RESUMEN
Somatic mutations have been identified in 10% to 63% of focal cortical dysplasia type II samples, primarily linked to the mTOR pathway. When the causative genetic mutations are not identified, this opens the possibility of discovering new pathogenic genes or pathways that could be contributing to the condition. In our previous study, we identified a novel candidate pathogenic somatic variant of IRS-1 c.1791dupG in the brain tissue of a child with focal cortical dysplasia type II. This study further explored the variant's role in causing type II focal cortical dysplasia through in vitro overexpression in 293T and SH-SY5Y cells and in vivo evaluation via in utero electroporation in fetal brains, assessing effects on neuronal migration, morphology, and network integrity. It was found that the mutant IRS-1 variant led to hyperactivity of p-ERK, increased cell volume, and was predominantly associated with the MAPK signaling pathway. In vivo, the IRS-1 c.1791dupG variant induced abnormal neuron migration, cytomegaly, and network hyperexcitability. Notably, the ERK inhibitor GDC-0994, rather than the mTOR inhibitor rapamycin, effectively rescued the neuronal defects. This study directly highlighted the ERK signaling pathway's role in the pathogenesis of focal cortical dysplasia II and provided a new therapeutic target for cases of focal cortical dysplasia II that are not treatable by rapamycin analogs.
Asunto(s)
Proteínas Sustrato del Receptor de Insulina , Sistema de Señalización de MAP Quinasas , Mutación , Humanos , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Animales , Malformaciones del Desarrollo Cortical de Grupo I/genética , Malformaciones del Desarrollo Cortical de Grupo I/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Neuronas/metabolismo , Neuronas/patología , Movimiento Celular/genética , Células HEK293 , Femenino , Displasia Cortical Focal , EpilepsiaRESUMEN
OBJECTIVE: Genetic variations in proteins of the mechanistic target of rapamycin (mTOR) pathway cause a spectrum of neurodevelopmental disorders often associated with brain malformations and with intractable epilepsy. The mTORopathies are characterized by hyperactive mTOR pathway and comprise tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) type II. How hyperactive mTOR translates into abnormal neuronal activity and hypersynchronous network remains to be better understood. Previously, the role of upregulated GluN2C-containing glutamate-gated N-methyl-D-aspartate receptors (NMDARs) has been demonstrated for germline defects in the TSC genes. Here, we questioned whether this mechanism would expand to other mTORopathies in the different context of a somatic genetic variation of the MTOR protein recurrently found in FCD type II. METHODS: We used a rat model of FCD created by in utero electroporation of neural progenitors of dorsal telencephalon with expression vectors encoding either the wild-type or the pathogenic MTOR variant (p.S2215F). In this mosaic configuration, patch-clamp whole-cell recordings of the electroporated, spiny stellate neurons and extracellular recordings of the electroporated areas were performed in neocortical slices. Selective inhibitors were used to target mTOR activity and GluN2C-mediated currents. RESULTS: Neurons expressing the mutant protein displayed an excessive activation of GluN2C NMDAR-mediated spontaneous excitatory postsynaptic currents. GluN2C-dependent increase in spontaneous spiking activity was detected in the area of electroporated neurons in the mutant condition and was restricted to a critical time window between postnatal days P9 and P20. SIGNIFICANCE: Somatic MTOR pathogenic variant recurrently found in FCD type II resulted in overactivation of GluN2C-mediated neuronal NMDARs in neocortices of rat pups. The related and time-restricted local hyperexcitability was sensitive to subunit GluN2C-specific blockade. Our study suggests that GluN2C-related pathomechanisms might be shared in common by mTOR-related brain disorders.
Asunto(s)
Neuronas , Receptores de N-Metil-D-Aspartato , Serina-Treonina Quinasas TOR , Animales , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Serina-Treonina Quinasas TOR/genética , Ratas , Neuronas/metabolismo , Femenino , Malformaciones del Desarrollo Cortical/genética , Malformaciones del Desarrollo Cortical/fisiopatología , Modelos Animales de Enfermedad , Ratas Sprague-Dawley , Técnicas de Placa-Clamp , Malformaciones del Desarrollo Cortical de Grupo I/genética , Displasia Cortical Focal , EpilepsiaRESUMEN
Focal cortical dysplasia (FCD) represents a group of diverse localized cortical lesions that are highly epileptogenic and occur due to abnormal brain development caused by genetic mutations, involving the mammalian target of rapamycin (mTOR). These somatic mutations lead to mosaicism in the affected brain, posing challenges to unravel the direct and indirect functional consequences of these mutations. To comprehensively characterize the impact of mTOR mutations on the brain, we employed here a multimodal approach in a preclinical mouse model of FCD type II (Rheb), focusing on spatial omics techniques to define the proteomic and lipidomic changes. Mass Spectrometry Imaging (MSI) combined with fluorescence imaging and label free proteomics, revealed insight into the brain's lipidome and proteome within the FCD type II affected region in the mouse model. MSI visualized disrupted neuronal migration and differential lipid distribution including a reduction in sulfatides in the FCD type II-affected region, which play a role in brain myelination. MSI-guided laser capture microdissection (LMD) was conducted on FCD type II and control regions, followed by label free proteomics, revealing changes in myelination pathways by oligodendrocytes. Surgical resections of FCD type IIb and postmortem human cortex were analyzed by bulk transcriptomics to unravel the interplay between genetic mutations and molecular changes in FCD type II. Our comparative analysis of protein pathways and enriched Gene Ontology pathways related to myelination in the FCD type II-affected mouse model and human FCD type IIb transcriptomics highlights the animal model's translational value. This dual approach, including mouse model proteomics and human transcriptomics strengthens our understanding of the functional consequences arising from somatic mutations in FCD type II, as well as the identification of pathways that may be used as therapeutic strategies in the future.
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Epilepsia , Malformaciones del Desarrollo Cortical de Grupo I , Proteómica , Animales , Humanos , Malformaciones del Desarrollo Cortical de Grupo I/genética , Malformaciones del Desarrollo Cortical de Grupo I/metabolismo , Malformaciones del Desarrollo Cortical de Grupo I/patología , Ratones , Masculino , Serina-Treonina Quinasas TOR/metabolismo , Serina-Treonina Quinasas TOR/genética , Femenino , Modelos Animales de Enfermedad , Encéfalo/metabolismo , Encéfalo/patología , Proteoma/metabolismo , Displasia Cortical FocalRESUMEN
Focal cortical dysplasia is the most common malformation during cortical development, sometimes excised by epilepsy surgery and often caused by somatic variants of the mTOR pathway genes. In this study, we performed a genetic analysis of epileptogenic brain malformed lesions from 64 patients with focal cortical dysplasia, hemimegalencephy, brain tumors, or hippocampal sclerosis. Targeted sequencing, whole-exome sequencing, and single nucleotide polymorphism microarray detected four germline and 35 somatic variants, comprising three copy number variants and 36 single nucleotide variants and indels in 37 patients. One of the somatic variants in focal cortical dysplasia type IIB was an in-frame deletion in MTOR, in which only gain-of-function missense variants have been reported. In focal cortical dysplasia type I, somatic variants of MAP2K1 and PTPN11 involved in the RAS/MAPK pathway were detected. The in-frame deletions of MTOR and MAP2K1 in this study resulted in the activation of the mTOR pathway in transiently transfected cells. In addition, the PTPN11 missense variant tended to elongate activation of the mTOR or RAS/MAPK pathway, depending on culture conditions. We demonstrate that epileptogenic brain malformed lesions except for focal cortical dysplasia type II arose from somatic variants of diverse genes but were eventually linked to the mTOR pathway.
Asunto(s)
Neoplasias Encefálicas , Displasia Cortical Focal , Malformaciones del Desarrollo Cortical de Grupo I , Malformaciones del Sistema Nervioso , Humanos , Malformaciones del Desarrollo Cortical de Grupo I/genética , EncéfaloRESUMEN
PURPOSE: Focal cortical dysplasia (FCD) is the most common developmental malformation that causes refractory epilepsy. FCD II is a common neuropathological finding in tissues resected therapeutically from patients with drug-resistant epilepsy. However, its molecular genetic etiology remains unclear. This study aimed to identify potential molecular markers of FCD II using bioinformatics analysis. METHODS: We downloaded two datasets for FCD II from the Gene Expression Omnibus data repository. Differentially expressed genes (DEGs) between FCD II and normal brain tissues were identified, and functional enrichment analysis was performed. A protein-protein interaction network was constructed, and hub genes were identified from the DEGs. The hub gene expression was validated using WB in vitro. IHC staining was performed to verify the feasibility of the target molecular markers identified in the bioinformatics analysis. RESULTS: One hundred sixty-seven common DEGs were identified between the datasets. The GO and KEGG analyses showed that variations were prominently enriched in some functions associated with gene expression. Five hub genes (i.e., FANCI, FANCA, BRCA2, RAD18, and KEAP1) were identified. Western blotting confirmed that all hub gene expressions were higher in the FCD II tissue than in the normal brain tissue. IHC staining showed that the FANCI expression significantly increased in the FCD II tissue. CONCLUSION: There are DEGs between FCD II and normal brain tissues, which may be considered biomarkers for FCD II, along with FANCI. The DEGs and hub genes identified in the bioinformatics analysis could serve as candidate targets for diagnosing and treating FCD II.
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Epilepsia , Malformaciones del Desarrollo Cortical de Grupo I , Biomarcadores de Tumor/genética , Biología Computacional , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Malformaciones del Desarrollo Cortical de Grupo I/genética , Factor 2 Relacionado con NF-E2/genética , Ubiquitina-Proteína LigasasRESUMEN
Type II focal cortical dysplasia (FCD) is a neuropathological entity characterised by cortical dyslamination with the presence of dysmorphic neurons only (FCDIIA) or the presence of both dysmorphic neurons and balloon cells (FCDIIB). The year 2021 marks the 50th anniversary of the recognition of FCD as a cause of drug resistant epilepsy, and it is now the most common reason for epilepsy surgery. The causes of FCD remained unknown until relatively recently. The study of resected human FCD tissue using novel genomic technologies has led to remarkable advances in understanding the genetic basis of FCD. Mechanistic parallels have emerged between these non-neoplastic lesions and neoplastic disorders of cell growth and differentiation, especially through perturbations of the mammalian target of rapamycin (mTOR) signalling pathway. This narrative review presents the advances through which the aetiology of FCDII has been elucidated in chronological order, from recognition of an association between FCD and the mTOR pathway to the identification of somatic mosaicism within FCD tissue. We discuss the role of a two-hit mechanism, highlight current challenges and future directions in detecting somatic mosaicism in brain and discuss how knowledge of FCD may inform novel precision treatments of these focal epileptogenic malformations of human cortical development.
Asunto(s)
Epilepsia Refractaria/etiología , Epilepsia/metabolismo , Malformaciones del Desarrollo Cortical de Grupo I/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Encéfalo/metabolismo , Epilepsia Refractaria/genética , Epilepsia Refractaria/fisiopatología , Epilepsia/etiología , Epilepsia/genética , Epilepsia/fisiopatología , Humanos , Malformaciones del Desarrollo Cortical/genética , Malformaciones del Desarrollo Cortical de Grupo I/genética , Malformaciones del Desarrollo Cortical de Grupo I/fisiopatología , Mutación/genética , Neuronas/metabolismo , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/genéticaRESUMEN
BACKGROUND: The role of PI3K/AKT/mTOR pathway hyperactivation in localized brain overgrowth is evolving. We describe two patients with focal cortical dysplasia (FCD) who demonstrated somatic mutations in TSC1 and TSC2 genes in the dysplastic brain tissue but not peripheral blood. METHODS: Paired whole-exome sequencing was performed on genomic DNA extracted from blood and excised brain tissue in two children with FCD who underwent excision of dysplastic tissue. RESULTS: Patient 1, a 14-year boy, had drug-resistant focal epilepsy with onset at 20 months. His brain MRI showed abnormalities suggestive of FCD in the left superior and middle frontal lobes. Patient 2 presented at the age of 10 years with pharmaco-resistant focal epilepsy (onset at six years). His MRI suggested FCD in the left insular lobe. Both patients underwent surgical excision of FCD, and excised tissues were pathologically confirmed to have type IIb FCD. For patient 1, a missense mutation (c.64C > T; p.Arg22Trp) was detected in the TSC1 gene in DNA of dysplastic brain tissue but not peripheral blood lymphocytes. Similarly, for patient 2, a frameshift mutation (c.4258_4261delCAGT; p.Ser1420GlyfsTer55) in the TSC2 gene was identified in the brain tissue but not blood. Both gene variants are likely pathogenic and cause mTOR pathway activation. CONCLUSION: Our report of TSC1/TSC2 somatic mutations in patients with non-syndromic FCD suggests that localized hyperactivation of the mTOR pathway can cause focal malformations during cortical development and presents pharmacological targets for precision therapy in FCD management.
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Epilepsia Refractaria/etiología , Epilepsia/genética , Malformaciones del Desarrollo Cortical de Grupo I/genética , Proteína 1 del Complejo de la Esclerosis Tuberosa/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Adolescente , Niño , Epilepsia/complicaciones , Epilepsia/patología , Epilepsia/cirugía , Humanos , Masculino , Malformaciones del Desarrollo Cortical de Grupo I/complicaciones , Malformaciones del Desarrollo Cortical de Grupo I/patología , Malformaciones del Desarrollo Cortical de Grupo I/cirugíaRESUMEN
Focal cortical dysplasia (FCD) is a malformation of the cerebral cortex with poorly-defined epileptogenic zones (EZs), and poor surgical outcome in FCD is associated with inaccurate localization of the EZ. Hence, identifying novel epileptogenic markers to aid in the localization of EZ in patients with FCD is very much needed. High-throughput gene expression studies of FCD samples have the potential to uncover molecular changes underlying the epileptogenic process and identify novel markers for delineating the EZ. For this purpose, we, for the first time performed RNA sequencing of surgically resected paired tissue samples obtained from electrocorticographically graded high (MAX) and low spiking (MIN) regions of FCD type II patients and autopsy controls. We identified significant changes in the MAX samples of the FCD type II patients when compared to non-epileptic controls, but not in the case of MIN samples. We found significant enrichment for myelination, oligodendrocyte development and differentiation, neuronal and axon ensheathment, phospholipid metabolism, cell adhesion and cytoskeleton, semaphorins, and ion channels in the MAX region. Through the integration of both MAX vs non-epileptic control and MAX vs MIN RNA sequencing (RNA Seq) data, PLP1, PLLP, UGT8, KLK6, SOX10, MOG, MAG, MOBP, ANLN, ERMN, SPP1, CLDN11, TNC, GPR37, SLC12A2, ABCA2, ABCA8, ASPA, P2RX7, CERS2, MAP4K4, TF, CTGF, Semaphorins, Opalin, FGFs, CALB2, and TNC were identified as potential key regulators of multiple pathways related to FCD type II pathology. We have identified novel epileptogenic marker elements that may contribute to epileptogenicity in patients with FCD and could be possible markers for the localization of EZ.
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Potenciales de Acción/fisiología , Epilepsia/genética , Epilepsia/fisiopatología , Perfilación de la Expresión Génica , Malformaciones del Desarrollo Cortical de Grupo I/genética , Malformaciones del Desarrollo Cortical de Grupo I/fisiopatología , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Redes Reguladoras de Genes , Humanos , Masculino , Reproducibilidad de los Resultados , Transducción de Señal/genética , Adulto JovenRESUMEN
AIMS: Focal cortical dysplasia (FCD) type 2 is an epileptogenic malformation of the neocortex associated with somatic mutations in the mammalian target of rapamycin (mTOR) pathway. Histopathologically, FCD 2 is subdivided into FCD 2a and FCD 2b, the only discriminator being the presence of balloon cells (BCs) in FCD 2b. While pro-epileptogenic immune system activation and inflammatory responses are commonly detected in both subtypes, it is unknown what contextual role BCs play. METHODS: The present study employed RNA sequencing of surgically resected brain tissue from FCD 2a (n = 11) and FCD 2b (n = 20) patients compared to autopsy control (n = 9) focusing on three immune system processes: adaptive immunity, innate immunity and cytokine production. This analysis was followed by immunohistochemistry on a clinically well-characterised FCD 2 cohort. RESULTS: Differential expression analysis revealed stronger expression of components of innate immunity, adaptive immunity and cytokine production in FCD 2b than in FCD 2a, particularly complement activation and antigen presentation. Immunohistochemical analysis confirmed these findings, with strong expression of leukocyte antigen I and II in FCD 2b as compared to FCD 2a. Moreover, T-lymphocyte tissue infiltration was elevated in FCD 2b. Expression of markers of immune system activation in FCD 2b was concentrated in subcortical white matter. Lastly, antigen presentation was strongly correlated with BC load in FCD 2b lesions. CONCLUSION: We conclude that, next to mutation-driven mTOR activation and seizure activity, BCs are crucial drivers of inflammation in FCD 2b. Our findings indicate that therapies targeting inflammation may be beneficial in FCD 2b.
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Epilepsia/patología , Sistema Inmunológico/metabolismo , Malformaciones del Desarrollo Cortical de Grupo I/patología , Malformaciones del Desarrollo Cortical/patología , Serina-Treonina Quinasas TOR/metabolismo , Adolescente , Niño , Epilepsia/genética , Epilepsia/inmunología , Humanos , Masculino , Malformaciones del Desarrollo Cortical/genética , Malformaciones del Desarrollo Cortical/inmunología , Malformaciones del Desarrollo Cortical de Grupo I/genética , Malformaciones del Desarrollo Cortical de Grupo I/inmunología , Persona de Mediana Edad , Mutación/genética , Neocórtex/patología , Neuronas/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/inmunología , Sustancia Blanca/metabolismoRESUMEN
Stem cells are characterized by self-renewal and by their ability to differentiate into cells of various organs. With massive progress in 2D and 3D cell culture techniques, in vitro generation of various types of such organoids from patient-derived stem cells is now possible. As in vitro differentiation protocols are usually made to resemble human developmental processes, organogenesis of patient-derived stem cells can provide key information regarding a range of developmental diseases. Human stem cell-based in vitro modeling as opposed to using animal models can particularly benefit the evaluation of neurological diseases because of significant differences in structure and developmental processes between the human and the animal brain. This review focuses on stem cell-based in vitro modeling of neurodevelopmental disorders, more specifically, the fundamentals and technical advancements in monolayer neuron and brain organoid cultures. Furthermore, we discuss the drawbacks of the conventional culture method and explore the advanced, cutting edge 3D organoid models for several neurodevelopmental diseases, including genetic diseases such as Down syndrome, Rett syndrome, and Miller-Dieker syndrome, as well as brain malformations like macrocephaly and microcephaly. Finally, we discuss the limitations of the current organoid techniques and some potential solutions that pave the way for accurate modeling of neurological disorders in a dish.
Asunto(s)
Encéfalo/citología , Técnicas de Cultivo de Célula/métodos , Malformaciones del Desarrollo Cortical de Grupo I/patología , Trastornos del Neurodesarrollo/patología , Neuronas/fisiología , Animales , Encéfalo/patología , Diferenciación Celular/fisiología , Modelos Animales de Enfermedad , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Malformaciones del Desarrollo Cortical de Grupo I/genética , Ratones , Trastornos del Neurodesarrollo/genética , Neurogénesis/genética , Neuronas/patología , Neuronas/trasplante , Organoides/patología , Organoides/fisiología , Quimera por TrasplanteRESUMEN
OBJECTIVE: Focal cortical dysplasia type II (FCDII) is a malformation of cortex development commonly found in children with drug-resistant epilepsy. FCDII has been associated with somatic mutations in mammalian target of rapamycin (mTOR)-related pathway genes and an upregulation of mTOR. Somatic mutations were found in 10%-63% of FCDII samples; the frequency of the mutant allele was 0.93%-33.5%. This study aimed to find new candidate genes involved in FCDII. METHODS: We collected resected FCD lesions, perilesional brain tissues, and peripheral blood from 17 children with pathologically confirmed FCDII. We performed whole exome sequencing and followed a set of screening and analysis strategies to identify potentially deleterious somatic variants (PDSVs) in brain-expressed genes. We performed site-specific amplicon sequencing to validate the results. We also performed an in vitro functional study on an IRS1 variant. RESULTS: In six of 17 samples, we identified seven PDSVs in seven genes, including two frameshift variants and five missense variants. The frequencies of the variant allele were 1.29%-5.50%. The genes were MTOR, TSC2, IRS1, RAB6B, RALA, HTR6, and ZNF337. PDSVs in IRS1, RAB6B, ZNF337, RALA, and HTR6 had not been previously associated with FCD. In one lesion, two PDSVs were found in two genes. In a transfected cell line, we demonstrated that the c.1791dupG (identified in FCDII from Patient 1) led to a truncated IRS1 and significant mTOR hyperactivation compared to cells that carried wild-type IRS1. mTOR was also activated in FCDII tissue from Patient 1. SIGNIFICANCE: Seven PDSVs were identified in FCDII lesions in six of 17 children. Five variant genes had not been previously associated with cortical malformations. We demonstrated that the IRS1 variant led to mTOR hyperactivation in vitro. Although functional experiments are needed, the results provide evidence for novel candidate genes in the pathogenesis of FCDII.
Asunto(s)
Epilepsia/genética , Predisposición Genética a la Enfermedad/genética , Malformaciones del Desarrollo Cortical de Grupo I/genética , Preescolar , Femenino , Humanos , Lactante , Masculino , MutaciónRESUMEN
AIMS: Oxidative stress is evident in resected epileptogenic brain tissue of patients with developmental brain malformations related to mammalian target of rapamycin activation: tuberous sclerosis complex (TSC) and focal cortical dysplasia type IIb (FCD IIb). Whether chronic activation of anti-oxidant pathways is beneficial or contributes to pathology is not clear. METHODS: We investigated oxidative stress markers, including haem oxygenase 1, ferritin and the inflammation associated microRNA-155 in surgically resected epileptogenic brain tissue of TSC (n = 10) and FCD IIb (n = 8) patients and in a TSC model (Tsc1GFAP-/- mice) using immunohistochemistry, in situ hybridization, real-time quantitative PCR and immunoblotting. Using human foetal astrocytes we performed an in vitro characterization of the anti-oxidant response to acute and chronic oxidative stress and evaluated overexpression of the disease-relevant pro-inflammatory microRNA-155. RESULTS: Resected TSC or FCD IIb tissue displayed higher expression of oxidative stress markers and microRNA-155. Tsc1GFAP-/- mice expressed more microRNA-155 and haem oxygenase 1 in the brain compared to wild-type, preceding the typical development of spontaneous seizures in these animals. In vitro, chronic microRNA-155 overexpression induced haem oxygenase 1, iron regulatory elements and increased susceptibility to oxidative stress. Overexpression of iron regulatory genes was also detected in patients with TSC, FCD IIb and Tsc1GFAP-/- mice. CONCLUSION: Our results demonstrate that early and sustained activation of anti-oxidant signalling and dysregulation of iron metabolism are a pathological hallmark of FCD IIb and TSC. Our findings suggest novel therapeutic strategies aimed at controlling the pathological link between both processes.
Asunto(s)
Antioxidantes/metabolismo , Epilepsia/metabolismo , Hierro/metabolismo , Malformaciones del Desarrollo Cortical/complicaciones , Malformaciones del Desarrollo Cortical/metabolismo , Redes y Vías Metabólicas , Animales , Células Cultivadas , Encefalitis/genética , Encefalitis/metabolismo , Epilepsia/complicaciones , Epilepsia/genética , Femenino , Ferritinas/metabolismo , Proteína Ácida Fibrilar de la Glía/genética , Hemo-Oxigenasa 1/metabolismo , Humanos , Masculino , Malformaciones del Desarrollo Cortical/genética , Malformaciones del Desarrollo Cortical de Grupo I/genética , Malformaciones del Desarrollo Cortical de Grupo I/metabolismo , Ratones , Ratones Noqueados , MicroARNs/genética , Estrés Oxidativo , Esclerosis Tuberosa/genética , Esclerosis Tuberosa/metabolismoRESUMEN
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.
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Epilepsias Parciales/metabolismo , Epilepsia/metabolismo , Proteínas Activadoras de GTPasa/genética , Malformaciones del Desarrollo Cortical de Grupo I/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Animales , Epilepsias Parciales/genética , Epilepsia/genética , Proteínas Activadoras de GTPasa/metabolismo , Técnicas Genéticas , Células HEK293 , Humanos , Malformaciones del Desarrollo Cortical de Grupo I/genética , Ratones , Ratones Noqueados , MutaciónRESUMEN
Single germline or somatic activating mutations of mammalian target of rapamycin (mTOR) pathway genes are emerging as a major cause of type II focal cortical dysplasia (FCD), hemimegalencephaly (HME) and tuberous sclerosis complex (TSC). A double-hit mechanism, based on a primary germline mutation in one allele and a secondary somatic hit affecting the other allele of the same gene in a small number of cells, has been documented in some patients with TSC or FCD. In a patient with HME, severe intellectual disability, intractable seizures and hypochromic skin patches, we identified the ribosomal protein S6 (RPS6) p.R232H variant, present as somatic mosaicism at ~15.1% in dysplastic brain tissue and ~11% in blood, and the MTOR p.S2215F variant, detected as ~8.8% mosaicism in brain tissue, but not in blood. Overexpressing the two variants independently in animal models, we demonstrated that MTOR p.S2215F caused neuronal migration delay and cytomegaly, while RPS6 p.R232H prompted increased cell proliferation. Double mutants exhibited a more severe phenotype, with increased proliferation and migration defects at embryonic stage and, at postnatal stage, cytomegalic cells exhibiting eccentric nuclei and binucleation, which are typical features of balloon cells. These findings suggest a synergistic effect of the two variants. This study indicates that, in addition to single activating mutations and double-hit inactivating mutations in mTOR pathway genes, severe forms of cortical dysplasia can also result from activating mutations affecting different genes in this pathway. RPS6 is a potential novel disease-related gene.
Asunto(s)
Hemimegalencefalia/genética , Proteína S6 Ribosómica/genética , Serina-Treonina Quinasas TOR/genética , Animales , Encéfalo/metabolismo , Niño , Epilepsia Refractaria/genética , Epilepsia Refractaria/metabolismo , Epilepsia/genética , Femenino , Humanos , Malformaciones del Desarrollo Cortical/genética , Malformaciones del Desarrollo Cortical/metabolismo , Malformaciones del Desarrollo Cortical de Grupo I/genética , Ratones , Mosaicismo , Mutación , Neuronas/metabolismo , Proteína S6 Ribosómica/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
It is challenging to estimate genetic variant burden across different subtypes of epilepsy. Herein, we used a comparative approach to assess the genetic variant burden and genotype-phenotype correlations in four most common brain lesions in patients with drug-resistant focal epilepsy. Targeted sequencing analysis was performed for a panel of 161 genes with a mean coverage of >400×. Lesional tissue was histopathologically reviewed and dissected from hippocampal sclerosis (n = 15), ganglioglioma (n = 16), dysembryoplastic neuroepithelial tumors (n = 8), and focal cortical dysplasia type II (n = 15). Peripheral blood (n = 12) or surgical tissue samples histopathologically classified as lesion-free (n = 42) were available for comparison. Variants were classified as pathogenic or likely pathogenic according to American College of Medical Genetics and Genomics guidelines. Overall, we identified pathogenic and likely pathogenic variants in 25.9% of patients with a mean coverage of 383×. The highest number of pathogenic/likely pathogenic variants was observed in patients with ganglioglioma (43.75%; all somatic) and dysembryoplastic neuroepithelial tumors (37.5%; all somatic), and in 20% of cases with focal cortical dysplasia type II (13.33% somatic, 6.67% germline). Pathogenic/likely pathogenic positive genes were disorder specific and BRAF V600E the only recurrent pathogenic variant. This study represents a reference for the genetic variant burden across the four most common lesion entities in patients with drug-resistant focal epilepsy. The observed large variability in variant burden by epileptic lesion type calls for whole exome sequencing of histopathologically well-characterized tissue in a diagnostic setting and in research to discover novel disease-associated genes.
Asunto(s)
Neoplasias Encefálicas/genética , Epilepsia Refractaria/genética , Epilepsia/genética , Predisposición Genética a la Enfermedad/genética , Variación Genética , Encéfalo , Ganglioglioma/genética , Estudios de Asociación Genética , Alemania , Glioma/genética , Humanos , Malformaciones del Desarrollo Cortical de Grupo I/genética , Esclerosis/genética , Secuenciación del ExomaRESUMEN
Focal cortical dysplasia type II (FCDII) is a cerebral cortex malformation characterized by local cortical structure disorganization, neuronal dysmorphology, and refractory epilepsy. Brain somatic mutations in several genes involved in the PI3K/AKT/mTOR pathway are associated with FCDII, but they are only found in a proportion of patients with FCDII. The genetic causes underlying the development FCDII in other patients remain unclear. Here, we carried out whole exome sequencing and targeted sequencing in paired brain-blood DNA from patients with FCDII and identified a brain somatic doublet mutation c.(A104T, C105A) in the Ras homolog, mTORC1 binding (RHEB) gene, which led to the RHEB p.Y35L mutation in one patient with FCDII. This RHEB mutation carrier had a dramatic increase of ribosomal protein S6 phosphorylation, indicating mTOR activation in the region of the brain lesion. The RHEB p.Y35L mutant protein had increased GTPλS-binding activity compared with wild-type RHEB. Overexpression of the RHEB p.Y35L variant in cultured cells also resulted in elevated S6 phosphorylation compared to wild-type RHEB. Importantly, in utero electroporation of the RHEB p.Y35L variant in mice induced S6 phosphorylation, cytomegalic neurons, dysregulated neuron migration, abnormal electroencephalogram, and seizures, all of which are found in patients with FCDII. Rapamycin treatment rescued abnormal electroencephalograms and alleviated seizures in these mice. These results demonstrate that brain somatic mutations in RHEB are also responsible for the pathogenesis of FCDII, indicating that aberrant activation of mTOR signaling is a primary driver and potential drug target for FCDII.
Asunto(s)
Epilepsia/genética , Malformaciones del Desarrollo Cortical de Grupo I/genética , Fosfatidilinositol 3-Quinasas/genética , Proteína Homóloga de Ras Enriquecida en el Cerebro/genética , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Epilepsia/diagnóstico por imagen , Epilepsia/patología , Femenino , Humanos , Masculino , Malformaciones del Desarrollo Cortical de Grupo I/diagnóstico por imagen , Malformaciones del Desarrollo Cortical de Grupo I/patología , Ratones , Mutación , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteína Homóloga de Ras Enriquecida en el Cerebro/metabolismo , Alineación de Secuencia , Serina-Treonina Quinasas TOR/metabolismo , Secuenciación del ExomaRESUMEN
Malformations of cortical development (MCDs) include many different Central Nervous System (CNS) disorders related to a complex process of cortex formation. In children with refractory epilepsy to drug treatment undergoing surgery, focal cortical dysplasia (FCD), one of the MCDs, is considered the most common structural brain lesion found. This study aimed to study the possible alterations in neural differentiation process of human induced pluripotent stem cells (hiPSCs) related to migration and synaptic aspects from fibroblasts of two individuals affected by FCD type IIb (45-year-old male and 12-year-old female) and normal individuals. At the days 14th, 22nd and 35th, hiPSCs were neural differentiated and analyzed. Using qRT-PCR approach, the expression of 9 genes associated with synaptic and neural migration were quantified. Diagnostic of both patients was consistent with FCD type IIb. Our results showed that in all processes and groups, individuals with dysplasia presented alterations in most part of the genes in relation to control individuals. According to our results, it is suggested that the different expressions are mainly involved in alterations of the expression of receptors and capture sites, timing, coupling of synaptic vesicles with the presynaptic membrane, regulation of ion channel and synaptic exocytosis, imbalance of the apoptosis process and abnormal microtubules that may also contribute to delays in synaptogenesis. Thus, brain formation with dysplasia is probably influenced by these genes studied.
Asunto(s)
Movimiento Celular/fisiología , Epilepsia/patología , Células Madre Pluripotentes Inducidas/patología , Malformaciones del Desarrollo Cortical de Grupo I/patología , Neurogénesis/fisiología , Neuronas/patología , Sinapsis/patología , Niño , Epilepsia/genética , Epilepsia/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Malformaciones del Desarrollo Cortical de Grupo I/genética , Malformaciones del Desarrollo Cortical de Grupo I/metabolismo , Persona de Mediana Edad , Neuronas/metabolismo , Sinapsis/metabolismoRESUMEN
Focal cortical dysplasia (FCD) is one of the most common pathologies associated with drug-resistant epilepsy (DRE). The pharmacological targets remain obscured, as the molecular mechanisms underlying FCD are unclear. Implications of epigenetically modulated aberrant gene expression in disease progression are reported in various DRE pathologies except FCD. Here we performed genome-wide CpG-DNA methylation profiling by methylated DNA immunoprecipitation (MeDIP) microarray and RNA sequencing (RNAseq) on cortical tissues resected from FCD type II patients. A total of 19088 sites showed altered DNA methylation in all the CpG islands. Of these, 5725 sites were present in the promoter regions, of which 176 genes showed an inverse correlation between methylation and gene expression. Many of these 176 genes were found to belong to a cohesive network of physically interacting proteins linked to several cellular functions. Pathway analysis revealed significant enrichment of receptor tyrosine kinases (RTK), EGFR, PDGFRA, NTRK3, and mTOR signalling pathways. This is the first study that investigates the epigenetic signature associated with FCD type II pathology. The candidate genes and pathways identified in this study may play a crucial role in the regulation of the pathogenic mechanisms of epileptogenesis associated with FCD type II pathologies.
Asunto(s)
Metilación de ADN , Epigénesis Genética , Epilepsia/genética , Epilepsia/metabolismo , Estudio de Asociación del Genoma Completo , Malformaciones del Desarrollo Cortical de Grupo I/genética , Malformaciones del Desarrollo Cortical de Grupo I/metabolismo , Transducción de Señal , Susceptibilidad a Enfermedades , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo/métodos , Humanos , Regiones Promotoras GenéticasRESUMEN
Epileptogenic mechanisms in focal cortical dysplasia (FCD) remain elusive, as no animal models faithfully recapitulate FCD seizures, which have distinct electrographic features and a wide range of semiologies. Given that DEPDC5 plays significant roles in focal epilepsies with FCD, we used in utero electroporation with clustered regularly interspaced short palindromic repeats gene deletion to create focal somatic Depdc5 deletion in the rat embryonic brain. Animals developed spontaneous seizures with focal pathological and electroclinical features highly clinically relevant to FCD IIA, paving the way toward understanding its pathogenesis and developing mechanistic-based therapies. Ann Neurol 2018;83:140-146.
Asunto(s)
Epilepsia/genética , Epilepsia/fisiopatología , Malformaciones del Desarrollo Cortical de Grupo I/genética , Malformaciones del Desarrollo Cortical de Grupo I/fisiopatología , Proteínas Represoras/genética , Eliminación de Secuencia/genética , Animales , Animales Recién Nacidos , Encéfalo/citología , Ondas Encefálicas/genética , Electroencefalografía , Electroporación , Embrión de Mamíferos , Epilepsia/patología , Femenino , Proteínas Activadoras de GTPasa , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Técnicas In Vitro , Imagen por Resonancia Magnética , Masculino , Malformaciones del Desarrollo Cortical de Grupo I/patología , Neuronas/fisiología , Ratas , Proteínas Represoras/metabolismo , Proteína S6 Ribosómica/metabolismoRESUMEN
Focal Cortical Dysplasia (FCD), a common type of Malformations of cortical development, may result from an early disturbance in the migration and final arrangement of the cortical architecture of immature neurons. FCD type II is now known to be due to a post-zygotic somatic mutation that involves the mTOR and AKT pathways. The aim of the present study was to investigate the possible differences in neurogenesis and neurodifferentiation of iPSCs (induced pluripotent stem cells) from fibroblasts of individuals affected by FCD type II (2) and normal individuals (2). iPSCs were generated from skin fibroblasts of FCD individuals and healthy individuals. The reprogramming was done through the fibroblasts exposure to viral vectors containing the OCT4, KLF4, SOX2, and c-MYC genes and the clones were characterized by immunohistochemistry. iPSCs were neurodifferentiated and analyzed at the 14th, 22nd and 35th days. We also analyzed the cerebral cortex tissue, fibroblasts and iPSCs cells from the individuals. Through qRT-PCR, the expression of 4 genes involved in Notch signaling process were quantified. In general, individuals with dysplasia presented increase and decrease in the relative quantification in the most genes analyzed compared to control individuals in all processes and study groups. We suggest that, during embryonic neurogenesis, the neural precursor cells of FCD type II individuals present increase and decrease in gene expression in the Notch signaling pathway causing cortical formation disorders and can be seen as a candidate for the developmental changes observed in the cerebral cortex of individuals with FCD type II. This altered gene expression may be related to brain formation with dysplasia.