Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Elife ; 82019 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-31498083

RESUMO

Most of our knowledge on human CNS circuitry and related disorders originates from model organisms. How well such data translate to the human CNS remains largely to be determined. Human brain slice cultures derived from neurosurgical resections may offer novel avenues to approach this translational gap. We now demonstrate robust preservation of the complex neuronal cytoarchitecture and electrophysiological properties of human pyramidal neurons in long-term brain slice cultures. Further experiments delineate the optimal conditions for efficient viral transduction of cultures, enabling 'high throughput' fluorescence-mediated 3D reconstruction of genetically targeted neurons at comparable quality to state-of-the-art biocytin fillings, and demonstrate feasibility of long term live cell imaging of human cells in vitro. This model system has implications toward a broad spectrum of translational studies, regarding the validation of data obtained in non-human model systems, for therapeutic screening and genetic dissection of human CNS circuitry.

2.
Int J Mol Med ; 44(4): 1484-1494, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432139

RESUMO

Virotherapy using oncolytic viruses is an upcoming therapy strategy for cancer treatment. A variety of preclinical and clinical trials have indicated that adenoviruses may be used as potent agents in the treatment of a variety of cancers, and also for the treatment of brain tumors. In these studies, it has also been shown that oncovirotherapy is safe in terms of toxicity and side effects. In addition, previous studies have presented evidence for a significant role of oncovirotherapy in the activation of anti­tumor immune responses. With regard to oncolytic adenoviruses, we have demonstrated previously that the multifunctional protein Y­box binding protein­1 (YB­1) is a potent factor that was used to develop an YB­1­dependent oncolytic adenovirus (XVir­N­31). XVir­N­31 provides the opportunity for tumor­selective replication and exhibited marked oncolytic properties in a mouse glioma tumor model using therapy­resistant brain tumor initiating cells (BTICs). In a number of, but not all, patients with glioma, YB­1 is primarily located in the nucleus; this promotes XVir­N­31­replication and subsequently tumor cell lysis. However, in certain BTICs, only a small amount of YB­1 has been identified to be nuclear, and therefore virus replication is suboptimal. YB­1 in BTICs was demonstrated to be translocated into the nucleus following irradiation, which was accompanied by an enhancement in XVir­N­31 production. R28 glioma spheres implanted in living organotypic human brain slices exhibited a significantly delayed growth rate when pre­irradiated prior to XVir­N­31­infection as compared with single treatment methods. Consistent with the in vitro data, R28 glioma­bearing mice exhibited a prolonged mean and median survival following single tumor irradiation prior to intratumoral XVir­N­31 injection, compared with the single treatment methods. In conclusion, the present study demonstrated that in an experimental glioma model, tumor irradiation strengthened the effect of an XVir­N­31­based oncovirotherapy.

3.
Stem Cell Res ; 40: 101543, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31465893

RESUMO

De novo mutations in the KCNA2 gene, encoding the voltage-gated potassium channel KV1.2, have been identified to cause early-onset developmental and epileptic encephalopathies (DEE). KV1.2 channels conduct delayed-rectifier type K+ currents and play a crucial role in action potential repolarization. In this study we reprogrammed fibroblasts from a 6-months-old male patient with DEE carrying a de novo point mutation (c.1120A > G, p.Thr374Ala) in KCNA2 to induced pluripotent stem cells. Their pluripotency was verified by the capability to differentiate into all three germ layers and the expression of several pluripotency markers on RNA and protein levels.

4.
Am J Hum Genet ; 104(6): 1060-1072, 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31104773

RESUMO

The developmental and epileptic encephalopathies (DEEs) are heterogeneous disorders with a strong genetic contribution, but the underlying genetic etiology remains unknown in a significant proportion of individuals. To explore whether statistical support for genetic etiologies can be generated on the basis of phenotypic features, we analyzed whole-exome sequencing data and phenotypic similarities by using Human Phenotype Ontology (HPO) in 314 individuals with DEEs. We identified a de novo c.508C>T (p.Arg170Trp) variant in AP2M1 in two individuals with a phenotypic similarity that was higher than expected by chance (p = 0.003) and a phenotype related to epilepsy with myoclonic-atonic seizures. We subsequently found the same de novo variant in two individuals with neurodevelopmental disorders and generalized epilepsy in a cohort of 2,310 individuals who underwent diagnostic whole-exome sequencing. AP2M1 encodes the µ-subunit of the adaptor protein complex 2 (AP-2), which is involved in clathrin-mediated endocytosis (CME) and synaptic vesicle recycling. Modeling of protein dynamics indicated that the p.Arg170Trp variant impairs the conformational activation and thermodynamic entropy of the AP-2 complex. Functional complementation of both the µ-subunit carrying the p.Arg170Trp variant in human cells and astrocytes derived from AP-2µ conditional knockout mice revealed a significant impairment of CME of transferrin. In contrast, stability, expression levels, membrane recruitment, and localization were not impaired, suggesting a functional alteration of the AP-2 complex as the underlying disease mechanism. We establish a recurrent pathogenic variant in AP2M1 as a cause of DEEs with distinct phenotypic features, and we implicate dysfunction of the early steps of endocytosis as a disease mechanism in epilepsy.

5.
Stem Cell Res ; 37: 101445, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31075689

RESUMO

Developmental and epileptic encephalopathies (DEE) can be caused by mutations in the KCNA2 gene, coding for the voltage-gated K+ channel Kv1.2. This ion channel belongs to the delayed rectifier class of potassium channels and plays a role during the repolarization phase of an action potential. In this study we reprogrammed fibroblasts from a 30-year-old male patient with DDE carrying a point mutation (c.890G > A, p.Arg297Gln) in KCNA2 to induced pluripotent stem cells. Pluripotency state of the cells was verified by the capability to differentiate into all three germ layers and the expression of several pluripotency markers on RNA and protein levels.

7.
Am J Hum Genet ; 103(5): 666-678, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30343943

RESUMO

Developmental and epileptic encephalopathies (DEEs) are severe neurodevelopmental disorders often beginning in infancy or early childhood that are characterized by intractable seizures, abundant epileptiform activity on EEG, and developmental impairment or regression. CACNA1E is highly expressed in the central nervous system and encodes the α1-subunit of the voltage-gated CaV2.3 channel, which conducts high voltage-activated R-type calcium currents that initiate synaptic transmission. Using next-generation sequencing techniques, we identified de novo CACNA1E variants in 30 individuals with DEE, characterized by refractory infantile-onset seizures, severe hypotonia, and profound developmental impairment, often with congenital contractures, macrocephaly, hyperkinetic movement disorders, and early death. Most of the 14, partially recurring, variants cluster within the cytoplasmic ends of all four S6 segments, which form the presumed CaV2.3 channel activation gate. Functional analysis of several S6 variants revealed consistent gain-of-function effects comprising facilitated voltage-dependent activation and slowed inactivation. Another variant located in the domain II S4-S5 linker results in facilitated activation and increased current density. Five participants achieved seizure freedom on the anti-epileptic drug topiramate, which blocks R-type calcium channels. We establish pathogenic variants in CACNA1E as a cause of DEEs and suggest facilitated R-type calcium currents as a disease mechanism for human epilepsy and developmental disorders.

8.
Stem Cell Res ; 33: 6-9, 2018 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-30292882

RESUMO

Mutations in the KCNA2 gene, coding for the voltage-gated K+ channel Kv1.2, can cause developmental and epileptic encephalopathies. Kv1.2 channels play an important role in the repolarization phase of an action potential in nerve cells. Here, we reprogrammed human skin fibroblasts from a 13-year-old male patient with developmental and epileptic encephalopathy carrying a point mutation (c.982T>G, p.Leu328Val) in KCNA2 to human induced pluripotent stem cells (iPSCs) (HIHDNEi001-A). The cells maintained a normal karyotype and their pluripotency state was verified by the expression and staining of several pluripotency markers and capability to differentiate into all three germ layers.

9.
Sci Rep ; 7(1): 12249, 2017 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-28947761

RESUMO

Pathophysiological investigation of CNS-related diseases, such as epilepsy or neurodegenerative disorders, largely relies on histological studies on human post mortem tissue, tissue obtained by biopsy or resective surgery and on studies using disease models including animal models, heterologous expression systems or cell culture based approaches. However, in general it remains elusive to what extent results obtained in model systems can be directly translated to the human brain, calling for strategies allowing validation or even primary investigation in live human CNS tissue. In the work reported here, we prepared human organotypic slice cultures from access tissue of resective epilepsy surgery. Employing different culture conditions, we systematically compared artificial culturing media versus human cerbrospinal fluid (hCSF) obtained from patients with normal pressure hydrocephalus (NPH). Presented data demonstrates sustained cortical neuronal survival including not only maintenance of typical cellular electrophysiological properties and activity, such as robust action potential generation and synaptic connectivity, but also preservation of tonic and phasic network activity up to several weeks in vitro. As clearly delineated by immunocytochemistry, single cell patch clamp and extracellular recordings, we find that in contrast to artificial culturing media, hCSF significantly enhances neuron viability and maintenance of network activity.

10.
Brain ; 140(5): 1316-1336, 2017 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-28379373

RESUMO

Mutations in SCN2A, a gene encoding the voltage-gated sodium channel Nav1.2, have been associated with a spectrum of epilepsies and neurodevelopmental disorders. Here, we report the phenotypes of 71 patients and review 130 previously reported patients. We found that (i) encephalopathies with infantile/childhood onset epilepsies (≥3 months of age) occur almost as often as those with an early infantile onset (<3 months), and are thus more frequent than previously reported; (ii) distinct phenotypes can be seen within the late onset group, including myoclonic-atonic epilepsy (two patients), Lennox-Gastaut not emerging from West syndrome (two patients), and focal epilepsies with an electrical status epilepticus during slow sleep-like EEG pattern (six patients); and (iii) West syndrome constitutes a common phenotype with a major recurring mutation (p.Arg853Gln: two new and four previously reported children). Other known phenotypes include Ohtahara syndrome, epilepsy of infancy with migrating focal seizures, and intellectual disability or autism without epilepsy. To assess the response to antiepileptic therapy, we retrospectively reviewed the treatment regimen and the course of the epilepsy in 66 patients for which well-documented medical information was available. We find that the use of sodium channel blockers was often associated with clinically relevant seizure reduction or seizure freedom in children with early infantile epilepsies (<3 months), whereas other antiepileptic drugs were less effective. In contrast, sodium channel blockers were rarely effective in epilepsies with later onset (≥3 months) and sometimes induced seizure worsening. Regarding the genetic findings, truncating mutations were exclusively seen in patients with late onset epilepsies and lack of response to sodium channel blockers. Functional characterization of four selected missense mutations using whole cell patch-clamping in tsA201 cells-together with data from the literature-suggest that mutations associated with early infantile epilepsy result in increased sodium channel activity with gain-of-function, characterized by slowing of fast inactivation, acceleration of its recovery or increased persistent sodium current. Further, a good response to sodium channel blockers clinically was found to be associated with a relatively small gain-of-function. In contrast, mutations in patients with late-onset forms and an insufficient response to sodium channel blockers were associated with loss-of-function effects, including a depolarizing shift of voltage-dependent activation or a hyperpolarizing shift of channel availability (steady-state inactivation). Our clinical and experimental data suggest a correlation between age at disease onset, response to sodium channel blockers and the functional properties of mutations in children with SCN2A-related epilepsy.


Assuntos
Epilepsia/tratamento farmacológico , Epilepsia/genética , Epilepsia/fisiopatologia , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Canal de Sódio Disparado por Voltagem NAV1.2/fisiologia , Transtornos do Neurodesenvolvimento/genética , Bloqueadores dos Canais de Sódio/uso terapêutico , Adolescente , Adulto , Idade de Início , Criança , Pré-Escolar , Dinamarca/epidemiologia , Epilepsia/epidemiologia , Feminino , Humanos , Lactente , Masculino , Mutação , Fenótipo , Adulto Jovem
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA