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1.
Ann Neurol ; 91(1): 101-116, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34693554

RESUMO

OBJECTIVE: Germline loss-of-function mutations in DEPDC5, and in its binding partners (NPRL2/3) of the mammalian target of rapamycin (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. METHODS: Clinical cardiac investigations were performed in 16 patients with pathogenic variants in DEPDC5, NPRL2, or NPRL3. Two novel Depdc5 mouse strains, a human HA-tagged Depdc5 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. RESULTS: Holter, echocardiographic, and electrocardiographic (ECG) examinations provided no evidence for altered clinical cardiac function in the patient cohort, of whom 3 DEPDC5 patients succumbed to SUDEP and 6 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 electroencephalographic-ECG records on Depdc5c/- mice showed that spontaneous epileptic seizures resulting in a SUDEP-like event are not preceded by cardiac arrhythmia. INTERPRETATION: Mouse 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. ANN NEUROL 2022;91:101-116.


Assuntos
Epilepsias Parciais/complicações , Proteínas Ativadoras de GTPase/genética , Coração , Morte Súbita Inesperada na Epilepsia/etiologia , Adolescente , Adulto , Animais , Eletrocardiografia , Eletroencefalografia , Epilepsias Parciais/genética , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Proteínas Supressoras de Tumor/genética , Adulto Jovem
2.
Acta Neuropathol ; 138(6): 885-900, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31444548

RESUMO

Genetic 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.


Assuntos
Encéfalo/patologia , Epilepsia/patologia , Hemimegalencefalia/patologia , Malformações do Desenvolvimento Cortical/patologia , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , Epilepsia/genética , Feminino , Hemimegalencefalia/genética , Humanos , Lactente , Masculino , Malformações do Desenvolvimento Cortical/genética , Mutação/genética , Neurônios/patologia
3.
Nat Neurosci ; 27(6): 1125-1136, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38710875

RESUMO

Cortical malformations such as focal cortical dysplasia type II (FCDII) are associated with pediatric drug-resistant epilepsy that necessitates neurosurgery. FCDII results from somatic mosaicism due to post-zygotic mutations in genes of the PI3K-AKT-mTOR pathway, which produce a subset of dysmorphic cells clustered within healthy brain tissue. Here we show a correlation between epileptiform activity in acute cortical slices obtained from human surgical FCDII brain tissues and the density of dysmorphic neurons. We uncovered multiple signatures of cellular senescence in these pathological cells, including p53/p16 expression, SASP expression and senescence-associated ß-galactosidase activity. We also show that administration of senolytic drugs (dasatinib/quercetin) decreases the load of senescent cells and reduces seizure frequency in an MtorS2215F FCDII preclinical mouse model, providing proof of concept that senotherapy may be a useful approach to control seizures. These findings pave the way for therapeutic strategies selectively targeting mutated senescent cells in FCDII brain tissue.


Assuntos
Convulsões , Serina-Treonina Quinases TOR , Animais , Serina-Treonina Quinases TOR/metabolismo , Camundongos , Humanos , Convulsões/tratamento farmacológico , Senoterapia/farmacologia , Senescência Celular/efeitos dos fármacos , Dasatinibe/farmacologia , Epilepsia/tratamento farmacológico , Masculino , Malformações do Desenvolvimento Cortical/tratamento farmacológico , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Feminino
4.
Epileptic Disord ; 21(4): 359-365, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31368437

RESUMO

Unverricht-Lundborg disease (ULD), an autosomal recessive progressive myoclonus epilepsy, is due to an expansion, or less commonly a mutation, of the cystatin B (CSTB) gene. We report a clinical and molecular study of a Tunisian ULD family with five affected members presenting with a juvenile myoclonic epilepsy (JME)-like phenotype. The expansion of dodecamers was detected by a deamination/PCR assay. The expression profiles of CSTB and other candidate modifying genes, cathepsin B and cystatin C, were established by quantitative RT-PCR, and their respective transcription levels were compared with those from patients with a classic picture of ULD. Three patients had a fixed phenotype mimicking JME after 29 years of evolution. Only a discrete dysarthria was noticed in the two other patients. No correlation was observed between transcription level and severity of disease. Genetic screening should be performed in patients with a JME-like phenotype, when careful examination reveals discrete atypical signs of JME. This particular phenotype may be due to modifying genes and/or gene-environment interactions which require further clarification.


Assuntos
Mutação/genética , Epilepsias Mioclônicas Progressivas/genética , Epilepsia Mioclônica Juvenil/genética , Síndrome de Unverricht-Lundborg/genética , Adolescente , Adulto , Feminino , Testes Genéticos/métodos , Humanos , Masculino , Fenótipo
5.
J Clin Invest ; 128(6): 2452-2458, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29708508

RESUMO

DEP 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.


Assuntos
Epilepsias Parciais , Proteínas Ativadoras de GTPase , Mutação em Linhagem Germinativa , Malformações do Desenvolvimento Cortical , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas Repressoras , Animais , Sistemas CRISPR-Cas , Dendritos/metabolismo , Dendritos/patologia , Epilepsias Parciais/genética , Epilepsias Parciais/metabolismo , Epilepsias Parciais/patologia , Feminino , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Masculino , Malformações do Desenvolvimento Cortical/genética , Malformações do Desenvolvimento Cortical/metabolismo , Malformações do Desenvolvimento Cortical/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Mutantes , Neurônios/metabolismo , Neurônios/patologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Coluna Vertebral/metabolismo , Coluna Vertebral/patologia
6.
J Clin Invest ; 125(6): 2347-62, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25938788

RESUMO

Chronic infections induce a complex immune response that controls pathogen replication, but also causes pathology due to sustained inflammation. Ca2+ influx mediates T cell function and immunity to infection, and patients with inherited mutations in the gene encoding the Ca2+ channel ORAI1 or its activator stromal interaction molecule 1 (STIM1) are immunodeficient and prone to chronic infection by various pathogens, including Mycobacterium tuberculosis (Mtb). Here, we demonstrate that STIM1 is required for T cell-mediated immune regulation during chronic Mtb infection. Compared with WT animals, mice with T cell-specific Stim1 deletion died prematurely during the chronic phase of infection and had increased bacterial burdens and severe pulmonary inflammation, with increased myeloid and lymphoid cell infiltration. Although STIM1-deficient T cells exhibited markedly reduced IFN-γ production during the early phase of Mtb infection, bacterial growth was not immediately exacerbated. During the chronic phase, however, STIM1-deficient T cells displayed enhanced IFN-γ production in response to elevated levels of IL-12 and IL-18. The lack of STIM1 in T cells was associated with impaired activation-induced cell death upon repeated TCR engagement and pulmonary lymphocytosis and hyperinflammation in Mtb-infected mice. Chronically Mtb-infected, STIM1-deficient mice had reduced levels of inducible regulatory T cells (iTregs) due to a T cell-intrinsic requirement for STIM1 in iTreg differentiation and excessive production of IFN-γ and IL-12, which suppress iTreg differentiation and maintenance. Thus, STIM1 controls multiple aspects of T cell-mediated immune regulation to limit injurious inflammation during chronic infection.


Assuntos
Canais de Cálcio/imunologia , Imunidade Celular , Mycobacterium tuberculosis/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Tuberculose Pulmonar/imunologia , Animais , Canais de Cálcio/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Doença Crônica , Citocinas/genética , Citocinas/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/microbiologia , Inflamação/patologia , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos T/genética , Molécula 1 de Interação Estromal , Linfócitos T Reguladores/patologia , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/patologia
8.
Oncotarget ; 8(4): 5674-5675, 2017 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-28030824
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