Functional screening of GATOR1 complex variants reveals a role for mTORC1 deregulation in FCD and focal epilepsy

Ruby E. Dawson, Alvaro F. Nieto Guil, Louise J. Robertson, Sandra Piltz, James N. Hughes, Paul Thomas

Research output: Contribution to journalArticle

Abstract

Mutations in the GAP activity toward RAGs 1 (GATOR1) complex genes (DEPDC5, NPRL2 and NPRL3) have been associated with focal epilepsy and focal cortical dysplasia (FCD). GATOR1 functions as an inhibitor of the mTORC1 signalling pathway, indicating that the downstream effects of mTORC1 deregulation underpin the disease. However, the vast majority of putative disease-causing variants have not been functionally assessed for mTORC1 repression activity. Here, we develop a novel in vitro functional assay that enables rapid assessment of GATOR1-gene variants. Surprisingly, of the 17 variants tested, we show that only six showed significantly impaired mTORC1 inhibition. To further investigate variant function in vivo, we generated a conditional Depdc5 mouse which modelled a ‘second-hit’ mechanism of disease. Generation of Depdc5 null ‘clones’ in the embryonic brain resulted in mTORC1 hyperactivity and modelled epilepsy and FCD symptoms including large dysmorphic neurons, defective migration and lower seizure thresholds. Using this model, we validated DEPDC5 variant F164del to be loss-of-function. We also show that Q542P is not functionally compromised in vivo, consistent with our in vitro findings. Overall, our data show that mTORC1 deregulation is the central pathological mechanism for GATOR1 variants and also indicates that a significant proportion of putative disease variants are pathologically inert, highlighting the importance of GATOR1 variant functional assessment.

LanguageEnglish
Article number104640
JournalNeurobiology of Disease
Volume134
DOIs
Publication statusPublished - 1 Feb 2020

Keywords

  • CRISPR/CAS9
  • Developmental genetics
  • Disease model
  • Epilepsy
  • Focal cortical dysplasia
  • Functional testing
  • Molecular genetics
  • Mouse model
  • Neurodevelopment
  • mTOR

ASJC Scopus subject areas

  • Neurology

Cite this

@article{88c145a223f7480fb061e0ae88190e76,
title = "Functional screening of GATOR1 complex variants reveals a role for mTORC1 deregulation in FCD and focal epilepsy",
abstract = "Mutations in the GAP activity toward RAGs 1 (GATOR1) complex genes (DEPDC5, NPRL2 and NPRL3) have been associated with focal epilepsy and focal cortical dysplasia (FCD). GATOR1 functions as an inhibitor of the mTORC1 signalling pathway, indicating that the downstream effects of mTORC1 deregulation underpin the disease. However, the vast majority of putative disease-causing variants have not been functionally assessed for mTORC1 repression activity. Here, we develop a novel in vitro functional assay that enables rapid assessment of GATOR1-gene variants. Surprisingly, of the 17 variants tested, we show that only six showed significantly impaired mTORC1 inhibition. To further investigate variant function in vivo, we generated a conditional Depdc5 mouse which modelled a ‘second-hit’ mechanism of disease. Generation of Depdc5 null ‘clones’ in the embryonic brain resulted in mTORC1 hyperactivity and modelled epilepsy and FCD symptoms including large dysmorphic neurons, defective migration and lower seizure thresholds. Using this model, we validated DEPDC5 variant F164del to be loss-of-function. We also show that Q542P is not functionally compromised in vivo, consistent with our in vitro findings. Overall, our data show that mTORC1 deregulation is the central pathological mechanism for GATOR1 variants and also indicates that a significant proportion of putative disease variants are pathologically inert, highlighting the importance of GATOR1 variant functional assessment.",
keywords = "CRISPR/CAS9, Developmental genetics, Disease model, Epilepsy, Focal cortical dysplasia, Functional testing, Molecular genetics, Mouse model, Neurodevelopment, mTOR",
author = "Dawson, {Ruby E.} and {Nieto Guil}, {Alvaro F.} and Robertson, {Louise J.} and Sandra Piltz and Hughes, {James N.} and Paul Thomas",
year = "2020",
month = "2",
day = "1",
doi = "10.1016/j.nbd.2019.104640",
language = "English",
volume = "134",
journal = "Neurobiology of Disease",
issn = "0969-9961",
publisher = "Academic Press Inc.",

}

Functional screening of GATOR1 complex variants reveals a role for mTORC1 deregulation in FCD and focal epilepsy. / Dawson, Ruby E.; Nieto Guil, Alvaro F.; Robertson, Louise J.; Piltz, Sandra; Hughes, James N.; Thomas, Paul.

In: Neurobiology of Disease, Vol. 134, 104640, 01.02.2020.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Functional screening of GATOR1 complex variants reveals a role for mTORC1 deregulation in FCD and focal epilepsy

AU - Dawson, Ruby E.

AU - Nieto Guil, Alvaro F.

AU - Robertson, Louise J.

AU - Piltz, Sandra

AU - Hughes, James N.

AU - Thomas, Paul

PY - 2020/2/1

Y1 - 2020/2/1

N2 - Mutations in the GAP activity toward RAGs 1 (GATOR1) complex genes (DEPDC5, NPRL2 and NPRL3) have been associated with focal epilepsy and focal cortical dysplasia (FCD). GATOR1 functions as an inhibitor of the mTORC1 signalling pathway, indicating that the downstream effects of mTORC1 deregulation underpin the disease. However, the vast majority of putative disease-causing variants have not been functionally assessed for mTORC1 repression activity. Here, we develop a novel in vitro functional assay that enables rapid assessment of GATOR1-gene variants. Surprisingly, of the 17 variants tested, we show that only six showed significantly impaired mTORC1 inhibition. To further investigate variant function in vivo, we generated a conditional Depdc5 mouse which modelled a ‘second-hit’ mechanism of disease. Generation of Depdc5 null ‘clones’ in the embryonic brain resulted in mTORC1 hyperactivity and modelled epilepsy and FCD symptoms including large dysmorphic neurons, defective migration and lower seizure thresholds. Using this model, we validated DEPDC5 variant F164del to be loss-of-function. We also show that Q542P is not functionally compromised in vivo, consistent with our in vitro findings. Overall, our data show that mTORC1 deregulation is the central pathological mechanism for GATOR1 variants and also indicates that a significant proportion of putative disease variants are pathologically inert, highlighting the importance of GATOR1 variant functional assessment.

AB - Mutations in the GAP activity toward RAGs 1 (GATOR1) complex genes (DEPDC5, NPRL2 and NPRL3) have been associated with focal epilepsy and focal cortical dysplasia (FCD). GATOR1 functions as an inhibitor of the mTORC1 signalling pathway, indicating that the downstream effects of mTORC1 deregulation underpin the disease. However, the vast majority of putative disease-causing variants have not been functionally assessed for mTORC1 repression activity. Here, we develop a novel in vitro functional assay that enables rapid assessment of GATOR1-gene variants. Surprisingly, of the 17 variants tested, we show that only six showed significantly impaired mTORC1 inhibition. To further investigate variant function in vivo, we generated a conditional Depdc5 mouse which modelled a ‘second-hit’ mechanism of disease. Generation of Depdc5 null ‘clones’ in the embryonic brain resulted in mTORC1 hyperactivity and modelled epilepsy and FCD symptoms including large dysmorphic neurons, defective migration and lower seizure thresholds. Using this model, we validated DEPDC5 variant F164del to be loss-of-function. We also show that Q542P is not functionally compromised in vivo, consistent with our in vitro findings. Overall, our data show that mTORC1 deregulation is the central pathological mechanism for GATOR1 variants and also indicates that a significant proportion of putative disease variants are pathologically inert, highlighting the importance of GATOR1 variant functional assessment.

KW - CRISPR/CAS9

KW - Developmental genetics

KW - Disease model

KW - Epilepsy

KW - Focal cortical dysplasia

KW - Functional testing

KW - Molecular genetics

KW - Mouse model

KW - Neurodevelopment

KW - mTOR

UR - http://www.scopus.com/inward/record.url?scp=85073967042&partnerID=8YFLogxK

U2 - 10.1016/j.nbd.2019.104640

DO - 10.1016/j.nbd.2019.104640

M3 - Article

VL - 134

JO - Neurobiology of Disease

T2 - Neurobiology of Disease

JF - Neurobiology of Disease

SN - 0969-9961

M1 - 104640

ER -