RESUMO
The most frequent genetic cause of focal epilepsies is variations in the GAP activity toward RAGs 1 complex genes DEP domain containing 5 (DEPDC5), nitrogen permease regulator 2-like protein (NPRL2) and nitrogen permease regulator 3-like protein (NPRL3). Because these variations are frequent and associated with a broad spectrum of focal epilepsies, a unique pathology categorized as GATORopathy can be conceptualized. Animal models recapitulating the clinical features of patients are essential to decipher GATORopathy. Although several genetically modified animal models recapitulate DEPDC5-related epilepsy, no models have been reported for NPRL2- or NPRL3-related epilepsies. Here, we conditionally deleted Nprl2 and Nprl3 from the dorsal telencephalon in mice [Emx1cre/+; Nprl2f/f (Nprl2-cKO) and Emx1cre/+; Nprl3f/f (Nprl3-cKO)] and compared their phenotypes with Nprl2+/-, Nprl3+/- and Emx1cre/+; Depdc5f/f (Depdc5-cKO) mice. Nprl2-cKO and Nprl3-cKO mice recapitulated the major abnormal features of patients-spontaneous seizures, and dysmorphic enlarged neuronal cells with increased mechanistic target of rapamycin complex 1 signaling-similar to Depdc5-cKO mice. Chronic postnatal rapamycin administration dramatically prolonged the survival period and inhibited seizure occurrence but not enlarged neuronal cells in Nprl2-cKO and Nprl3-cKO mice. However, the benefit of rapamycin after withdrawal was less durable in Nprl2- and Nprl3-cKO mice compared with Depdc5-cKO mice. Further studies using these conditional knockout mice will be useful for understanding GATORopathy and for the identification of novel therapeutic targets.
Assuntos
Epilepsias Parciais , Epilepsia , Animais , Modelos Animais de Doenças , Epilepsias Parciais/genética , Epilepsia/genética , Proteínas Ativadoras de GTPase/genética , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Knockout , Mutação , Nitrogênio/metabolismo , Convulsões , Sirolimo , Telencéfalo/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Thioredoxin, encoded by Txn1, acts as a critical antioxidant in the defense against oxidative stress by regulating the dithiol/disulfide balance of interacting proteins. The role of thioredoxin in the central nervous system (CNS) is largely unknown. A phenotype-driven study of N-ethyl-N-nitrosourea-mutated rats with wild-running seizures revealed the importance of Txn1 mutations in CNS degeneration. Genetic mapping identified Txn1-F54L in the epileptic rats. The insulin-reducing activity of Txn1-F54L was approximately one-third of that of the wild-type (WT). Bilateral symmetrical vacuolar degeneration in the midbrain, mainly in the thalamus and the inferior colliculus, was observed in the Txn1-F54L rats. The lesions displayed neuronal and oligodendrocytic cell death. Neurons in Txn1-F54L rats showed morphological changes in the mitochondria. Vacuolar degeneration peaked at five weeks of age, and spontaneous repair began at seven weeks. The TUNEL assay showed that fibroblasts derived from homozygotes were susceptible to cell death under oxidative stress. In five-week-old WT rats, energy metabolism in the thalamus was significantly higher than that in the cerebral cortex. In conclusion, in juvenile rats, Txn1 seems to play an essential role in reducing oxidative stress in the midbrains with high energy metabolism.
Assuntos
Estresse Oxidativo , Tiorredoxinas , Animais , Ratos , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Estresse Oxidativo/fisiologia , Mesencéfalo/metabolismo , Antioxidantes , OxirreduçãoRESUMO
Thioredoxin, encoded by Txn1, is a critical antioxidant that protects against oxidative damage by regulating the dithiol/disulfide balance of interacting proteins. We recently discovered the Adem rat, an epileptic rat harboring the Txn1-F54L mutation, characterized by wild running and vacuolar degeneration in the midbrain. This study aimed to characterize the classification of epilepsy in Adem rats. We performed simultaneous video-electroencephalographic recordings, magnetic resonance imaging, neurotransmitter measurements using gas chromatography-mass spectrometry (GC-MS), and immunohistochemistry. Adem rats exhibited absence, tonic, and focal seizures. The type of epilepsy was classified as combined generalized and focal epilepsy. Neurotransmitters in the midbrain and cortex were measured at 3 weeks of age, when neuronal cell death occurs in the midbrain. The results of GC-MS ruled out the dominance of the excitatory system in the midbrain and cortex of Adem rats. Activation of astrocytes and microglia was more pronounced at 5 weeks of age, at which time epileptic seizures occurred frequently. The underlying pathology in Adem rats remains unknown. However, glial cell activation and inflammation may play a significant role in the occurrence of epilepsy.
Assuntos
Epilepsias Parciais , Epilepsia Generalizada , Epilepsia , Animais , Modelos Animais de Doenças , Eletroencefalografia , Epilepsias Parciais/genética , Epilepsia/patologia , Ratos , ConvulsõesRESUMO
The importance of epigenetic control in the development of the central nervous system has recently been attracting attention. Methylation patterns of lysine 4 and lysine 36 in histone H3 (H3K4 and H3K36) in the central nervous system are highly conserved among species. Numerous complications of body malformations and neuropsychiatric disorders are due to abnormal histone H3 methylation modifiers. In this study, we analyzed a Japanese family with a dominant inheritance of symptoms including Marfan syndrome-like minor physical anomalies (MPAs), intellectual disability, and schizophrenia (SCZ). We performed positional cloning for this family using a single nucleotide polymorphism (SNP) array and whole-exome sequencing, which revealed a missense coding strand mutation (rs1555289644, NM_032590.4: c.2173G>A, p.A725T) in exon 15 on the plant homeodomain of the KDM2B gene as a possible cause of the disease in the family. The exome sequencing revealed that within the coding region, only a point mutation in KDM2B was present in the region with the highest logarithm of odds score of 2.41 resulting from whole genome linkage analysis. Haplotype analysis revealed co-segregation with four affected family members (IV-9, III-4, IV-5, and IV-8). Lymphoblastoid cell lines from the proband with this mutation showed approximately halved KDM2B expression in comparison with healthy controls. KDM2B acts as an H3K4 and H3K36 histone demethylase. Our findings suggest that haploinsufficiency of KDM2B in the process of development, like other H3K4 and H3K36 methylation modifiers, may have caused MPAs, intellectual disability, and SCZ in this Japanese family.
Assuntos
Proteínas F-Box/genética , Deficiência Intelectual/genética , Histona Desmetilases com o Domínio Jumonji/genética , Síndrome de Marfan/genética , Esquizofrenia/genética , Clonagem Molecular/métodos , Análise Mutacional de DNA , Exoma/genética , Feminino , Ligação Genética , Predisposição Genética para Doença , Haplótipos/genética , Histona Desmetilases/genética , Histonas/genética , Humanos , Deficiência Intelectual/epidemiologia , Deficiência Intelectual/patologia , Japão/epidemiologia , Masculino , Síndrome de Marfan/epidemiologia , Síndrome de Marfan/patologia , Metilação , Mutação/genética , Linhagem , Esquizofrenia/epidemiologia , Esquizofrenia/patologia , Sequenciamento do ExomaAssuntos
Antipsicóticos , Eletroconvulsoterapia , Esquizofrenia , Antipsicóticos/farmacologia , Antipsicóticos/uso terapêutico , Haloperidol/farmacologia , Haloperidol/uso terapêutico , Humanos , Piperazinas , Piperidinas , Esquizofrenia/tratamento farmacológico , Esquizofrenia Resistente ao TratamentoRESUMO
DEP-domain containing 5 (DEPDC5), encoding a repressor of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, has recently emerged as a major gene mutated in familial focal epilepsies and focal cortical dysplasia. Here we established a global knockout rat using TALEN technology to investigate in vivo the impact of Depdc5-deficiency. Homozygous Depdc5(-/-) embryos died from embryonic day 14.5 due to a global growth delay. Constitutive mTORC1 hyperactivation was evidenced in the brains and in cultured fibroblasts of Depdc5(-/-) embryos, as reflected by enhanced phosphorylation of its downstream effectors S6K1 and rpS6. Consistently, prenatal treatment with mTORC1 inhibitor rapamycin rescued the phenotype of Depdc5(-/-) embryos. Heterozygous Depdc5(+/-) rats developed normally and exhibited no spontaneous electroclinical seizures, but had altered cortical neuron excitability and firing patterns. Depdc5(+/-) rats displayed cortical cytomegalic dysmorphic neurons and balloon-like cells strongly expressing phosphorylated rpS6, indicative of mTORC1 upregulation, and not observed after prenatal rapamycin treatment. These neuropathological abnormalities are reminiscent of the hallmark brain pathology of human focal cortical dysplasia. Altogether, Depdc5 knockout rats exhibit multiple features of rodent models of mTORopathies, and thus, stand as a relevant model to study their underlying pathogenic mechanisms.
Assuntos
Córtex Cerebral/anormalidades , Modelos Animais de Doenças , Desenvolvimento Embrionário/genética , Complexos Multiproteicos/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Animais Geneticamente Modificados , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiopatologia , Desenvolvimento Embrionário/efeitos dos fármacos , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Genótipo , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/antagonistas & inibidores , Neurônios/patologia , Neurônios/fisiologia , Fosforilação , Ratos , Ratos Endogâmicos F344 , Ratos Wistar , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/administração & dosagem , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
OBJECTIVE: The DEPDC5 (DEP domain-containing protein 5) gene, encoding a repressor of the mTORC1 signaling pathway, has recently emerged as a major gene mutated in familial focal epilepsies. We aimed to further extend the role of DEPDC5 to focal cortical dysplasias (FCDs). METHODS: Seven patients from 4 families with DEPDC5 mutations and focal epilepsy associated with FCD were recruited and investigated at the clinical, neuroimaging, and histopathological levels. The DEPDC5 gene was sequenced from genomic blood and brain DNA. RESULTS: All patients had drug-resistant focal epilepsy, 5 of them underwent surgery, and 1 had a brain biopsy. Electroclinical phenotypes were compatible with FCD II, although magnetic resonance imaging (MRI) was typical in only 4 cases. Histopathology confirmed FCD IIa in 2 patients (including 1 MRI-negative case) and showed FCD I in 2 other patients, and remained inconclusive in the last 2 patients. Three patients were seizure-free postsurgically, and 1 had a worthwhile improvement. Sequencing of blood DNA revealed truncating DEPDC5 mutations in all 4 families; 1 mutation was found to be mosaic in an asymptomatic father. A brain somatic DEPDC5 mutation was identified in 1 patient in addition to the germline mutation. INTERPRETATION: Germline, germline mosaic, and brain somatic DEPDC5 mutations may cause epilepsy associated with FCD, reinforcing the link between mTORC1 pathway and FCDs. Similarly to other mTORopathies, a "2-hit" mutational model could be responsible for cortical lesions. Our study also indicates that epilepsy surgery is a valuable alternative in the treatment of drug-resistant DEPDC5-positive focal epilepsies, even if the MRI is unremarkable.
Assuntos
Epilepsias Parciais/diagnóstico , Epilepsias Parciais/genética , Malformações do Desenvolvimento Cortical/diagnóstico , Malformações do Desenvolvimento Cortical/genética , Mutação/genética , Proteínas Repressoras/genética , Adolescente , Adulto , Criança , Feminino , Proteínas Ativadoras de GTPase , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Adulto JovemRESUMO
Leucin-rich, glioma inactivated 1 (LGI1) is a secreted protein linked to human seizures of both genetic and autoimmune aetiology. Mutations in the LGI1 gene are responsible for autosomal dominant temporal lobe epilepsy with auditory features, whereas LGI1 autoantibodies are involved in limbic encephalitis, an acquired epileptic disorder associated with cognitive impairment. We and others previously reported that Lgi1-deficient mice have early-onset spontaneous seizures leading to premature death at 2-3 weeks of age. Yet, where and when Lgi1 deficiency causes epilepsy remains unknown. To address these questions, we generated Lgi1 conditional knockout (cKO) mice using a set of universal Cre-driver mouse lines. Selective deletion of Lgi1 was achieved in glutamatergic pyramidal neurons during embryonic (Emx1-Lgi1cKO) or late postnatal (CaMKIIα-Lgi1cKO) developmental stages, or in gamma amino butyric acidergic (GABAergic) parvalbumin interneurons (PV-Lgi1cKO). Emx1-Lgi1cKO mice displayed early-onset and lethal seizures, whereas CaMKIIα-Lgi1cKO mice presented late-onset occasional seizures associated with variable reduced lifespan. In contrast, neither spontaneous seizures nor increased seizure susceptibility to convulsant were observed when Lgi1 was deleted in parvalbumin interneurons. Together, these data showed that LGI1 depletion restricted to pyramidal cells is sufficient to generate seizures, whereas seizure thresholds were unchanged after depletion in gamma amino butyric acidergic parvalbumin interneurons. We suggest that LGI1 secreted from excitatory neurons, but not parvalbumin inhibitory neurons, makes a major contribution to the pathogenesis of LGI1-related epilepsies. Our data further indicate that LGI1 is required from embryogenesis to adulthood to achieve proper circuit functioning.
Assuntos
Encéfalo/metabolismo , Neurônios/metabolismo , Proteínas/fisiologia , Convulsões/etiologia , Fatores Etários , Animais , Animais Recém-Nascidos , Encéfalo/fisiopatologia , Eletroencefalografia , Embrião de Mamíferos/metabolismo , Neurônios GABAérgicos/metabolismo , Interneurônios/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Proteínas/genética , Células Piramidais/metabolismo , Convulsões/genéticaRESUMO
Mutations of the leucine-rich glioma-inactivated 1 (LGI1) gene cause an autosomal dominant partial epilepsy with auditory features also known as autosomal-dominant lateral temporal lobe epilepsy. LGI1 is also the main antigen present in sera and cerebrospinal fluids of patients with limbic encephalitis and seizures, highlighting its importance in a spectrum of epileptic disorders. LGI1 encodes a neuronal secreted protein, whose brain function is still poorly understood. Here, we generated, by ENU (N-ethyl-N-nitrosourea) mutagenesis, Lgi1-mutant rats carrying a missense mutation (L385R). We found that the L385R mutation prevents the secretion of Lgi1 protein by COS7 transfected cells. However, the L385R-Lgi1 protein was found at low levels in the brains and cultured neurons of Lgi1-mutant rats, suggesting that mutant protein may be destabilized in vivo. Studies on the behavioral phenotype and intracranial electroencephalographic signals from Lgi1-mutant rats recalled several features of the human genetic disorder. We show that homozygous Lgi1-mutant rats (Lgi1(L385R/L385R)) generated early-onset spontaneous epileptic seizures from P10 and died prematurely. Heterozygous Lgi1-mutant rats (Lgi1(+/L385R)) were more susceptible to sound-induced, generalized tonic-clonic seizures than control rats. Audiogenic seizures were suppressed by antiepileptic drugs such as carbamazepine, phenytoin and levetiracetam, which are commonly used to treat partial seizures, but not by the prototypic absence seizure drug, ethosuximide. Our findings provide the first rat model with a missense mutation in Lgi1 gene, an original model complementary to knockout mice. This study revealed that LGI1 disease-causing missense mutations might cause a depletion of the protein in neurons, and not only a failure of Lgi1 secretion.
Assuntos
Epilepsia/etiologia , Proteínas/genética , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Anticonvulsivantes/farmacologia , Encéfalo/metabolismo , Células COS , Carbamazepina/farmacologia , Células Cultivadas , Chlorocebus aethiops , Modelos Animais de Doenças , Eletroencefalografia , Epilepsias Parciais/tratamento farmacológico , Epilepsias Parciais/genética , Epilepsia/tratamento farmacológico , Epilepsia/genética , Epilepsia Reflexa/tratamento farmacológico , Epilepsia Reflexa/genética , Etossuximida/farmacologia , Heterozigoto , Homozigoto , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Levetiracetam , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Neurônios/metabolismo , Fenitoína/farmacologia , Piracetam/análogos & derivados , Piracetam/farmacologia , Ratos MutantesRESUMO
Hematopoietic stem cell (HSC) transplantation is extensively studied in mouse models, but their limited scale presents challenges for effective engraftment and comprehensive evaluations. Rats, owing to their larger size and anatomical similarity to humans, offer a promising alternative. In this study, we establish a rat model with the KitV834M mutation, mirroring KitW41 mice often used in KIT signaling and HSC research. KitV834M rats are viable and fertile, displaying anemia and mast cell depletion similar to KitW41 mice. The colony-forming unit assay revealed that the KitV834M mutation leads to reduced proliferation and loss of or decreased pluripotency of hematopoietic stem and progenitor cells (HSPCs), resulting in diminished competitive repopulating capacity of KitV834M HSPCs in competitive transplantation assays. Importantly, KitV834M rats support donor rat-HSC engraftment without irradiation. Leveraging the larger scale of this rat model enhances our understanding of HSC biology and transplantation dynamics, potentially advancing our knowledge in this field.
Assuntos
Anemia , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Animais , Ratos , Células-Tronco Hematopoéticas , Ensaio de Unidades Formadoras de Colônias , Anemia/genética , Mutação , Camundongos Endogâmicos C57BLRESUMO
The type I CRISPR system has recently emerged as a promising tool, especially for large-scale genomic modification, but its application to generate model animals by editing zygotes had not been established. In this study, we demonstrate genome editing in zygotes using the type I-E CRISPR-Cas3 system, which efficiently generates deletions of several thousand base pairs at targeted loci in mice with 40%-70% editing efficiency without off-target mutations. To overcome the difficulties associated with detecting the variable deletions, we used a newly long-read sequencing-based multiplex genotyping approach. Demonstrating remarkable versatility, our Cas3-based technique was successfully extended to rats as well as mice, even by zygote electroporation methods. Knockin for SNP exchange and genomic replacement with a donor plasmid were also achieved in mice. This pioneering work with the type I CRISPR zygote editing system offers increased flexibility and broader applications in genetic engineering across different species.
RESUMO
Background: Restless legs syndrome (RLS) is a neurological sensorimotor disorder characterized by an uncontrollable urge to move the legs. In the perioperative period, patients with RLS may experience an acute exacerbation of symptoms. Although studies on the exacerbation of RLS after brain surgery are limited, we present a case wherein symptoms worsened following left amygdalohippocampectomy. Case Presentation: A 58-year-old woman diagnosed with mesiotemporal lobe epilepsy accompanied by left hippocampal sclerosis underwent a left amygdalohippocampectomy. The patient reported uncomfortable sensations in the lower limbs preoperatively. However, the urge to move her legs was manageable and not distinctly diagnosed with RLS. The symptoms began to deteriorate on the fifth postoperative day primarily affecting the legs and back, with a notable emphasis on the right side. Pramipexole treatment effectively ameliorated these symptoms. Conclusion: No reports are available highlighting the exacerbation of RLS after amygdalohippocampectomy. Perioperative factors, such as anesthesia and iron deficiency due to hemorrhage, have been proposed as aggravating factors for RLS; however, the asymmetry of RLS, particularly the atypical right-sided exacerbation in this case, makes it unlikely that this was the primary cause. A negative correlation between opioid receptor availability in the amygdala and RLS severity has been reported, suggesting that amygdalohippocampectomy contributes to the exacerbation of RLS symptoms. This case provides valuable insights into the possible involvement of the amygdala in the pathophysiology of RLS and practical considerations for the clinical management of the condition.
RESUMO
Immunodeficient animals are valuable models for the engraftment of exogenous tissues; they are widely used in many fields, including the creation of humanized animal models, as well as regenerative medicine and oncology. Compared with mice, laboratory rats have a larger body size and can more easily undergo transplantation of various tissues and organs. Considering the absence of high-quality resources of immunodeficient rats, we used the CRISPR/Cas9 genome editing system to knock out the interleukin-2 receptor gamma chain gene (Il2rg) in F344/Jcl rats-alone or together with recombination activating gene 2 (Rag2)-to create a high-quality bioresource that researchers can freely use: severe combined immunodeficiency (SCID) rats. We selected one founder rat with frame-shift mutations in both Il2rg (5-bp del) and Rag2 ([1-bp del+2-bp ins]/[7-bp del+2-bp ins]), then conducted mating to establish a line of immunodeficient rats. The immunodeficiency phenotype was preliminarily confirmed by the presence of severe thymic hypoplasia in Il2rg-single knockout (sKO) and Il2rg/Rag2-double knockout (dKO) rats. Assessment of blood cell counts in peripheral blood showed that the white blood cell count was significantly decreased in sKO and dKO rats, while the red blood cell count was unaffected. The decrease in white blood cell count was mainly caused by a decrease in lymphocytes. Furthermore, analyses of lymphocyte populations via flow cytometry showed that the numbers of B cells (CD3- CD45+) and natural killer cells (CD3- CD161+) were markedly reduced in both knockout rats. In contrast, T cells were markedly reduced but showed slightly different results between sKO and dKO rats. Notably, our immunodeficient rats do not exhibit growth retardation or gametogenesis defects. This high-quality SCID rat resource is now managed by the National BioResource Project in Japan. Our SCID rat model has been used in various research fields, demonstrating its importance as a bioresource.
Assuntos
Imunodeficiência Combinada Severa , Animais , Edição de Genes , Subunidade gama Comum de Receptores de Interleucina/genética , Camundongos , Camundongos SCID , Ratos , Ratos Endogâmicos F344 , Imunodeficiência Combinada Severa/genética , Linfócitos TRESUMO
Humic substances are formed during the decomposition of organic matter in humus, and are found in many natural environments in which organic materials and microorganisms are present. Oral administration of humus extract to mice successfully induced effective protection against experimental challenge by the two subspecies, Trypanosoma brucei brucei and T. brucei gambiense. Mortality was most reduced among mice who received a 3% humus extract for 21 days in drinking water ad libitum. Spleen cells from humus-administered mice exhibited significant non-specific cytotoxic activity against L1210 mouse leukemia target cells. Also, spleen cells produced significantly higher amounts of Interferon-gamma when stimulated in vitro with Concanavalin A than cells from normal controls. These results clearly show that administration to mice of humus extract induced effective resistance against Trypanosoma infection. Enhancement of the innate immune system may be involved in host defense against trypanosomiasis.
Assuntos
Solo , Tripanossomicidas/uso terapêutico , Tripanossomíase Africana/prevenção & controle , Animais , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/biossíntese , Feminino , L-Lactato Desidrogenase/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Leucemia L1210/tratamento farmacológico , Leucemia L1210/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Baço/efeitos dos fármacos , Baço/patologia , Baço/fisiopatologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei gambiense/efeitos dos fármacosRESUMO
Epilepsy is one of the most frequent neurological disorders characterized by spontaneous and recurrent seizures. Most seizures last for the lifetime and the patients require long term therapies. However, about 30% of the patients are refractory to antiepileptic drugs. Therefore, the need for newer and more effective therapies is urgent. Focal epilepsies, in which the abnormal electrical discharges occur within neuronal networks limited to one hemisphere, accounts for about 60% of all adult idiopathic epilepsy cases. Recently, mutations of DEPDC5 gene has been reported in wide spectrum of focal epilepsy syndromes. Most epilepsy genes encode ion channel or transmitter receptor, but DEPDC5 has no homology with them. DEPDC5 forms a complex, named GATOR1, together with other focal epilepsy related proteins NPRL2 and NPRL3. GATOR1 inhibits the mTORC1 pathway, regulating multiple cellular processes including cell growth and proliferation. The role of DEPDC5 in neuronal system is becoming clear from recent studies using the animal models. Because DEPDC5 is the most common causative gene in focal epilepsies and different from other epilepsy genes, DEPDC5 will be a key to understand epileptogenesis of various epilepsies, and provide new insight to develop new versatile therapies.
Assuntos
Epilepsia , Animais , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Mutação , Proteínas Repressoras , Proteínas Supressoras de TumorRESUMO
OBJECTIVE: DEPDC5 was identified as a major genetic cause of focal epilepsy with deleterious mutations found in a wide range of inherited forms of focal epilepsy, associated with malformation of cortical development in certain cases. Identification of frameshift, truncation, and deletion mutations implicates haploinsufficiency of DEPDC5 in the etiology of focal epilepsy. DEPDC5 is a component of the GATOR1 complex, acting as a negative regulator of mTOR signaling. METHODS: Zebrafish represents a vertebrate model suitable for genetic analysis and drug screening in epilepsy-related disorders. In this study, we defined the expression of depdc5 during development and established an epilepsy model with reduced Depdc5 expression. RESULTS: Here we report a zebrafish model of Depdc5 loss-of-function that displays a measurable behavioral phenotype, including hyperkinesia, circular swimming, and increased neuronal activity. These phenotypic features persisted throughout embryonic development and were significantly reduced upon treatment with the mTORC1 inhibitor, rapamycin, as well as overexpression of human WT DEPDC5 transcript. No phenotypic rescue was obtained upon expression of epilepsy-associated DEPDC5 mutations (p.Arg487* and p.Arg485Gln), indicating that these mutations cause a loss of function of the protein. INTERPRETATION: This study demonstrates that Depdc5 knockdown leads to early-onset phenotypic features related to motor and neuronal hyperactivity. Restoration of phenotypic features by WT but not epilepsy-associated Depdc5 mutants, as well as by mTORC1 inhibition confirm the role of Depdc5 in the mTORC1-dependent molecular cascades, defining this pathway as a potential therapeutic target for DEPDC5-inherited forms of focal epilepsy.
RESUMO
OBJECTIVE: To identify the genetic cause in a large family with febrile seizures (FS) and temporal lobe epilepsy (TLE) and subsequently search for additional mutations in a cohort of 107 families with FS, with or without epilepsy. METHODS: The cohort consisted of 1 large family with FS and TLE, 64 smaller French families recruited through a national French campaign, and 43 Italian families. Molecular analyses consisted of whole-exome sequencing and mutational screening. RESULTS: Exome sequencing revealed a p.Glu402fs*3 mutation in the γ2 subunit of the GABAA receptor gene (GABRG2) in the large family with FS and TLE. Three additional nonsense and frameshift GABRG2 mutations (p.Arg136*, p.Val462fs*33, and p.Pro59fs*12), 1 missense mutation (p.Met199Val), and 1 exonic deletion were subsequently identified in 5 families of the follow-up cohort. CONCLUSIONS: We report GABRG2 mutations in 5.6% (6/108) of families with FS, with or without associated epilepsy. This study provides evidence that GABRG2 mutations are linked to the FS phenotype, rather than epilepsy, and that loss-of-function of GABAA receptor γ2 subunit is the probable underlying pathogenic mechanism.
RESUMO
Mutations in the leucine-rich, glioma inactivated 1 (LGI1) gene have been identified in patients with autosomal dominant lateral temporal lobe epilepsy (ADLTE). We previously reported that Lgi1 mutant rats, carrying a missense mutation (L385R) generated by gene-driven N-ethyl-N-nitrosourea (ENU) mutagenesis, showed generalized tonic-clonic seizures (GTCS) in response to acoustic stimuli. In the present study, we assessed clinically relevant features of Lgi1 heterozygous mutant rats (Lgi1(L385R/+)) as an animal model of ADLTE. First, to explore the focus of the audiogenic seizures, we performed electroencephalography (EEG) and brain Fos immunohistochemistry in Lgi1(L385R/+) and wild type rats. EEG showed unique seizure patterns (e.g., bilateral rhythmic spikes) in Lgi1(L385R/+) rats with GTCS. An elevated level of Fos expression indicated greater neural excitability to acoustic stimuli in Lgi1(L385R/+) rats, especially in the temporal lobe, thalamus and subthalamic nucleus. Finally, microarray analysis revealed a number of differentially expressed genes that may be involved in epilepsy. These results suggest that Lgi1(L385R/+) rats are useful as an animal model of human ADLTE.
Assuntos
Epilepsia Reflexa/genética , Mutação de Sentido Incorreto/genética , Proteínas/genética , Estimulação Acústica/efeitos adversos , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Eletroencefalografia , Epilepsia Reflexa/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Endogâmicos F344RESUMO
OBJECTIVE: To study the prevalence of DEPDC5 mutations in a series of 30 small European families with a phenotype compatible with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). METHODS: Thirty unrelated families referred with ADNFLE were recruited in France, Italy, Germany, Belgium, and Norway. Whole-exome sequencing was performed in 10 probands and direct sequencing of the DEPDC5 coding sequence in 20 probands. Testing for nonsense-mediated messenger RNA decay (NMD) was performed in lymphoblastic cells. RESULTS: Exome sequencing revealed a splice acceptor mutation (c.2355-2A>G) in DEPDC5 in the proband of a German family. In addition, 3 nonsense DEPDC5 mutations (p.Arg487*, p.Arg1087*, and p.Trp1369*) were detected in the probands of 2 French and one Belgian family. The nonsense mutations p.Arg487* and p.Arg1087* were targeted by NMD, leading to the degradation of the mutated transcripts. At the clinical level, 78% of the patients with DEPDC5 mutations were drug resistant. CONCLUSIONS: DEPDC5 loss-of-function mutations were found in 13% of the families with a presentation of ADNFLE. The rate of drug resistance was high in patients with DEPDC5 mutations. Small ADNFLE pedigrees with DEPDC5 mutations might actually represent a part of the broader familial focal epilepsy with variable foci phenotype.
Assuntos
Mutação/genética , Proteínas Repressoras/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Cromossomos Humanos Par 22/genética , Resistência a Medicamentos/genética , Epilepsia do Lobo Frontal/genética , Europa (Continente) , Exoma/genética , Feminino , Proteínas Ativadoras de GTPase , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , FenótipoRESUMO
The main familial focal epilepsies are autosomal dominant nocturnal frontal lobe epilepsy, familial temporal lobe epilepsy and familial focal epilepsy with variable foci. A frameshift mutation in the DEPDC5 gene (encoding DEP domain-containing protein 5) was identified in a family with focal epilepsy with variable foci by linkage analysis and exome sequencing. Subsequent pyrosequencing of DEPDC5 in a cohort of 15 additional families with focal epilepsies identified 4 nonsense mutations and 1 missense mutation. Our findings provided evidence of frequent (37%) loss-of-function mutations in DEPDC5 associated with a broad spectrum of focal epilepsies. The implication of a DEP (Dishevelled, Egl-10 and Pleckstrin) domain-containing protein that may be involved in membrane trafficking and/or G protein signaling opens new avenues for research.