Investigations génétiques et physiopathologiques des dysplasies corticales focales de type 2

Focal cortical dysplasia type 2 (FCD2) is a neurodevelopmental disorder due to mutations in genes of the mechanistic target of rapamycin complex 1 (mTORC1) pathway. It is frequently associated with pediatric drug-resistant epilepsies and represents 9% of the epilepsy surgery population. Most cases are sporadic with the occurrence of a low-allele frequency brain-specific somatic variants, especially activating somatic MTOR variants. However, FCD2 is also described in familial cases of focal epilepsy due to germline loss-of-function mutations in DEPDC5, a gene encoding a protein repressor of mTOR. Interestingly, only a subset of patients develops FCD2 while other relatives have apparently non-lesional focal epilepsy. In this manuscript, I describe the work that I have achieved over the course of my 4 years of PhD training: two main projects that I led and two projects for which I contributed. First main project. We describe the occurrence of a somatic mosaic second-hit in DEPDC5, explaining the development of FCD2 in a patient with familial focal epilepsy, following a similar hypothesis to the Knudson’s two-hit model in cancer. Subsequently, we describe a mouse model of focal Depdc5 biallelic inactivation recapitulating the main hallmarks of FCD2. Second main project. We describe the occurrence of a novel pathological mechanism in pathogenic cells of FCD2: premature cellular senescence. We further demonstrate a non-cell autonomous effect of cellular senescence mediated by the secretion of mTOR-activating cytokines. Finally, we provide initial evidence that targeting senescent cells can improve the epileptic phenotype in a preclinical mouse model of the pathology. Overall, my work has contributed to expand our knowledge about the mutational mechanisms underlying the development of FCD2. Furthermore, it has unveiled a druggable pathological mechanism with promising results on the epileptic phenotype of mouse model of the pathology.La dysplasie corticale focale de type 2 (DCF2) est une pathologie neurodéveloppementale associée à des formes focales d’épilepsies pédiatriques pharmaco-résistantes, et représente 9% des chirurgies de l’épilepsie. La plupart des cas de DCF2 sont dû à des variants somatiques de faible fréquence allélique dans des gènes de la voie mTORC1. Toutefois, on retrouve également des cas de DCF2 dans des formes familiales d’épilepsie focale due à une haploinsuffisance de DEPDC5, un gène codant une protéine répresseur de mTOR. Dans ce cas, seuls quelques individus présentent une DCF2 tandis que les autres membres de la famille présentent une épilepsie focale non-lésionnelle. Dans ce manuscrit, je présente le travail accompli durant mon doctorat en neurosciences : deux projets principaux, et deux projets auxquels j’ai participé. Premier projet principal. Nous décrivons un nouveau mécanisme mutationnel de type ‘second-hit’ dans DEPDC5 expliquant le développement d’une DCF2 dans les formes familiales d’épilepsie focale. Nous avons également généré un premier modèle murin présentant les principales caractéristiques de DCF2 par inactivation focale biallélique de Depdc5 au cours du neurodéveloppement. Second projet principal. Nous révélons un nouveau mécanisme pathogène des DCF2 : la sénescence cellulaire prématurée, qui exerce un effet non-autonome cellulaire au travers de la sécrétion de cytokines capables de stimuler la voie mTORC1. Nous apportons la preuve de concept que le ciblage médicamenteux de la sénescence cellulaire permet d’améliorer le phénotype épileptique d’un modèle murin de la pathologie. Dans son ensemble, mon travail de doctorat aura permis d’étendre le spectre des mécanismes mutationnels responsables des DCF2, et d’apporter de nouvelles pistes thérapeutiques en décrivant un nouveau mécanisme pathogène spécifique des cellules pathogéniques de la pathologie

Investigations génétiques et physiopathologiques des dysplasies corticales focales de type 2

Focal cortical dysplasia type 2 (FCD2) is a neurodevelopmental disorder due to mutations in genes of the mechanistic target of rapamycin complex 1 (mTORC1) pathway. It is frequently associated with pediatric drug-resistant epilepsies and represents 9% of the epilepsy surgery population. Most cases are sporadic with the occurrence of a low-allele frequency brain-specific somatic variants, especially activating somatic MTOR variants. However, FCD2 is also described in familial cases of focal epilepsy due to germline loss-of-function mutations in DEPDC5, a gene encoding a protein repressor of mTOR. Interestingly, only a subset of patients develops FCD2 while other relatives have apparently non-lesional focal epilepsy. In this manuscript, I describe the work that I have achieved over the course of my 4 years of PhD training: two main projects that I led and two projects for which I contributed. First main project. We describe the occurrence of a somatic mosaic second-hit in DEPDC5, explaining the development of FCD2 in a patient with familial focal epilepsy, following a similar hypothesis to the Knudson’s two-hit model in cancer. Subsequently, we describe a mouse model of focal Depdc5 biallelic inactivation recapitulating the main hallmarks of FCD2. Second main project. We describe the occurrence of a novel pathological mechanism in pathogenic cells of FCD2: premature cellular senescence. We further demonstrate a non-cell autonomous effect of cellular senescence mediated by the secretion of mTOR-activating cytokines. Finally, we provide initial evidence that targeting senescent cells can improve the epileptic phenotype in a preclinical mouse model of the pathology. Overall, my work has contributed to expand our knowledge about the mutational mechanisms underlying the development of FCD2. Furthermore, it has unveiled a druggable pathological mechanism with promising results on the epileptic phenotype of mouse model of the pathology.La dysplasie corticale focale de type 2 (DCF2) est une pathologie neurodéveloppementale associée à des formes focales d’épilepsies pédiatriques pharmaco-résistantes, et représente 9% des chirurgies de l’épilepsie. La plupart des cas de DCF2 sont dû à des variants somatiques de faible fréquence allélique dans des gènes de la voie mTORC1. Toutefois, on retrouve également des cas de DCF2 dans des formes familiales d’épilepsie focale due à une haploinsuffisance de DEPDC5, un gène codant une protéine répresseur de mTOR. Dans ce cas, seuls quelques individus présentent une DCF2 tandis que les autres membres de la famille présentent une épilepsie focale non-lésionnelle. Dans ce manuscrit, je présente le travail accompli durant mon doctorat en neurosciences : deux projets principaux, et deux projets auxquels j’ai participé. Premier projet principal. Nous décrivons un nouveau mécanisme mutationnel de type ‘second-hit’ dans DEPDC5 expliquant le développement d’une DCF2 dans les formes familiales d’épilepsie focale. Nous avons également généré un premier modèle murin présentant les principales caractéristiques de DCF2 par inactivation focale biallélique de Depdc5 au cours du neurodéveloppement. Second projet principal. Nous révélons un nouveau mécanisme pathogène des DCF2 : la sénescence cellulaire prématurée, qui exerce un effet non-autonome cellulaire au travers de la sécrétion de cytokines capables de stimuler la voie mTORC1. Nous apportons la preuve de concept que le ciblage médicamenteux de la sénescence cellulaire permet d’améliorer le phénotype épileptique d’un modèle murin de la pathologie. Dans son ensemble, mon travail de doctorat aura permis d’étendre le spectre des mécanismes mutationnels responsables des DCF2, et d’apporter de nouvelles pistes thérapeutiques en décrivant un nouveau mécanisme pathogène spécifique des cellules pathogéniques de la pathologie

Investigations génétiques et physiopathologiques des dysplasies corticales focales de type 2

Focal cortical dysplasia type 2 (FCD2) is a neurodevelopmental disorder due to mutations in genes of the mechanistic target of rapamycin complex 1 (mTORC1) pathway. It is frequently associated with pediatric drug-resistant epilepsies and represents 9% of the epilepsy surgery population. Most cases are sporadic with the occurrence of a low-allele frequency brain-specific somatic variants, especially activating somatic MTOR variants. However, FCD2 is also described in familial cases of focal epilepsy due to germline loss-of-function mutations in DEPDC5, a gene encoding a protein repressor of mTOR. Interestingly, only a subset of patients develops FCD2 while other relatives have apparently non-lesional focal epilepsy. In this manuscript, I describe the work that I have achieved over the course of my 4 years of PhD training: two main projects that I led and two projects for which I contributed. First main project. We describe the occurrence of a somatic mosaic second-hit in DEPDC5, explaining the development of FCD2 in a patient with familial focal epilepsy, following a similar hypothesis to the Knudson’s two-hit model in cancer. Subsequently, we describe a mouse model of focal Depdc5 biallelic inactivation recapitulating the main hallmarks of FCD2. Second main project. We describe the occurrence of a novel pathological mechanism in pathogenic cells of FCD2: premature cellular senescence. We further demonstrate a non-cell autonomous effect of cellular senescence mediated by the secretion of mTOR-activating cytokines. Finally, we provide initial evidence that targeting senescent cells can improve the epileptic phenotype in a preclinical mouse model of the pathology. Overall, my work has contributed to expand our knowledge about the mutational mechanisms underlying the development of FCD2. Furthermore, it has unveiled a druggable pathological mechanism with promising results on the epileptic phenotype of mouse model of the pathology.La dysplasie corticale focale de type 2 (DCF2) est une pathologie neurodéveloppementale associée à des formes focales d’épilepsies pédiatriques pharmaco-résistantes, et représente 9% des chirurgies de l’épilepsie. La plupart des cas de DCF2 sont dû à des variants somatiques de faible fréquence allélique dans des gènes de la voie mTORC1. Toutefois, on retrouve également des cas de DCF2 dans des formes familiales d’épilepsie focale due à une haploinsuffisance de DEPDC5, un gène codant une protéine répresseur de mTOR. Dans ce cas, seuls quelques individus présentent une DCF2 tandis que les autres membres de la famille présentent une épilepsie focale non-lésionnelle. Dans ce manuscrit, je présente le travail accompli durant mon doctorat en neurosciences : deux projets principaux, et deux projets auxquels j’ai participé. Premier projet principal. Nous décrivons un nouveau mécanisme mutationnel de type ‘second-hit’ dans DEPDC5 expliquant le développement d’une DCF2 dans les formes familiales d’épilepsie focale. Nous avons également généré un premier modèle murin présentant les principales caractéristiques de DCF2 par inactivation focale biallélique de Depdc5 au cours du neurodéveloppement. Second projet principal. Nous révélons un nouveau mécanisme pathogène des DCF2 : la sénescence cellulaire prématurée, qui exerce un effet non-autonome cellulaire au travers de la sécrétion de cytokines capables de stimuler la voie mTORC1. Nous apportons la preuve de concept que le ciblage médicamenteux de la sénescence cellulaire permet d’améliorer le phénotype épileptique d’un modèle murin de la pathologie. Dans son ensemble, mon travail de doctorat aura permis d’étendre le spectre des mécanismes mutationnels responsables des DCF2, et d’apporter de nouvelles pistes thérapeutiques en décrivant un nouveau mécanisme pathogène spécifique des cellules pathogéniques de la pathologie

Le mosaïcisme somatique en cause dans les épilepsies neurodéveloppementales

International audienceNo abstract availabl

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

International audienceDEP 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

Dissecting the genetic basis of focal cortical dysplasia: a large cohort study

International audienceGenetic malformations of cortical development (MCDs), such as mild MCDs (mMCD), focal cortical dysplasia (FCD), and hemimegalencephaly (HME), are major causes of severe pediatric refractory epilepsies subjected to neurosurgery. FCD2 are characterized by neuropathological hallmarks that include enlarged dysmorphic neurons (DNs) and balloon cells (BCs). Here, we provide a comprehensive assessment of the contribution of germline and somatic variants in a large cohort of surgical MCD cases. We enrolled in a monocentric study 80 children with drug-resistant epilepsy and a postsurgical neuropathological diagnosis of mMCD, FCD1, FCD2, or HME. We performed targeted gene sequencing ( ≥ 2000X read depth) on matched blood-brain samples to search for low-allele frequency variants in mTOR pathway and FCD genes. We were able to elucidate 29% of mMCD/FCD1 patients and 63% of FCD2/HME patients. Somatic loss-of-function variants in the N-glycosylation pathway-associated SLC35A2 gene were found in mMCD/FCD1 cases. Somatic gain-of-function variants in MTOR and its activators (AKT3, PIK3CA, RHEB), as well as germline, somatic and two-hit loss-of-function variants in its repressors (DEPDC5, TSC1, TSC2) were found exclusively in FCD2/HME cases. We show that panel-negative FCD2 cases display strong pS6-immunostaining, stressing that all FCD2 are mTORopathies. Analysis of microdissected cells demonstrated that DNs and BCs carry the pathogenic variants. We further observed a correlation between the density of pathological cells and the variant-detection likelihood. Single-cell microdissection followed by sequencing of enriched pools of DNs unveiled a somatic second-hit loss-of-heterozygosity in a DEPDC5 germline case. In conclusion, this study indicates that mMCD/FCD1 and FCD2/HME are two distinct genetic entities: while all FCD2/HME are mosaic mTORopathies, mMCD/FCD1 are not caused by mTOR-pathway-hyperactivating variants, and ~ 30% of the cases are related to glycosylation defects. We provide a framework for efficient genetic testing in FCD/HME, linking neuropathology to genetic findings and emphasizing the usefulness of molecular evaluation in the pediatric epileptic neurosurgical population

Cardiac investigations in sudden unexpected death in DEPDC5 ‐related epilepsy

International audienceObjectiveGermline loss-of-function mutations in DEPDC5, and in its binding partners (NPRL2/3) of the mTOR repressor GATOR1 complex, cause focal epilepsies and increase the risk of sudden unexpected death in epilepsy (SUDEP). Here, we asked whether DEPDC5 haploinsufficiency predisposes to primary cardiac defects that could contribute to SUDEP and therefore impact the clinical management of patients at high risk of SUDEP.MethodsClinical cardiac investigations were performed in sixteen patients with pathogenic variants in DEPDC5, NPRL2 or NPRL3. Two novel Depdc5 mouse strains, a HA-tagged Depdc5 mouse strain and a Depdc5 heterozygous knockout with a neuron-specific deletion of the second allele (Depdc5c/−) were generated to investigate the role of Depdc5 in SUDEP and cardiac activity during seizures.ResultsHolter, echocardiography and ECG exams provided no evidence for altered clinical cardiac function in the patient cohort, of whom three DEPDC5-patients succumbed to a SUDEP and six had a family history of SUDEP. There was no cardiac injury at autopsy in a postmortem DEPDC5-SUDEP case. The HA-tagged Depdc5 mouse revealed expression of Depdc5 in the brain, heart and lungs. Simultaneous EEG-ECG records on Depdc5c/− mice showed that spontaneous epileptic seizures resulting in a SUDEP-like event, are not preceded by cardiac arrhythmia.InterpretationMouse and human data show neither structural nor functional cardiac damage that might underlie a primary contribution to SUDEP in the spectrum of DEPDC5-related epilepsies