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2.
Neurooncol Pract ; 9(3): 193-200, 2022 May.
Artículo en Inglés | MEDLINE | ID: mdl-35601970

RESUMEN

Background: Gliomas are the most common primary brain tumor in adults. Current treatments involve surgery, radiation, and temozolomide (TMZ) chemotherapy; however, prognosis remains poor and new approaches are required. Circadian medicine aims to maximize treatment efficacy and/or minimize toxicity by timed delivery of medications in accordance with the daily rhythms of the patient. We published a retrospective study showing greater anti-tumor efficacy for the morning, relative to the evening, administration of TMZ in patients with glioblastoma. We conducted this prospective randomized trial to determine the feasibility, and potential clinical impact, of TMZ chronotherapy in patients with gliomas (NCT02781792). Methods: Adult patients with gliomas (WHO grade II-IV) were enrolled prior to initiation of monthly TMZ therapy and were randomized to receive TMZ either in the morning (AM) before 10 am or in the evening (PM) after 8 pm. Pill diaries were recorded to measure compliance and FACT-Br quality of life (QoL) surveys were completed throughout treatment. Study compliance, adverse events (AE), and overall survival were compared between the two arms. Results: A total of 35 evaluable patients, including 21 with GBM, were analyzed (18 AM patients and 17 PM patients). Compliance data demonstrated the feasibility of timed TMZ dosing. There were no significant differences in AEs, QoL, or survival between the arms. Conclusions: Chronotherapy with TMZ is feasible. A larger study is needed to validate the effect of chronotherapy on clinical efficacy.

3.
J Neurosci ; 29(7): 2027-42, 2009 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-19228957

RESUMEN

The beta subunits of voltage-gated Na channels (Scnxb) regulate the gating of pore-forming alpha subunits, as well as their trafficking and localization. In heterologous expression systems, beta1, beta2, and beta3 subunits influence inactivation and persistent current in different ways. To test how the beta4 protein regulates Na channel gating, we transfected beta4 into HEK (human embryonic kidney) cells stably expressing Na(V)1.1. Unlike a free peptide with a sequence from the beta4 cytoplasmic domain, the full-length beta4 protein did not block open channels. Instead, beta4 expression favored open states by shifting activation curves negative, decreasing the slope of the inactivation curve, and increasing the percentage of noninactivating current. Consequently, persistent current tripled in amplitude. Expression of beta1 or chimeric subunits including the beta1 extracellular domain, however, favored inactivation. Coexpressing Na(V)1.1 and beta4 with beta1 produced tiny persistent currents, indicating that beta1 overcomes the effects of beta4 in heterotrimeric channels. In contrast, beta1(C121W), which contains an extracellular epilepsy-associated mutation, did not counteract the destabilization of inactivation by beta4 and also required unusually large depolarizations for channel opening. In cultured hippocampal neurons transfected with beta4, persistent current was slightly but significantly increased. Moreover, in beta4-expressing neurons from Scn1b and Scn1b/Scn2b null mice, entry into inactivated states was slowed. These data suggest that beta1 and beta4 have antagonistic roles, the former favoring inactivation, and the latter favoring activation. Because increased Na channel availability may facilitate action potential firing, these results suggest a mechanism for seizure susceptibility of both mice and humans with disrupted beta1 subunits.


Asunto(s)
Potenciales de Acción/genética , Encéfalo/metabolismo , Membrana Celular/metabolismo , Activación del Canal Iónico/genética , Neuronas/metabolismo , Canales de Sodio/metabolismo , Animales , Línea Celular , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Canal de Sodio Activado por Voltaje NAV1.1 , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Sodio/metabolismo , Canales de Sodio/química , Canales de Sodio/genética , Transfección , Subunidad beta-1 de Canal de Sodio Activado por Voltaje , Subunidad beta-4 de Canal de Sodio Activado por Voltaje
4.
J Neurosci ; 29(34): 10764-78, 2009 Aug 26.
Artículo en Inglés | MEDLINE | ID: mdl-19710327

RESUMEN

Dravet syndrome (also called severe myoclonic epilepsy of infancy) is one of the most severe forms of childhood epilepsy. Most patients have heterozygous mutations in SCN1A, encoding voltage-gated sodium channel Na(v)1.1 alpha subunits. Sodium channels are modulated by beta1 subunits, encoded by SCN1B, a gene also linked to epilepsy. Here we report the first patient with Dravet syndrome associated with a recessive mutation in SCN1B (p.R125C). Biochemical characterization of p.R125C in a heterologous system demonstrated little to no cell surface expression despite normal total cellular expression. This occurred regardless of coexpression of Na(v)1.1 alpha subunits. Because the patient was homozygous for the mutation, these data suggest a functional SCN1B null phenotype. To understand the consequences of the lack of beta1 cell surface expression in vivo, hippocampal slice recordings were performed in Scn1b(-/-) versus Scn1b(+/+) mice. Scn1b(-/-) CA3 neurons fired evoked action potentials with a significantly higher peak voltage and significantly greater amplitude compared with wild type. However, in contrast to the Scn1a(+/-) model of Dravet syndrome, we found no measurable differences in sodium current density in acutely dissociated CA3 hippocampal neurons. Whereas Scn1b(-/-) mice seize spontaneously, the seizure susceptibility of Scn1b(+/-) mice was similar to wild type, suggesting that, like the parents of this patient, one functional SCN1B allele is sufficient for normal control of electrical excitability. We conclude that SCN1B p.R125C is an autosomal recessive cause of Dravet syndrome through functional gene inactivation.


Asunto(s)
Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/fisiopatología , Polimorfismo de Nucleótido Simple/genética , Canales de Sodio/genética , Animales , Arginina/genética , Biofisica , Línea Celular Transformada , Cisteína/genética , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Estimulación Eléctrica , Epilepsias Mioclónicas/mortalidad , Femenino , Proteínas Fluorescentes Verdes/genética , Hipocampo/patología , Humanos , Técnicas In Vitro , Lactante , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Moleculares , Canal de Sodio Activado por Voltaje NAV1.1 , Proteínas del Tejido Nervioso/deficiencia , Oocitos , Canales de Sodio/deficiencia , Temperatura , Transfección , Gemelos , Subunidad beta-1 de Canal de Sodio Activado por Voltaje , Xenopus laevis
5.
J Neurosci ; 28(47): 12510-22, 2008 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-19020043

RESUMEN

Voltage-gated Na(+) channels initiate and propagate action potentials in excitable cells. Mammalian Na(+) channels are composed of one pore-forming alpha-subunit and two beta-subunits. SCN1B encodes the Na(+) channel beta1-subunit that modulates channel gating and voltage dependence, regulates channel cell surface expression, and functions as a cell adhesion molecule (CAM). We recently identified scn1ba, a zebrafish ortholog of SCN1B. Here we report that zebrafish express a second beta1-like paralog, scn1bb. In contrast to the restricted expression of scn1ba mRNA in excitable cells, we detected scn1bb transcripts and protein in several ectodermal derivatives including neurons, glia, the lateral line, peripheral sensory structures, and tissues derived from other germ layers such as the pronephros. As expected for beta1-subunits, elimination of Scn1bb protein in vivo by morpholino knock-down reduced Na(+) current amplitudes in Rohon-Beard neurons of zebrafish embryos, consistent with effects observed in heterologous systems. Further, after Scn1bb knock-down, zebrafish embryos displayed defects in Rohon-Beard mediated touch sensitivity, demonstrating the significance of Scn1bb modulation of Na(+) current to organismal behavior. In addition to effects associated with Na(+) current modulation, Scn1bb knockdown produced phenotypes consistent with CAM functions. In particular, morpholino knock-down led to abnormal development of ventrally projecting spinal neuron axons, defasciculation of the olfactory nerve, and increased hair cell number in the inner ear. We propose that, in addition to modulation of electrical excitability, Scn1bb plays critical developmental roles by functioning as a CAM in the zebrafish embryonic nervous system.


Asunto(s)
Axones/fisiología , Neuronas Motoras/citología , Neuronas Motoras/fisiología , Neuroglía/fisiología , Canales de Sodio/metabolismo , Tacto/fisiología , Proteínas de Pez Cebra/metabolismo , Análisis de Varianza , Animales , Animales Modificados Genéticamente , Anticuerpos Monoclonales/farmacología , Axones/efectos de los fármacos , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Embrión no Mamífero , Células Epiteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Riñón/citología , Riñón/embriología , Riñón/metabolismo , Neuronas Motoras/clasificación , Neuronas Motoras/efectos de los fármacos , Alineación de Secuencia/métodos , Canales de Sodio/inmunología , Médula Espinal/citología , Tubulina (Proteína)/metabolismo , Subunidad beta-1 de Canal de Sodio Activado por Voltaje , Pez Cebra , Proteínas de Pez Cebra/inmunología
6.
J Neurosci ; 28(12): 3246-56, 2008 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-18354028

RESUMEN

Voltage-gated Na(+) channel beta1 subunits are multifunctional, participating in channel modulation and cell adhesion in vitro. We previously demonstrated that beta1 promotes neurite outgrowth of cultured cerebellar granule neurons (CGNs) via homophilic adhesion. Both lipid raft-associated kinases and nonraft fibroblast growth factor (FGF) receptors are implicated in cell adhesion molecule-mediated neurite extension. In the present study, we reveal that beta1-mediated neurite outgrowth is abrogated in Fyn and contactin (Cntn) null CGNs. beta1 protein levels are unchanged in Fyn null brains, whereas levels are significantly reduced in Cntn null brain lysates. FGF or EGF (epidermal growth factor) receptor kinase inhibitors have no effect on beta1-mediated neurite extension. These results suggest that beta1-mediated neurite outgrowth occurs through a lipid raft signaling mechanism that requires the presence of both fyn kinase and contactin. In vivo, Scn1b null mice show defective CGN axon extension and fasciculation indicating that beta1 plays a role in cerebellar microorganization. In addition, we find that axonal pathfinding and fasciculation are abnormal in corticospinal tracts of Scn1b null mice consistent with the suggestion that beta1 may have widespread effects on postnatal neuronal development. These data are the first to demonstrate a cell-adhesive role for beta1 in vivo. We conclude that voltage-gated Na(+) channel beta1 subunits signal via multiple pathways on multiple timescales and play important roles in the postnatal development of the CNS.


Asunto(s)
Sistema Nervioso Central/crecimiento & desarrollo , Neuritas/fisiología , Neuronas/citología , Proteínas Proto-Oncogénicas c-fyn/fisiología , Canales de Sodio/fisiología , Aminoácidos , Análisis de Varianza , Animales , Animales Recién Nacidos , Bromodesoxiuridina/metabolismo , Proliferación Celular , Células Cultivadas , Sistema Nervioso Central/citología , Queratinocitos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/fisiología , Proteínas Proto-Oncogénicas c-fyn/deficiencia , Canales de Sodio/deficiencia , Subunidad beta-1 de Canal de Sodio Activado por Voltaje
7.
BMC Genomics ; 8: 226, 2007 Jul 10.
Artículo en Inglés | MEDLINE | ID: mdl-17623064

RESUMEN

BACKGROUND: Voltage-gated Na+ channel beta1 (Scn1b) subunits are multi-functional proteins that play roles in current modulation, channel cell surface expression, cell adhesion, cell migration, and neurite outgrowth. We have shown previously that beta1 modulates electrical excitability in vivo using a mouse model. Scn1b null mice exhibit spontaneous seizures and ataxia, slowed action potential conduction, decreased numbers of nodes of Ranvier in myelinated axons, alterations in nodal architecture, and differences in Na+ channel alpha subunit localization. The early death of these mice at postnatal day 19, however, make them a challenging model system to study. As a first step toward development of an alternative model to investigate the physiological roles of beta1 subunits in vivo we cloned two beta1-like subunit cDNAs from D. rerio. RESULTS: Two beta1-like subunit mRNAs from zebrafish, scn1ba_tv1 and scn1ba_tv2, arise from alternative splicing of scn1ba. The deduced amino acid sequences of Scn1ba_tv1 and Scn1ba_tv2 are identical except for their C-terminal domains. The C-terminus of Scn1ba_tv1 contains a tyrosine residue similar to that found to be critical for ankyrin association and Na+ channel modulation in mammalian beta1. In contrast, Scn1ba_tv2 contains a unique, species-specific C-terminal domain that does not contain a tyrosine. Immunohistochemical analysis shows that, while the expression patterns of Scn1ba_tv1 and Scn1ba_tv2 overlap in some areas of the brain, retina, spinal cord, and skeletal muscle, only Scn1ba_tv1 is expressed in optic nerve where its staining pattern suggests nodal expression. Both scn1ba splice forms modulate Na+ currents expressed by zebrafish scn8aa, resulting in shifts in channel gating mode, increased current amplitude, negative shifts in the voltage dependence of current activation and inactivation, and increases in the rate of recovery from inactivation, similar to the function of mammalian beta1 subunits. In contrast to mammalian beta1, however, neither zebrafish subunit produces a complete shift to the fast gating mode and neither subunit produces complete channel inactivation or recovery from inactivation. CONCLUSION: These data add to our understanding of structure-function relationships in Na+ channel beta1 subunits and establish zebrafish as an ideal system in which to determine the contribution of scn1ba to electrical excitability in vivo.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Canales de Sodio/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Anticuerpos/química , Anticuerpos/farmacología , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Cricetinae , Cricetulus , Electrofisiología , Embrión no Mamífero , Modelos Moleculares , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Ratas , Homología de Secuencia de Aminoácido , Canales de Sodio/inmunología , Canales de Sodio/metabolismo , Canales de Sodio/fisiología , Especificidad de la Especie , Distribución Tisular , Subunidad beta-1 de Canal de Sodio Activado por Voltaje , Xenopus laevis , Pez Cebra/embriología , Proteínas de Pez Cebra/inmunología , Proteínas de Pez Cebra/metabolismo
8.
J Biol Rhythms ; 32(2): 121-129, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28470120

RESUMEN

The safety and efficacy of chemotherapeutics can vary as a function of the time of their delivery during the day. This study aimed to improve the treatment of glioblastoma (GBM), the most common brain cancer, by testing whether the efficacy of the DNA alkylator temozolomide (TMZ) varies with the time of its administration. We found cell-intrinsic, daily rhythms in both human and mouse GBM cells. Circadian time of treatment affected TMZ sensitivity of murine GBM tumor cells in vitro. The maximum TMZ-induced DNA damage response, activation of apoptosis, and growth inhibition occurred near the daily peak in expression of the core clock gene Bmal1. Deletion of Bmal1 (Arntl) abolished circadian rhythms in gene expression and TMZ-induced activation of apoptosis and growth inhibition. These data indicate that tumor cell-intrinsic circadian rhythms are common to GBM tumors and can regulate TMZ cytotoxicity. Optimization of GBM treatment by timing TMZ administration to daily rhythms should be evaluated in prospective clinical trials.


Asunto(s)
Factores de Transcripción ARNTL/genética , Antineoplásicos Alquilantes/farmacología , Ritmo Circadiano/efectos de los fármacos , Dacarbazina/análogos & derivados , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción ARNTL/deficiencia , Factores de Transcripción ARNTL/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Reparación del ADN/efectos de los fármacos , Dacarbazina/farmacología , Esquema de Medicación , Glioblastoma/tratamiento farmacológico , Humanos , Ratones , Proteínas Circadianas Period/metabolismo , Temozolomida
9.
Cell Rep ; 7(3): 609-22, 2014 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-24767996

RESUMEN

Vertebrate circadian rhythms are organized by the hypothalamic suprachiasmatic nucleus (SCN). Despite its physiological importance, SCN development is poorly understood. Here, we show that Lim homeodomain transcription factor 1 (Lhx1) is essential for terminal differentiation and function of the SCN. Deletion of Lhx1 in the developing SCN results in loss of SCN-enriched neuropeptides involved in synchronization and coupling to downstream oscillators, among other aspects of circadian function. Intact, albeit damped, clock gene expression rhythms persist in Lhx1-deficient SCN; however, circadian activity rhythms are highly disorganized and susceptible to surprising changes in period, phase, and consolidation following neuropeptide infusion. Our results identify a factor required for SCN terminal differentiation. In addition, our in vivo study of combinatorial SCN neuropeptide disruption uncovered synergies among SCN-enriched neuropeptides in regulating normal circadian function. These animals provide a platform for studying the central oscillator's role in physiology and cognition.


Asunto(s)
Diferenciación Celular , Ritmo Circadiano/fisiología , Proteínas con Homeodominio LIM/metabolismo , Núcleo Supraquiasmático/citología , Factores de Transcripción/metabolismo , Animales , Apoptosis , Femenino , Expresión Génica , Genotipo , Proteínas con Homeodominio LIM/deficiencia , Proteínas con Homeodominio LIM/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuropéptidos/metabolismo , Núcleo Supraquiasmático/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genética
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