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1.
Cell ; 187(4): 962-980.e19, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38309258

RESUMO

Microglia (MG), the brain-resident macrophages, play major roles in health and disease via a diversity of cellular states. While embryonic MG display a large heterogeneity of cellular distribution and transcriptomic states, their functions remain poorly characterized. Here, we uncovered a role for MG in the maintenance of structural integrity at two fetal cortical boundaries. At these boundaries between structures that grow in distinct directions, embryonic MG accumulate, display a state resembling post-natal axon-tract-associated microglia (ATM) and prevent the progression of microcavities into large cavitary lesions, in part via a mechanism involving the ATM-factor Spp1. MG and Spp1 furthermore contribute to the rapid repair of lesions, collectively highlighting protective functions that preserve the fetal brain from physiological morphogenetic stress and injury. Our study thus highlights key major roles for embryonic MG and Spp1 in maintaining structural integrity during morphogenesis, with major implications for our understanding of MG functions and brain development.


Assuntos
Encéfalo , Microglia , Axônios , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Macrófagos/fisiologia , Microglia/patologia , Morfogênese
2.
Brain ; 147(3): 996-1010, 2024 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-37724593

RESUMO

Grey matter heterotopia (GMH) are neurodevelopmental disorders associated with abnormal cortical function and epilepsy. Subcortical band heterotopia (SBH) and periventricular nodular heterotopia (PVNH) are two well-recognized GMH subtypes in which neurons are misplaced, either forming nodules lining the ventricles in PVNH, or forming bands in the white matter in SBH. Although both PVNH and SBH are commonly associated with epilepsy, it is unclear whether these two GMH subtypes differ in terms of pathological consequences or, on the contrary, share common altered mechanisms. Here, we studied two robust preclinical models of SBH and PVNH, and performed a systematic comparative assessment of the physiological and morphological diversity of heterotopia neurons, as well as the dynamics of epileptiform activity and input connectivity. We uncovered a complex set of altered properties, including both common and distinct physiological and morphological features across heterotopia subtypes, and associated with specific dynamics of epileptiform activity. Taken together, these results suggest that pro-epileptic circuits in GMH are, at least in part, composed of neurons with distinct, subtype-specific, physiological and morphological properties depending on the heterotopia subtype. Our work supports the notion that GMH represent a complex set of disorders, associating both shared and diverging pathological consequences, and contributing to forming epileptogenic networks with specific properties. A deeper understanding of these properties may help to refine current GMH classification schemes by identifying morpho-electric signatures of GMH subtypes, to potentially inform new treatment strategies.


Assuntos
Vermis Cerebelar , Epilepsia , Transtornos do Neurodesenvolvimento , Humanos , Substância Cinzenta , Neurônios
3.
Neurobiol Dis ; 198: 106558, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38852754

RESUMO

Periventricular nodular heterotopia (PNH), the most common brain malformation diagnosed in adulthood, is characterized by the presence of neuronal nodules along the ventricular walls. PNH is mainly associated with mutations in the FLNA gene - encoding an actin-binding protein - and patients often develop epilepsy. However, the molecular mechanisms underlying the neuronal failure still remain elusive. It has been hypothesized that dysfunctional cortical circuitry, rather than ectopic neurons, may explain the clinical manifestations. To address this issue, we depleted FLNA from cortical pyramidal neurons of a conditional Flnaflox/flox mice by timed in utero electroporation of Cre recombinase. We found that FLNA regulates dendritogenesis and spinogenesis thus promoting an appropriate excitatory/inhibitory inputs balance. We demonstrated that FLNA modulates RAC1 and cofilin activity through its interaction with the Rho-GTPase Activating Protein 24 (ARHGAP24). Collectively, we disclose an uncharacterized role of FLNA and provide strong support for neural circuit dysfunction being a consequence of FLNA mutations.


Assuntos
Córtex Cerebral , Filaminas , Proteínas rac1 de Ligação ao GTP , Animais , Camundongos , Fatores de Despolimerização de Actina/metabolismo , Córtex Cerebral/metabolismo , Filaminas/metabolismo , Filaminas/genética , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/genética , Camundongos Transgênicos , Neurogênese/fisiologia , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Neuropeptídeos/genética , Heterotopia Nodular Periventricular/genética , Heterotopia Nodular Periventricular/metabolismo , Heterotopia Nodular Periventricular/patologia , Células Piramidais/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética
4.
Epilepsia ; 65(7): 2111-2126, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38717560

RESUMO

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.


Assuntos
Neurônios , Receptores de N-Metil-D-Aspartato , Serina-Treonina Quinases TOR , Animais , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Ratos , Neurônios/metabolismo , Feminino , Malformações do Desenvolvimento Cortical/genética , Malformações do Desenvolvimento Cortical/fisiopatologia , Modelos Animais de Doenças , Ratos Sprague-Dawley , Técnicas de Patch-Clamp , Malformações do Desenvolvimento Cortical do Grupo I/genética , Displasia Cortical Focal , Epilepsia
5.
Neurobiol Dis ; 177: 106002, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36649744

RESUMO

Malformations of cortical development represent a major cause of epilepsy in childhood. However, the pathological substrate and dynamic changes leading to the development and progression of epilepsy remain unclear. Here, we characterized an etiology-relevant rat model of subcortical band heterotopia (SBH), a diffuse type of cortical malformation associated with drug-resistant seizures in humans. We used longitudinal electrographic recordings to monitor the age-dependent evolution of epileptiform discharges during the course of epileptogenesis in this model. We found both quantitative and qualitative age-related changes in seizures properties and patterns, accompanying a gradual progression towards a fully developed seizure pattern seen in adulthood. We also dissected the relative contribution of the band heterotopia and the overlying cortex to the development and age-dependent progression of epilepsy using timed and spatially targeted manipulation of neuronal excitability. We found that an early suppression of neuronal excitability in SBH slows down epileptogenesis in juvenile rats, whereas epileptogenesis is paradoxically exacerbated when excitability is suppressed in the overlying cortex. However, in rats with active epilepsy, similar manipulations of excitability have no effect on chronic spontaneous seizures. Together, our data support the notion that complex developmental alterations occurring in both the SBH and the overlying cortex concur to creating pathogenic circuits prone to generate seizures. Our study also suggests that early and targeted interventions could potentially influence the course of these altered developmental trajectories, and favorably modify epileptogenesis in malformations of cortical development.


Assuntos
Lissencefalias Clássicas e Heterotopias Subcorticais em Banda , Epilepsia , Humanos , Ratos , Animais , Córtex Cerebral/patologia , Epilepsia/patologia , Convulsões/complicações , Neurônios/patologia
6.
Hum Mol Genet ; 28(22): 3755-3765, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31411685

RESUMO

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.


Assuntos
Hemimegalencefalia/genética , Proteína S6 Ribossômica/genética , Serina-Treonina Quinases TOR/genética , Animais , Encéfalo/metabolismo , Criança , Epilepsia Resistente a Medicamentos/genética , Epilepsia Resistente a Medicamentos/metabolismo , Epilepsia/genética , Feminino , Humanos , Malformações do Desenvolvimento Cortical/genética , Malformações do Desenvolvimento Cortical/metabolismo , Malformações do Desenvolvimento Cortical do Grupo I/genética , Camundongos , Mosaicismo , Mutação , Neurônios/metabolismo , Proteína S6 Ribossômica/metabolismo , Serina-Treonina Quinases TOR/metabolismo
7.
Cereb Cortex ; 29(10): 4253-4262, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-30534979

RESUMO

Subcortical band heterotopia (SBH), also known as double-cortex syndrome, is a neuronal migration disorder characterized by an accumulation of neurons in a heterotopic band below the normotopic cortex. The majority of patients with SBH have mild to moderate intellectual disability and intractable epilepsy. However, it is still not clear how cortical networks are organized in SBH patients and how this abnormal organization contributes to improper brain function. In this study, cortical networks were investigated in the barrel cortex in an animal model of SBH induced by in utero knockdown of Dcx, main causative gene of this condition in human patients. When the SBH was localized below the Barrel Field (BF), layer (L) four projection to correctly positioned L2/3 pyramidal cells was weakened due to lower connectivity. Conversely, when the SBH was below an adjacent cortical region, the excitatory L4 to L2/3 projection was stronger due to increased L4 neuron excitability, synaptic strength and excitation/inhibition ratio of L4 to L2/3 connection. We propose that these developmental alterations contribute to the spectrum of clinical dysfunctions reported in patients with SBH.


Assuntos
Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/fisiopatologia , Neurônios/fisiologia , Córtex Somatossensorial/fisiopatologia , Sinapses/fisiologia , Animais , Modelos Animais de Doenças , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Técnicas de Silenciamento de Genes , Potenciais da Membrana , Proteínas Associadas aos Microtúbulos/genética , Neuropeptídeos/genética , Ratos Wistar , Córtex Somatossensorial/patologia
8.
Epilepsia ; 60(2): 337-348, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30597542

RESUMO

OBJECTIVE: Malformations of cortical development are common causes of intellectual disability and epilepsy, yet there is a crucial lack of relevant preclinical models associating seizures and cortical malformations. Here, we describe a novel rat model with bilateral subcortical band heterotopia (SBH) and examine whether this model develops spontaneous epileptic seizures. METHODS: To generate bilateral SBH in rats, we combined RNAi-mediated knockdown of Dcx and in utero electroporation with a tripolar electrode configuration enabling simultaneous transfection of the two brain hemispheres. To determine whether bilateral SBH leads to epileptiform activity, rats of various ages were implanted for telemetric electrocorticographic recordings and histopathological examination was carried out at the end of the recording sessions. RESULTS: By 2 months, rats with bilateral SBH showed nonconvulsive spontaneous seizures consisting of spike-and-wave discharges (SWDs) with dominant frequencies in the alpha and theta bands and secondarily in higher-frequency bands. SWDs occurred during both the dark and the light period, but were more frequent during quiet awake state than during sleep. Also, SWDs were more frequent and lasted longer at older ages. No sex differences were found. Although frequencies and durations of SWDs were found to be uncorrelated with the size of SBH, SWDs were initiated in some occasions from brain hemispheres comprising a larger SBH. Lastly, SWDs exhibited absence-like pharmacological properties, being temporarily alleviated by ethosuximide administration. SIGNIFICANCE: This novel model of bilateral SBH with spontaneous epilepsy may potentially provide valuable new insights into causality between cortical malformations and seizures, and help translational research aiming at designing novel treatment strategies for epilepsy.


Assuntos
Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/fisiopatologia , Convulsões/fisiopatologia , Vigília/fisiologia , Animais , Modelos Animais de Doenças , Proteína Duplacortina , Eletrocorticografia/métodos , Eletroencefalografia/métodos , Feminino , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Ratos Wistar , Convulsões/complicações
9.
Cereb Cortex ; 28(8): 2976-2990, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29788228

RESUMO

The neocortex is a 6-layered laminated structure with a precise anatomical and functional organization ensuring proper function. Laminar positioning of cortical neurons, as determined by termination of neuronal migration, is a key determinant of their ability to assemble into functional circuits. However, the exact contribution of laminar placement to dendrite morphogenesis and synapse formation remains unclear. Here we manipulated the laminar position of cortical neurons by knocking down doublecortin (Dcx), a crucial effector of migration, and show that misplaced neurons fail to properly form dendrites, spines, and functional glutamatergic and GABAergic synapses. We further show that knocking down Dcx in properly positioned neurons induces similar but milder defects, suggesting that the laminar misplacement is the primary cause of altered neuronal development. Thus, the specific laminar environment of their fated layers is crucial for the maturation of cortical neurons, and influences their functional integration into developing cortical circuits.


Assuntos
Dendritos/fisiologia , Neurônios/citologia , Córtex Somatossensorial/citologia , Sinapses/fisiologia , Animais , Animais Recém-Nascidos , Proteína 4 Homóloga a Disks-Large/genética , Proteína 4 Homóloga a Disks-Large/metabolismo , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Estimulação Elétrica , Embrião de Mamíferos , Ácido Glutâmico/metabolismo , Técnicas In Vitro , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neurogênese/genética , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Técnicas de Patch-Clamp , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Córtex Somatossensorial/crescimento & desenvolvimento , Transdução Genética
10.
Proc Natl Acad Sci U S A ; 111(6): 2337-42, 2014 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-24469796

RESUMO

Alterations in the formation of brain networks are associated with several neurodevelopmental disorders. Mutations in TBC1 domain family member 24 (TBC1D24) are responsible for syndromes that combine cortical malformations, intellectual disability, and epilepsy, but the function of TBC1D24 in the brain remains unknown. We report here that in utero TBC1D24 knockdown in the rat developing neocortex affects the multipolar-bipolar transition of neurons leading to delayed radial migration. Furthermore, we find that TBC1D24-knockdown neurons display an abnormal maturation and retain immature morphofunctional properties. TBC1D24 interacts with ADP ribosylation factor (ARF)6, a small GTPase crucial for membrane trafficking. We show that in vivo, overexpression of the dominant-negative form of ARF6 rescues the neuronal migration and dendritic outgrowth defects induced by TBC1D24 knockdown, suggesting that TBC1D24 prevents ARF6 activation. Overall, our findings demonstrate an essential role of TBC1D24 in neuronal migration and maturation and highlight the physiological relevance of the ARF6-dependent membrane-trafficking pathway in brain development.


Assuntos
Fatores de Ribosilação do ADP/fisiologia , Proteínas de Transporte/fisiologia , Movimento Celular/fisiologia , Neurônios/citologia , Fator 6 de Ribosilação do ADP , Animais , Encéfalo/fisiologia , Proteínas de Transporte/genética , Células Cultivadas , Dendritos/fisiologia , Proteínas Ativadoras de GTPase , Técnicas de Silenciamento de Genes , Ácido Glutâmico/metabolismo , Proteínas de Membrana , Proteínas do Tecido Nervoso , Ratos , Sinapses/metabolismo
11.
Epilepsia ; 57(4): 648-59, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26873267

RESUMO

OBJECTIVE: To describe the epileptic phenotype of Tsc1(+/-) mice pups in comparison with age-related seizures in human tuberous sclerosis complex (TSC). METHODS: Tsc1(+/-) and control mice underwent intracranial electroencephalography (EEG) recording at postnatal ages (P)8 to P33, with linear silicon probe implanted in the somatosensory cortex of one or both hemispheres for 8-24 h. Ictal events were classified visually by independent analyzers; distinct EEG patterns were related to age and analyzed to quantify field potential characteristics and signal dynamics between hemispheres. We collected retrospectively 20 infants with prenatally diagnosed TSC and EEG before seizure onset, and analyzed the electroclinical course of epilepsy, taking into account a first-line treatment by vigabatrin. RESULTS: Spontaneous seizures were disclosed in 55% of Tsc1(+/-) mice at P9-18. Three ictal patterns were identified: from P9 to P12 "spike clusters" consisted of recurring large spikes without clinical correlate; "spasm-like" discharges dominated from P13 to P16 consisting of high amplitude large field potential superimposed with or followed by fast activity repeated every 2-10 s for at least 20 s, accompanied by rhythmic limb contractions; from P14 to P18 a "tonic-clonic like" pattern comprised rhythmic spikes of increasing amplitude with tonic-clonic movements. Early onset "spike clusters" were mainly unilateral, whereas "spasm-like" and "tonic-clonic like" patterns were bilateral. Interhemispheric propagation was significantly faster for "tonic-clonic like" than for "spasm-like" events. In infants diagnosed prenatally with TSC, clusters of sharp waves or spikes preceded the first seizure, and vigabatrin prevented the development of seizures. Patients treated after seizure onset developed spasms or focal seizures that were pharmacoresistant in 66.7% of cases. SIGNIFICANCE: Tsc1(+/-) mice pups exhibit an age-dependent seizure pattern sequence mimicking early human TSC epilepsy features. Spike clusters before seizure onset in TSC should be considered as a first stage of epilepsy reinforcing the concept of preventive antiepileptic therapy.


Assuntos
Epilepsia/metabolismo , Esclerose Tuberosa/metabolismo , Proteínas Supressoras de Tumor/biossíntese , Adolescente , Fatores Etários , Animais , Criança , Pré-Escolar , Epilepsia/genética , Epilepsia/patologia , Feminino , Seguimentos , Regulação da Expressão Gênica , Humanos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estudos Retrospectivos , Esclerose Tuberosa/genética , Esclerose Tuberosa/patologia , Proteína 1 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética
12.
PLoS Genet ; 9(9): e1003752, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24039599

RESUMO

Genomic imprinting is a process that causes genes to be expressed from one allele only according to parental origin, the other allele being silent. Diseases can arise when the normally active alleles are not expressed. In this context, low level of expression of the normally silent alleles has been considered as genetic noise although such expression has never been further studied. Prader-Willi Syndrome (PWS) is a neurodevelopmental disease involving imprinted genes, including NDN, which are only expressed from the paternally inherited allele, with the maternally inherited allele silent. We present the first in-depth study of the low expression of a normally silent imprinted allele, in pathological context. Using a variety of qualitative and quantitative approaches and comparing wild-type, heterozygous and homozygous mice deleted for Ndn, we show that, in absence of the paternal Ndn allele, the maternal Ndn allele is expressed at an extremely low level with a high degree of non-genetic heterogeneity. The level of this expression is sex-dependent and shows transgenerational epigenetic inheritance. In about 50% of mutant mice, this expression reduces birth lethality and severity of the breathing deficiency, correlated with a reduction in the loss of serotonergic neurons. In wild-type brains, the maternal Ndn allele is never expressed. However, using several mouse models, we reveal a competition between non-imprinted Ndn promoters which results in monoallelic (paternal or maternal) Ndn expression, suggesting that Ndn allelic exclusion occurs in the absence of imprinting regulation. Importantly, specific expression of the maternal NDN allele is also detected in post-mortem brain samples of PWS individuals. Our data reveal an unexpected epigenetic flexibility of PWS imprinted genes that could be exploited to reactivate the functional but dormant maternal alleles in PWS. Overall our results reveal high non-genetic heterogeneity between genetically identical individuals that might underlie the variability of the phenotype.


Assuntos
Epigênese Genética/genética , Impressão Genômica , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Síndrome de Prader-Willi/genética , Alelos , Animais , Apneia/genética , Apneia/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Heterozigoto , Humanos , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Síndrome de Prader-Willi/patologia , Regiões Promotoras Genéticas
13.
Ann Neurol ; 76(3): 428-42, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25074818

RESUMO

OBJECTIVE: Subcortical band heterotopia (SBH) is a cortical malformation formed when neocortical neurons prematurely stop their migration in the white matter, forming a heterotopic band below the normotopic cortex, and is generally associated with intractable epilepsy. Although it is clear that the band heterotopia and the overlying cortex both contribute to creating an abnormal circuit prone to generate epileptic discharges, it is less understood which part of this circuitry is the most critical. Here, we sought to identify the origin of epileptiform activity in a targeted genetic model of SBH in rats. METHODS: Rats with SBH (Dcx-KD rats) were generated by knocking down the Dcx gene using shRNA vectors transfected into neocortical progenitors of rat embryos. Origin, spatial extent, and laminar profile of bicuculline-induced interictal-like activity on neocortical slices were analyzed by using extracellular recordings from 60-channel microelectrode arrays. Susceptibility to pentylenetetrazole-induced seizures was assessed by electrocorticography in head-restrained nonanesthetized rats. RESULTS: We show that the band heterotopia does not constitute a primary origin for interictal-like epileptiform activity in vitro and is dispensable for generating induced seizures in vivo. Furthermore, we report that most interictal-like discharges originating in the overlying cortex secondarily propagate to the band heterotopia. Importantly, we found that in vivo suppression of neuronal excitability in SBH does not alter the higher propensity of Dcx-KD rats to display seizures. INTERPRETATION: These results suggest a major role of the normotopic cortex over the band heterotopia in generating interictal epileptiform activity and seizures in brains with SBH.


Assuntos
Córtex Cerebral/fisiopatologia , Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/fisiopatologia , Epilepsia/etiologia , Epilepsia/fisiopatologia , Neocórtex/fisiopatologia , Animais , Bicuculina/farmacologia , Córtex Cerebral/efeitos dos fármacos , Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/complicações , Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/patologia , Convulsivantes/farmacologia , Modelos Animais de Doenças , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Eletroencefalografia , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Fenômenos Eletrofisiológicos/fisiologia , Epilepsia/induzido quimicamente , Técnicas de Silenciamento de Genes , Proteínas Associadas aos Microtúbulos/genética , Neocórtex/efeitos dos fármacos , Rede Nervosa/anormalidades , Rede Nervosa/fisiopatologia , Neuropeptídeos/genética , Pentilenotetrazol/farmacologia , Ratos , Ratos Transgênicos , Convulsões/induzido quimicamente , Convulsões/fisiopatologia , Córtex Somatossensorial/efeitos dos fármacos , Córtex Somatossensorial/fisiopatologia
14.
Brain ; 136(Pt 8): 2457-73, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23831613

RESUMO

Altered development of the human cerebral cortex can cause severe malformations with often intractable focal epileptic seizures and may participate in common pathologies, notably epilepsy. This raises important conceptual and therapeutic issues. Two missense mutations in the sushi repeat-containing protein SRPX2 had been previously identified in epileptic disorders with or without structural developmental alteration of the speech cortex. In the present study, we aimed to decipher the precise developmental role of SRPX2, to have a better knowledge on the consequences of its mutations, and to start addressing therapeutic issues through the design of an appropriate animal model. Using an in utero Srpx2 silencing approach, we show that SRPX2 influences neuronal migration in the developing rat cerebral cortex. Wild-type, but not the mutant human SRPX2 proteins, rescued the neuronal migration phenotype caused by Srpx2 silencing in utero, and increased alpha-tubulin acetylation. Following in utero Srpx2 silencing, spontaneous epileptiform activity was recorded post-natally. The neuronal migration defects and the post-natal epileptic consequences were prevented early in embryos by maternal administration of tubulin deacetylase inhibitor tubacin. Hence epileptiform manifestations of developmental origin could be prevented in utero, using a transient and drug-based therapeutic protocol.


Assuntos
Anilidas/farmacologia , Movimento Celular/genética , Córtex Cerebral/metabolismo , Epilepsia/genética , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Proteínas de Membrana/genética , Neurônios/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Epilepsia/metabolismo , Inativação Gênica , Humanos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ratos , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
15.
Brain ; 136(Pt 11): 3378-94, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24056535

RESUMO

Periventricular nodular heterotopia is caused by defective neuronal migration that results in heterotopic neuronal nodules lining the lateral ventricles. Mutations in filamin A (FLNA) or ADP-ribosylation factor guanine nucleotide-exchange factor 2 (ARFGEF2) cause periventricular nodular heterotopia, but most patients with this malformation do not have a known aetiology. Using comparative genomic hybridization, we identified 12 patients with developmental brain abnormalities, variably combining periventricular nodular heterotopia, corpus callosum dysgenesis, colpocephaly, cerebellar hypoplasia and polymicrogyria, harbouring a common 1.2 Mb minimal critical deletion in 6q27. These anatomic features were mainly associated with epilepsy, ataxia and cognitive impairment. Using whole exome sequencing in 14 patients with isolated periventricular nodular heterotopia but no copy number variants, we identified one patient with periventricular nodular heterotopia, developmental delay and epilepsy and a de novo missense mutation in the chromosome 6 open reading frame 70 (C6orf70) gene, mapping in the minimal critical deleted region. Using immunohistochemistry and western blots, we demonstrated that in human cell lines, C6orf70 shows primarily a cytoplasmic vesicular puncta-like distribution and that the mutation affects its stability and subcellular distribution. We also performed in utero silencing of C6orf70 and of Phf10 and Dll1, the two additional genes mapping in the 6q27 minimal critical deleted region that are expressed in human and rodent brain. Silencing of C6orf70 in the developing rat neocortex produced periventricular nodular heterotopia that was rescued by concomitant expression of wild-type human C6orf70 protein. Silencing of the contiguous Phf10 or Dll1 genes only produced slightly delayed migration but not periventricular nodular heterotopia. The complex brain phenotype observed in the 6q terminal deletion syndrome likely results from the combined haploinsufficiency of contiguous genes mapping to a small 1.2 Mb region. Our data suggest that, of the genes within this minimal critical region, C6orf70 plays a major role in the control of neuronal migration and its haploinsufficiency or mutation causes periventricular nodular heterotopia.


Assuntos
Anormalidades Múltiplas/genética , Encéfalo/anormalidades , Malformações do Desenvolvimento Cortical do Grupo II/genética , Heterotopia Nodular Periventricular/genética , Anormalidades Múltiplas/patologia , Anormalidades Múltiplas/fisiopatologia , Adolescente , Adulto , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Criança , Deleção Cromossômica , Cromossomos Humanos Par 6/genética , Estudos de Coortes , Deficiências do Desenvolvimento/genética , Epilepsia/genética , Exoma/genética , Feminino , Haploinsuficiência/genética , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Malformações do Desenvolvimento Cortical do Grupo II/patologia , Malformações do Desenvolvimento Cortical do Grupo II/fisiopatologia , Mutação/genética , Heterotopia Nodular Periventricular/patologia , Heterotopia Nodular Periventricular/fisiopatologia , Ratos , Ratos Wistar , Síndrome
16.
Hum Mol Genet ; 19(24): 4895-905, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-20876615

RESUMO

The onset of feeding at birth is a vital step for the adaptation of the neonate to extra uterine life. Prader-Willi syndrome (PWS) is a complex neurogenetic disorder caused by the alteration of several imprinted contiguous genes including MAGEL2. PWS presents with various clinical manifestations, including poor suckling behaviour and feeding problems in neonates. Hypothalamic defects have been proposed, but the pathophysiological mechanisms remain poorly understood. Here, we report that a Magel2-deficient mouse with 50% neonatal mortality had an altered onset of suckling activity and subsequent impaired feeding, suggesting a role of MAGEL2 in the suckling deficit seen in PW newborns. The hypothalamus of Magel2 mutant neonates showed a significant reduction in oxytocin (OT). Furthermore, injection of a specific OT receptor antagonist in wild-type neonates recapitulated the feeding deficiency seen in Magel2 mutants, and a single injection of OT, 3-5 h after birth, rescued the phenotype of Magel2 mutant pups, allowing all of them to survive. Our study illustrates the crucial role of feeding onset behaviour after birth. We propose that OT supply might constitute a promising avenue for the treatment of feeding difficulties in PW neonates and potentially of other newborns with impaired feeding onset.


Assuntos
Antígenos de Neoplasias/genética , Comportamento Alimentar/efeitos dos fármacos , Impressão Genômica/efeitos dos fármacos , Ocitocina/administração & dosagem , Ocitocina/farmacologia , Proteínas/genética , Animais , Animais Recém-Nascidos , Animais Lactentes/metabolismo , Antígenos de Neoplasias/metabolismo , Feminino , Marcação de Genes , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Imuno-Histoquímica , Injeções Subcutâneas , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mutação/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Sistemas Neurossecretores/efeitos dos fármacos , Sistemas Neurossecretores/fisiologia , Orexinas , Fenótipo , Proteínas/metabolismo , Receptores de Ocitocina/antagonistas & inibidores , Vasopressinas/metabolismo
17.
Development ; 136(20): 3413-21, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19762426

RESUMO

The imprinted H19 gene produces a non-coding RNA of unknown function. Mice lacking H19 show an overgrowth phenotype, due to a cis effect of the H19 locus on the adjacent Igf2 gene. To explore the function of the RNA itself, we produced transgenic mice overexpressing H19. We observed postnatal growth reduction in two independent transgenic lines and detected a decrease of Igf2 expression in embryos. An extensive analysis of several other genes from the newly described imprinted gene network (IGN) was performed in both loss- and gain-of-function animals. We found that H19 deletion leads to the upregulation of several genes of the IGN. This overexpression is restored to the wild-type level by transgenic expression of H19. We therefore propose that the H19 gene participates as a trans regulator in the fine-tuning of this IGN in the mouse embryo. This is the first in vivo evidence of a functional role for the H19 RNA. Our results also bring further experimental evidence for the existence of the IGN and open new perspectives in the comprehension of the role of genomic imprinting in embryonic growth and in human imprinting pathologies.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , RNA não Traduzido/genética , Sequências Reguladoras de Ácido Nucleico , Animais , Feminino , Fator de Crescimento Insulin-Like II/genética , Masculino , Camundongos , Camundongos Transgênicos , Fenótipo , RNA Longo não Codificante
18.
J Neurosci ; 28(7): 1745-55, 2008 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-18272695

RESUMO

Prader-Willi syndrome is a neurogenetic disease resulting from the absence of paternal expression of several imprinted genes, including NECDIN. Prader-Willi children and adults have severe breathing defects with irregular rhythm, frequent sleep apneas, and blunted respiratory regulations. For the first time, we show that Prader-Willi infants have sleep apneas already present at birth. In parallel, in wild-type and Necdin-deficient mice, we studied the respiratory system with in vivo plethysmography, in vitro electrophysiology, and pharmacology. Because serotonin is known to contribute to CNS development and to affect maturation and function of the brainstem respiratory network, we also investigated the serotonergic system with HPLC, immunohistochemistry, Rabies virus tracing approaches, and primary culture experiments. We report first that Necdin-deficiency in mice induces central respiratory deficits reminiscent of Prader-Willi syndrome (irregular rhythm, frequent apneas, and blunted respiratory regulations), second that Necdin is expressed by medullary serotonergic neurons, and third that Necdin deficiency alters the serotonergic metabolism, the morphology of serotonin vesicles in medullary serotonergic neurons but not the number of these cells. We also show that Necdin deficiency in neonatal mice alters the serotonergic modulation of the respiratory rhythm generator. Thus, we propose that the lack of Necdin expression induces perinatal serotonergic alterations that affect the maturation and function of the respiratory network, inducing breathing deficits in mice and probably in Prader-Willi patients.


Assuntos
Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Síndrome de Prader-Willi/genética , Síndrome de Prader-Willi/metabolismo , Respiração/genética , Serotonina/metabolismo , Síndromes da Apneia do Sono/fisiopatologia , Adulto , Animais , Animais Recém-Nascidos , Células Cultivadas , Pré-Escolar , Modelos Animais de Doenças , Humanos , Lactente , Bulbo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Neurônios/metabolismo , Medula Espinal/metabolismo
19.
Front Cell Neurosci ; 13: 473, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31680876

RESUMO

Studies conducted in human and rodent models have suggested that preexisting neurodevelopmental defects could predispose immature brains to febrile seizures (FS). However, the impact of the anatomical extent of preexisting cortical malformations on FS susceptibility was never assessed. Here, we induced hyperthermic seizures (HS) in rats with bilateral subcortical band heterotopia (SBH) and found variable degrees of HS susceptibility depending on inter-individual anatomical differences in size and extent of SBH. This indicates that an association exists between the overall extent or location of a cortical malformation, and the predisposition to FS. This also suggests that various predisposing factors and underlying causes may contribute to the etiology of complex FS.

20.
Brain Res ; 1711: 146-155, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30689978

RESUMO

Subcortical band heterotopia (SBH), also known as doublecortex syndrome, is a malformation of cortical development resulting from mutations in the doublecortin gene (DCX). It is characterized by a lack of migration of cortical neurons that accumulate in the white matter forming a heterotopic band. Patients with SBH may present mild to moderate intellectual disability as well as epilepsy. The SBH condition can be modeled in rats by in utero knockdown (KD) of Dcx. The affected cells form an SBH reminiscent of that observed in human patients and the animals develop a chronic epileptic condition in adulthood. Here, we investigated if the presence of a SBH is sufficient to induce cognitive impairment in juvenile Dcx-KD rats, before the onset of epilepsy. Using a wide range of behavioral tests, we found that the presence of SBH did not appear to affect motor control or somatosensory processing. In addition, cognitive abilities such as learning, short-term and long-term memory, were normal in pre-epileptic Dcx-KD rats. We suggest that the SBH presence is not sufficient to impair these behavioral functions.


Assuntos
Comportamento Animal , Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/psicologia , Cognição , Modelos Animais de Doenças , Epilepsia/genética , Deficiência Intelectual/genética , Animais , Ansiedade/genética , Doenças Assintomáticas , Movimento Celular , Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/complicações , Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/embriologia , Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/genética , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Eletroporação , Comportamento Exploratório , Substância Cinzenta/anormalidades , Substância Cinzenta/embriologia , Aprendizagem , Aprendizagem em Labirinto , Memória , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Mosaicismo , Neuropeptídeos/deficiência , Neuropeptídeos/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/toxicidade , Ratos , Teste de Desempenho do Rota-Rod , Sensação , Substância Branca/anormalidades , Substância Branca/embriologia
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