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1.
Biochim Biophys Acta Mol Basis Dis ; 1870(5): 167127, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38519006

RESUMEN

Mutations in the SCN8A gene, encoding the voltage-gated sodium channel NaV1.6, are associated with a range of neurodevelopmental syndromes. The p.(Gly1625Arg) (G1625R) mutation was identified in a patient diagnosed with developmental epileptic encephalopathy (DEE). While most of the characterized DEE-associated SCN8A mutations were shown to cause a gain-of-channel function, we show that the G1625R variant, positioned within the S4 segment of domain IV, results in complex effects. Voltage-clamp analyses of NaV1.6G1625R demonstrated a mixture of gain- and loss-of-function properties, including reduced current amplitudes, increased time constant of fast voltage-dependent inactivation, a depolarizing shift in the voltage dependence of activation and inactivation, and increased channel availability with high-frequency repeated depolarization. Current-clamp analyses in transfected cultured neurons revealed that these biophysical properties caused a marked reduction in the number of action potentials when firing was driven by the transfected mutant NaV1.6. Accordingly, computational modeling of mature cortical neurons demonstrated a mild decrease in neuronal firing when mimicking the patients' heterozygous SCN8A expression. Structural modeling of NaV1.6G1625R suggested the formation of a cation-π interaction between R1625 and F1588 within domain IV. Double-mutant cycle analysis revealed that this interaction affects the voltage dependence of inactivation in NaV1.6G1625R. Together, our studies demonstrate that the G1625R variant leads to a complex combination of gain and loss of function biophysical changes that result in an overall mild reduction in neuronal firing, related to the perturbed interaction network within the voltage sensor domain, necessitating personalized multi-tiered analysis for SCN8A mutations for optimal treatment selection.


Asunto(s)
Potenciales de Acción , Discapacidades del Desarrollo , Epilepsia , Canal de Sodio Activado por Voltaje NAV1.6 , Neuronas , Canal de Sodio Activado por Voltaje NAV1.6/genética , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Humanos , Neuronas/metabolismo , Neuronas/patología , Epilepsia/genética , Epilepsia/patología , Epilepsia/metabolismo , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/patología , Animales , Masculino , Femenino , Células HEK293 , Mutación
2.
Front Cell Neurosci ; 17: 1149391, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37206664

RESUMEN

Dravet syndrome (Dravet) is a severe congenital developmental genetic epilepsy caused by de novo mutations in the SCN1A gene. Nonsense mutations are found in ∼20% of the patients, and the R613X mutation was identified in multiple patients. Here we characterized the epileptic and non-epileptic phenotypes of a novel preclinical Dravet mouse model harboring the R613X nonsense Scn1a mutation. Scn1aWT/R613X mice, on a mixed C57BL/6J:129S1/SvImJ background, exhibited spontaneous seizures, susceptibility to heat-induced seizures, and premature mortality, recapitulating the core epileptic phenotypes of Dravet. In addition, these mice, available as an open-access model, demonstrated increased locomotor activity in the open-field test, modeling some non-epileptic Dravet-associated phenotypes. Conversely, Scn1aWT/R613X mice, on the pure 129S1/SvImJ background, had a normal life span and were easy to breed. Homozygous Scn1aR613X/R613X mice (pure 129S1/SvImJ background) died before P16. Our molecular analyses of hippocampal and cortical expression demonstrated that the premature stop codon induced by the R613X mutation reduced Scn1a mRNA and NaV1.1 protein levels to ∼50% in heterozygous Scn1aWT/R613X mice (on either genetic background), with marginal expression in homozygous Scn1aR613X/R613X mice. Together, we introduce a novel Dravet model carrying the R613X Scn1a nonsense mutation that can be used to study the molecular and neuronal basis of Dravet, as well as the development of new therapies associated with SCN1A nonsense mutations in Dravet.

3.
J Clin Invest ; 133(12)2023 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-37192002

RESUMEN

Dravet syndrome (DS), an intractable childhood epileptic encephalopathy with a high fatality rate, is typically caused by loss-of-function mutations in one allele of SCN1A, which encodes NaV1.1, a 250-kDa voltage-gated sodium channel. In contrast to other epilepsies, pharmaceutical treatment for DS is limited. Here, we demonstrate that viral vector-mediated delivery of a codon-modified SCN1A open reading frame into the brain improves DS comorbidities in juvenile and adolescent DS mice (Scn1aA1783V/WT). Notably, bilateral vector injections into the hippocampus and/or the thalamus of DS mice increased survival, reduced the occurrence of epileptic spikes, provided protection from thermally induced seizures, corrected background electrocorticographic activity and behavioral deficits, and restored hippocampal inhibition. Together, our results provide a proof of concept for the potential of SCN1A delivery as a therapeutic approach for infants and adolescents with DS-associated comorbidities.


Asunto(s)
Epilepsias Mioclónicas , Canal de Sodio Activado por Voltaje NAV1.1 , Ratones , Animales , Canal de Sodio Activado por Voltaje NAV1.1/genética , Canal de Sodio Activado por Voltaje NAV1.1/metabolismo , Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/terapia , Convulsiones/genética , Convulsiones/metabolismo , Hipocampo/metabolismo , Mutación
4.
Front Pharmacol ; 14: 1118216, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37021051

RESUMEN

Dravet syndrome (Dravet) is a rare and severe form of developmental epileptic encephalopathy. Antiseizure medications (ASMs) for Dravet patients include valproic acid (VA) or clobazam (CLB), with or without stiripentol (STP), while sodium channel blockers like carbamazepine (CBZ) or lamotrigine (LTG) are contraindicated. In addition to their effect on epileptic phenotypes, ASMs were shown to modify the properties of background neuronal activity. Nevertheless, little is known about these background properties alterations in Dravet. Here, utilizing Dravet mice (DS, Scn1a A1783V/WT), we tested the acute effect of several ASMs on background electrocorticography (ECoG) activity and frequency of interictal spikes. Compared to wild-type mice, background ECoG activity in DS mice had lower power and reduced phase coherence, which was not corrected by any of the tested ASMs. However, acute administration of Dravet-recommended drugs, VA, CLB, or a combination of CLB + STP, caused, in most mice, a reduction in the frequency of interictal spikes, alongside an increase in the relative contribution of the beta frequency band. Conversely, CBZ and LTG increased the frequency of interictal spikes, with no effect on background spectral properties. Moreover, we uncovered a correlation between the reduction in interictal spike frequency, the drug-induced effect on the power of background activity, and a spectral shift toward higher frequency bands. Together, these data provide a comprehensive analysis of the effect of selected ASMs on the properties of background neuronal oscillations, and highlight a possible correlation between their effect on epilepsy and background activity.

5.
Front Mol Neurosci ; 15: 823640, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35370551

RESUMEN

Dravet syndrome is severe childhood-onset epilepsy, caused by loss of function mutations in the SCN1A gene, encoding for the voltage-gated sodium channel NaV1.1. The leading hypothesis is that Dravet is caused by selective reduction in the excitability of inhibitory neurons, due to hampered activity of NaV1.1 channels in these cells. However, these initial neuronal changes can lead to further network alterations. Here, focusing on the CA1 microcircuit in hippocampal brain slices of Dravet syndrome (DS, Scn1a A1783V/WT) and wild-type (WT) mice, we examined the functional response to the application of Hm1a, a specific NaV1.1 activator, in CA1 stratum-oriens (SO) interneurons and CA1 pyramidal excitatory neurons. DS SO interneurons demonstrated reduced firing and depolarized threshold for action potential (AP), indicating impaired activity. Nevertheless, Hm1a induced a similar AP threshold hyperpolarization in WT and DS interneurons. Conversely, a smaller effect of Hm1a was observed in CA1 pyramidal neurons of DS mice. In these excitatory cells, Hm1a application resulted in WT-specific AP threshold hyperpolarization and increased firing probability, with no effect on DS neurons. Additionally, when the firing of SO interneurons was triggered by CA3 stimulation and relayed via activation of CA1 excitatory neurons, the firing probability was similar in WT and DS interneurons, also featuring a comparable increase in the firing probability following Hm1a application. Interestingly, a similar functional response to Hm1a was observed in a second DS mouse model, harboring the nonsense Scn1a R613X mutation. Furthermore, we show homeostatic synaptic alterations in both CA1 pyramidal neurons and SO interneurons, consistent with reduced excitation and inhibition onto CA1 pyramidal neurons and increased release probability in the CA1-SO synapse. Together, these results suggest global neuronal alterations within the CA1 microcircuit extending beyond the direct impact of NaV1.1 dysfunction.

6.
Neurobiol Dis ; 148: 105209, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33271326

RESUMEN

Dravet syndrome (Dravet) is a rare, severe childhood-onset epilepsy, caused by heterozygous de novo mutations in the SCN1A gene, encoding for the alpha subunit of the voltage-gated sodium channel, NaV1.1. The neuronal basis of Dravet is debated, with evidence favoring reduced function of inhibitory neurons, that might be transient, or enhanced activity of excitatory cells. Here, we utilized Dravet mice to trace developmental changes in the hippocampal CA1 circuit, examining the properties of CA1 horizontal stratum-oriens (SO) interneurons and pyramidal neurons, through the pre-epileptic, severe and stabilization stages of Dravet. Our data indicate that reduced function of SO interneurons persists from the pre-epileptic through the stabilization stages, with the greatest functional impairment observed during the severe stage. In contrast, opposing changes were detected in CA1 excitatory neurons, with a transient increase in their excitability during the pre-epileptic stage, followed by reduced excitability at the severe stage. Interestingly, alterations in the function of both inhibitory and excitatory neurons were more pronounced when the firing was evoked by synaptic stimulation, implying that loss of function of NaV1.1 may also affect somatodendritic functions. These results suggest a complex pathophysiological mechanism and indicate that the developmental trajectory of this disease is governed by reciprocal functional changes in both excitatory and inhibitory neurons.


Asunto(s)
Potenciales de Acción/fisiología , Región CA1 Hipocampal/metabolismo , Epilepsias Mioclónicas/metabolismo , Interneuronas/metabolismo , Células Piramidales/metabolismo , Animales , Región CA1 Hipocampal/fisiopatología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/fisiopatología , Interneuronas/fisiología , Ratones , Canal de Sodio Activado por Voltaje NAV1.1/genética , Neuronas , Células Piramidales/fisiología , Convulsiones/genética , Convulsiones/metabolismo , Convulsiones/fisiopatología
7.
Biochim Biophys Acta Mol Basis Dis ; 1865(9): 2210-2223, 2019 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-31055083

RESUMEN

Mutations in the type I procollagen C-propeptide occur in ~6.5% of Osteogenesis Imperfecta (OI) patients. They are of special interest because this region of procollagen is involved in α chain selection and folding, but is processed prior to fibril assembly and is absent in mature collagen fibrils in tissue. We investigated the consequences of seven COL1A1 C-propeptide mutations for collagen biochemistry in comparison to three probands with classical glycine substitutions in the collagen helix near the C-propeptide and a normal control. Procollagens with C-propeptide defects showed the expected delayed chain incorporation, slow folding and overmodification. Immunofluorescence microscopy indicated that procollagen with C-propeptide defects was mislocalized to the ER lumen, in contrast to the ER membrane localization of normal procollagen and procollagen with helical substitutions. Notably, pericellular processing of procollagen with C-propeptide mutations was defective, with accumulation of pC-collagen and/or reduced production of mature collagen. In vitro cleavage assays with BMP-1 ±â€¯PCPE-1 confirmed impaired C-propeptide processing of procollagens containing mutant proα1(I) chains. Overmodified collagens were incorporated into the matrix in culture. Dermal fibrils showed alterations in average diameter and diameter variability and bone fibrils were disorganized. Altered ER-localization and reduced pericellular processing of defective C-propeptides are expected to contribute to abnormal osteoblast differentiation and matrix function, respectively.


Asunto(s)
Colágeno Tipo I/genética , Retículo Endoplásmico/metabolismo , Procolágeno/metabolismo , Rastreo Diferencial de Calorimetría , Células Cultivadas , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Microscopía Fluorescente , Mutación Missense , Osteogénesis Imperfecta/metabolismo , Osteogénesis Imperfecta/patología , Estructura Terciaria de Proteína
8.
PLoS One ; 14(2): e0211901, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30735520

RESUMEN

Mutations in the SCN1A gene, which encodes for the voltage-gated sodium channel NaV1.1, cause Dravet syndrome, a severe developmental and epileptic encephalopathy. Genetic testing of this gene is recommended early in life. However, predicting the outcome of de novo missense SCN1A mutations is difficult, since milder epileptic syndromes may also be associated. In this study, we correlated clinical severity with functional in vitro electrophysiological testing of channel activity and bioinformatics prediction of damaging mutational effects. Three patients, bearing the mutations p.Gly177Ala, p.Ser259Arg and p.Glu1923Arg, showed frequent intractable seizures that had started early in life, with cognitive and behavioral deterioration, consistent with classical Dravet phenotypes. These mutations failed to produce measurable sodium currents in a mammalian expression system, indicating complete loss of channel function. A fourth patient, who harbored the mutation p.Met1267Ile, though presenting with seizures early in life, showed lower seizure burden and higher cognitive function, matching borderland Dravet phenotypes. In correlation with this, functional analysis demonstrated the presence of sodium currents, but with partial loss of function. In contrast, six bioinformatics tools for predicting mutational pathogenicity suggested similar impact for all mutations. Likewise, homology modeling of the secondary and tertiary structures failed to reveal misfolding. In conclusion, functional studies using patch clamp are suggested as a prognostic tool, whereby detectable currents imply milder phenotypes and absence of currents indicate an unfavorable prognosis. Future development of automated patch clamp systems will facilitate the inclusion of such functional testing as part of personalized patient diagnostic schemes.


Asunto(s)
Disfunción Cognitiva/diagnóstico , Epilepsias Mioclónicas/diagnóstico , Predisposición Genética a la Enfermedad , Mutación Missense , Canal de Sodio Activado por Voltaje NAV1.1/genética , Potenciales de Acción , Sustitución de Aminoácidos , Niño , Preescolar , Disfunción Cognitiva/genética , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/fisiopatología , Biología Computacional/métodos , Diagnóstico Precoz , Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/metabolismo , Epilepsias Mioclónicas/fisiopatología , Femenino , Expresión Génica , Células HEK293 , Humanos , Transporte Iónico , Masculino , Canal de Sodio Activado por Voltaje NAV1.1/química , Canal de Sodio Activado por Voltaje NAV1.1/metabolismo , Técnicas de Placa-Clamp , Medicina de Precisión , Pronóstico , Índice de Severidad de la Enfermedad , Homología Estructural de Proteína , Transfección
9.
Clin Exp Rheumatol ; 34 Suppl 100(5): 23-29, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26886502

RESUMEN

OBJECTIVES: γδ T cells of the Vγ9Vδ2 subtype secrete anti-fibrotic cytokines upon isopentenyl pyrophosphate (IPP) stimulation. In this study, we sought to compare IPP and Zoledronate, an up-regulator of IPP, effects on proliferation and cytokine secretion of Vγ9+ T cells from systemic sclerosis (SSc) patients and healthy controls (HCs). We also examined the effect of IPP-triggered peripheral blood mononuclear cells (PBMC) on fibroblast procolla- gen secretion. METHODS: PBMC from SSc patients and HCs were stimulated by increasing concentrations of Zoledronate, with or without IPP, and Vγ9+ T cell percentages were calculated using FACScan analysis. Subsequently, PBMC were cultured with IPP or toxic shock syndrome toxin-1 (TSST-1), and contents of the anti-fibrotic cytokines tumour necrosis factor (TNF)-α and interferon (IFN)-γ were measured by ELISA kits. Finally, supernatants of IPP-triggered Vγ9+ T cells from SSc patients were added to fibroblast cultures, and relative intensities of procollagen α1 chains were determined by densinometry. RESULTS: Higher concentrations of Zoledronate were required for maximal proliferation of Vγ9+ T cells in 9 SSc patients compared to 9 HCs, irrespective of exogenous IPP. When compared to stimulation by TSST-1, a non-Vγ9+ selective reagent, secretion of the anti-fibrotic cytokines TNF-α and IFN-γ in response to IPP was relatively diminished in SSc but not in HCs. Reduction of procollagen secretion by fibroblasts cultured with supernatants of IPP-stimulated PBMC was observed only in some SSc patients. CONCLUSIONS: Activated Vγ9+ T cells could act as anti-fibrotic mediators in SSc, although decreased responsiveness to IPP may play a role in the pathological fibrosis of this disease.


Asunto(s)
Activación de Linfocitos , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Esclerodermia Sistémica/inmunología , Subgrupos de Linfocitos T/inmunología , Estudios de Casos y Controles , Proliferación Celular , Células Cultivadas , Colágeno Tipo I/metabolismo , Difosfonatos/farmacología , Relación Dosis-Respuesta a Droga , Fibroblastos/inmunología , Fibroblastos/metabolismo , Fibrosis , Hemiterpenos/farmacología , Humanos , Imidazoles/farmacología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Activación de Linfocitos/efectos de los fármacos , Compuestos Organofosforados/farmacología , Fenotipo , Procolágeno/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Transducción de Señal , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Ácido Zoledrónico
10.
Int J Biochem Cell Biol ; 57: 45-53, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25286301

RESUMEN

Procollagen C-proteinase enhancer 1 (PCPE-1) is an extracellular matrix glycoprotein that can stimulate procollagen processing by procollagen C-proteinases (PCPs) such as bone morphogenetic protein-1 (BMP-1). PCPE-1 consists of two CUB domains that bind to the procollagen C-propeptide and are responsible for enhancing activity and a netrin-like (NTR) domain that binds to BMP-1 as well as heparin and heparan sulfate. The NTR domain also mediates binding of PCPE-1 to cells, an interaction inhibited by heparin, thus suggesting involvement of cell membrane heparan-sulfate proteoglycans (HSPGs). Using pull-down experiments and an ELISA type binding assay we show here that PCPE-1 binds to three cell membrane HSPGs, syndecans-1, -2 and -4. We also demonstrate that this binding is mediated by the NTR domain and depends on the glycosaminoglycan chains of the syndecans. Using co-immunoprecipitation and an ELISA type binding assay we show that PCPE-1 can also bind fibronectin (an established binding partner of BMP-1), another interaction involving the NTR domain. Consistently, fibronectin inhibits cell attachment to PCPE-1 although it does not affect PCPE-1 enhancing activity. PCPE-1 is not an adhesive protein since cell attachment to PCPE-1 is not associated with cell spreading and/or actin filaments formation. The results suggest that PCPE-1 binding to syndecans and/or fibronectin may control collagen fibril assembly on the cell surface. Further characterization of these interactions may pave the way for future design of new means to modulate collagen deposition in pathological conditions such as fibrosis.


Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Glicoproteínas/metabolismo , Sindecanos/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Unión Proteica , Estructura Terciaria de Proteína
11.
Hum Mutat ; 32(6): 598-609, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21344539

RESUMEN

Osteogenesis imperfecta (OI) is most often caused by mutations in the type I procollagen genes (COL1A1/COL1A2). We identified two children with substitutions in the type I procollagen C-propeptide cleavage site, which disrupt a unique processing step in collagen maturation and define a novel phenotype within OI. The patients have mild OI caused by mutations in COL1A1 (Patient 1: p.Asp1219Asn) or COL1A2 (Patient 2: p.Ala1119Thr), respectively. Patient 1 L1-L4 DXA Z-score was +3.9 and pQCT vBMD was+3.1; Patient 2 had L1-L4 DXA Z-score of 0.0 and pQCT vBMD of -1.8. Patient BMD contrasts with radiographic osteopenia and histomorphometry without osteosclerosis. Mutant procollagen processing is impaired in pericellular and in vitro assays. Patient dermal collagen fibrils have irregular borders. Incorporation of pC-collagen into matrix leads to increased bone mineralization. FTIR imaging confirms elevated mineral/matrix ratios in both patients, along with increased collagen maturation in trabecular bone, compared to normal or OI controls. Bone mineralization density distribution revealed a marked shift toward increased mineralization density for both patients. Patient 1 has areas of higher and lower bone mineralization than controls; Patient 2's bone matrix has a mineral content exceeding even classical OI bone. These patients define a new phenotype of high BMD OI and demonstrate that procollagen C-propeptide cleavage is crucial to normal bone mineralization.


Asunto(s)
Huesos/anomalías , Huesos/patología , Colágeno Tipo I/genética , Osteogénesis Imperfecta/genética , Osteogénesis Imperfecta/patología , Fragmentos de Péptidos/genética , Procolágeno/genética , Adolescente , Secuencia de Aminoácidos , Animales , Densidad Ósea/genética , Matriz Ósea , Calcificación Fisiológica/genética , Niño , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Femenino , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Mutación , Fragmentos de Péptidos/metabolismo , Fenotipo , Procolágeno/metabolismo
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