutic value of prenatal rapamycin treatment in a mouse brain model of tuberous sclerosis complex

    Hum. Mol. Genet.

    (2011)
  • R.D. Bagnall

    Exome-based analysis of cardiac arrhythmia, respiratory control, and epilepsy genes in sudden unexpected death in epilepsy

    Ann. Neurol.

    (2016)
  • A.J. Barkovich

    Malformations of cortical development and epilepsy

    Cold Spring Harb. Perspect. Med.

    (2015)
  • L. Bar-Peled

    A tumor suppressor complex with GAP activity for the rag GTPases that signal amino acid sufficiency to mTORC1

    Science

    (2013)
  • S. Baulac

    Familial focal epilepsy with focal cortical dysplasia due to DEPDC5 mutations

    Ann. Neurol.

    (2015)
  • G.L. Carvill

    Epileptic spasms are a feature of DEPDC5 mTORopathy

    Neurol Genet.

    (2015)
  • D.A. Coulter et al.

    Role of astrocytes in epilepsy

    Cold Spring Harb. Perspect. Med.

    (2015)
  • A.M. D'Gama

    Mammalian target of rapamycin pathway mutations cause hemimegalencephaly and focal cortical dysplasia

    Ann. Neurol.

    (2015)
  • A.M. D'Gama

    Mammalian target of rapamycin pathway mutations cause hemimegalencephaly and focal cortical dysplasia

    Ann. Neurol.

    (2015)
  • S.C. Dhamne

    Acute seizure suppression by transcranial direct current stimulation in rats

    Ann. Clin. Transl. Neurol.

    (2015)
  • S.C. Dhamne

    Replicable in vivo physiological and behavioral phenotypes of the Shank3B null mutant mouse model of autism

    Mol. Autism

    (2017)
  • A. Di Cristofano

    Pten is essential for embryonic development and tumour suppression

    Nat. Genet.

    (1998)
  • L.M. Dibbens

    Mutations in DEPDC5 cause familial focal epilepsy with variable foci

    Nat. Genet.

    (2013)
  • M.E. Dickinson

    High-throughput discovery of novel developmental phenotypes

    Nature

    (2016)
  • Epik4K consortium, Epilepsy Phenome/Genome Project

    Ultra-rare genetic variation in common epilepsies: a case-control sequencing study

    Lancet Neurol.

    (2017)
  • E. Ercan

    Neuronal CTGF/CCN2 negatively regulates myelination in a mouse model of tuberous sclerosis complex

    J. Exp. Med.

    (2017)
  • W.N. Frankel

    Szt2, a novel gene for seizure threshold in mice

    Genes Brain Behav.

    (2009)
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