RESUMO
Thioredoxin, encoded by Txn1, acts as a critical antioxidant in the defense against oxidative stress by regulating the dithiol/disulfide balance of interacting proteins. The role of thioredoxin in the central nervous system (CNS) is largely unknown. A phenotype-driven study of N-ethyl-N-nitrosourea-mutated rats with wild-running seizures revealed the importance of Txn1 mutations in CNS degeneration. Genetic mapping identified Txn1-F54L in the epileptic rats. The insulin-reducing activity of Txn1-F54L was approximately one-third of that of the wild-type (WT). Bilateral symmetrical vacuolar degeneration in the midbrain, mainly in the thalamus and the inferior colliculus, was observed in the Txn1-F54L rats. The lesions displayed neuronal and oligodendrocytic cell death. Neurons in Txn1-F54L rats showed morphological changes in the mitochondria. Vacuolar degeneration peaked at five weeks of age, and spontaneous repair began at seven weeks. The TUNEL assay showed that fibroblasts derived from homozygotes were susceptible to cell death under oxidative stress. In five-week-old WT rats, energy metabolism in the thalamus was significantly higher than that in the cerebral cortex. In conclusion, in juvenile rats, Txn1 seems to play an essential role in reducing oxidative stress in the midbrains with high energy metabolism.
Assuntos
Estresse Oxidativo , Tiorredoxinas , Animais , Ratos , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Estresse Oxidativo/fisiologia , Mesencéfalo/metabolismo , Antioxidantes , OxirreduçãoRESUMO
CUX2 gene encodes a transcription factor that controls neuronal proliferation, dendrite branching and synapse formation, locating at the epilepsy-associated chromosomal region 12q24 that we previously identified by a genome-wide association study (GWAS) in Japanese population. A CUX2 recurrent de novo variant p.E590K has been described in patients with rare epileptic encephalopathies and the gene is a candidate for the locus, however the mutation may not be enough to generate the genome-wide significance in the GWAS and whether CUX2 variants appear in other types of epilepsies and physiopathological mechanisms are remained to be investigated. Here in this study, we conducted targeted sequencings of CUX2, a paralog CUX1 and its short isoform CASP harboring a unique C-terminus on 271 Japanese patients with a variety of epilepsies, and found that multiple CUX2 missense variants, other than the p.E590K, and some CASP variants including a deletion, predominantly appeared in patients with temporal lobe epilepsy (TLE). The CUX2 variants showed abnormal localization in human cell culture analysis. While wild-type CUX2 enhances dendritic arborization in fly neurons, the effect was compromised by some of the variants. Cux2- and Casp-specific knockout mice both showed high susceptibility to kainate, increased excitatory cell number in the entorhinal cortex, and significant enhancement in glutamatergic synaptic transmission to the hippocampus. CASP and CUX2 proteins physiologically bound to each other and co-expressed in excitatory neurons in brain regions including the entorhinal cortex. These results suggest that CUX2 and CASP variants contribute to the TLE pathology through a facilitation of excitatory synaptic transmission from entorhinal cortex to hippocampus.
Assuntos
Epilepsia do Lobo Temporal , Epilepsia , Animais , Epilepsia/genética , Estudo de Associação Genômica Ampla , Hipocampo/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Ácido Caínico , Camundongos , Convulsões/genética , Transmissão SinápticaRESUMO
Thioredoxin, encoded by Txn1, is a critical antioxidant that protects against oxidative damage by regulating the dithiol/disulfide balance of interacting proteins. We recently discovered the Adem rat, an epileptic rat harboring the Txn1-F54L mutation, characterized by wild running and vacuolar degeneration in the midbrain. This study aimed to characterize the classification of epilepsy in Adem rats. We performed simultaneous video-electroencephalographic recordings, magnetic resonance imaging, neurotransmitter measurements using gas chromatography-mass spectrometry (GC-MS), and immunohistochemistry. Adem rats exhibited absence, tonic, and focal seizures. The type of epilepsy was classified as combined generalized and focal epilepsy. Neurotransmitters in the midbrain and cortex were measured at 3 weeks of age, when neuronal cell death occurs in the midbrain. The results of GC-MS ruled out the dominance of the excitatory system in the midbrain and cortex of Adem rats. Activation of astrocytes and microglia was more pronounced at 5 weeks of age, at which time epileptic seizures occurred frequently. The underlying pathology in Adem rats remains unknown. However, glial cell activation and inflammation may play a significant role in the occurrence of epilepsy.
Assuntos
Epilepsias Parciais , Epilepsia Generalizada , Epilepsia , Animais , Modelos Animais de Doenças , Eletroencefalografia , Epilepsias Parciais/genética , Epilepsia/patologia , Ratos , ConvulsõesRESUMO
This study aimed to examine the effects of Cacna1a mutation on the phenotype of Scn1a-associated epilepsy in rats. We used rats with an N1417H missense mutation in the Scn1a gene and others with an M251K mutation in the Cacna1a gene. Scn1a/Cacna1a double mutant rats were generated by mating both Scn1a and Cacna1a mutants. We investigated general health and the epileptic phenotype in all these genotypes. The onset threshold of hyperthermia-induced seizures was examined at 5 weeks and spontaneous seizures were monitored using video-EEG recordings from 6 to 12 weeks of age. Scn1a/Cacna1a double mutants showed significantly reduced threshold for hyperthermia-sensitive seizures onset compared with the Scn1a mutants and had absence seizures having 6-7 c/s spike-wave bursts with changes in the spike-wave pattern, whereas Cacna1a mutants had regular 6-7 c/s spike-wave bursts. In Scn1a/Cacna1a double mutants, 6-7 c/s spike-wave bursts were accompanied with eyelid myoclonia and continuously shifting generalized clonic seizures, which were not observed in either Scn1a or Cacna1a mutants. Although a curvature of the spine was observed in rats of all these genotypes, the degree of curvature was more pronounced in Scn1a/Cacna1a double mutants, followed by Cacna1a and Scn1a mutants. Our results indicate that Cacna1a and Scn1a mutations mutually alter their original phenotypes in rats. The phenotype of absence seizures with eyelid myoclonia, generalized clonic seizures, and of spine curvature in the Scn1a/Cacna1a double mutants were similar to that observed in patients with Dravet syndrome.
Assuntos
Canais de Cálcio/genética , Epilepsia/genética , Epilepsia/fisiopatologia , Mutação de Sentido Incorreto/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Animais , Eletroencefalografia , Epilepsias Mioclônicas/genética , Epilepsia Tônico-Clônica/genética , Genótipo , Hipertermia/complicações , Fenótipo , Ratos , Ratos Endogâmicos F344 , Convulsões/etiologia , Convulsões/genética , Coluna Vertebral/anormalidadesRESUMO
OBJECTIVE: Neurodevelopmental disorders (NDDs) often associate with epilepsy or craniofacial malformations. Recent large-scale DNA analyses identified hundreds of candidate genes for NDDs, but a large portion of the cases still remain unexplained. We aimed to identify novel candidate genes for NDDs. METHODS: We performed exome sequencing of 95 patients with NDDs including 51 with trigonocephaly and subsequent targeted sequencing of additional 463 NDD patients, functional analyses of variant in vitro, and evaluations of autism spectrum disorder (ASD)-like phenotypes and seizure-related phenotypes in vivo. RESULTS: We identified de novo truncation variants in nine novel genes; CYP1A1, C14orf119, FLI1, CYB5R4, SEL1L2, RAB11FIP2, ZMYND8, ZNF143, and MSX2. MSX2 variants have been described in patients with cranial malformations, and our present patient with the MSX2 de novo truncation variant showed cranial meningocele and partial epilepsy. MSX2 protein is known to be ubiquitinated by an E3 ubiquitin ligase PJA1, and interestingly we found a PJA1 hemizygous p.Arg376Cys variant recurrently in seven Japanese NDD patients; five with trigonocephaly and one with partial epilepsy, and the variant was absent in 886 Japanese control individuals. Pja1 knock-in mice carrying p.Arg365Cys, which is equivalent to p.Arg376Cys in human, showed a significant decrease in PJA1 protein amount, suggesting a loss-of-function effect of the variant. Pja1 knockout mice displayed moderate deficits in isolation-induced ultrasonic vocalizations and increased seizure susceptibility to pentylenetetrazole. INTERPRETATION: These findings propose novel candidate genes including PJA1 and MSX2 for NDDs associated with craniofacial abnormalities and/or epilepsy.
Assuntos
Craniossinostoses/genética , Epilepsia/genética , Transtornos do Neurodesenvolvimento/genética , Ubiquitina-Proteína Ligases/genética , Animais , Transtorno do Espectro Autista/genética , Modelos Animais de Doenças , Feminino , Proteínas de Homeodomínio/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Comportamento Social , Vocalização Animal/fisiologia , Sequenciamento do ExomaRESUMO
Homozygous Groggy dams, which carry a Cacna1a missense mutation, often show no interest in their offspring, leading to frequent offspring deaths due to lack of nurturing. The present study aimed to clarify whether the Cacna1a mutation contributes to impaired attachment behaviors between dam and offspring. The open field test showed that homozygous female rats exhibited markedly short travel distance, whereas no difference was found between the motor activity of heterozygous females and that of wild types (WT). A series of behavioral tests was performed to compare the mother-offspring relationship between WT and heterozygous rats. Performance in the pup retrieval test was significantly less successful in heterozygous than WT dams. During the experiment, heterozygous dams spent significantly less time licking and crouching than WT dams. The offspring dam-seeking behavior test revealed that heterozygous pups' vocalizations were significantly less frequent and shorter than those of WT pups. Although no significant difference was found between WT and heterozygous offspring in the olfactory sense test, using a piece of chocolate, heterozygous pups took significantly longer to reach a sample of the dam's bedding. Taken together, these findings suggest that the Cacna1a mutation impairs both the dam's maternal behavior and the offspring's attachment behavior toward the dam.
Assuntos
Canais de Cálcio/genética , Comportamento Materno , Mutação , Animais , RatosRESUMO
The recent development of various new antiepileptic drugs (AEDs) has provided a wide range of therapeutic strategies for epilepsy. Information regarding the mechanisms of the action of AEDs is valuable when selecting drugs for individual epilepsy patients. AEDs can be categorized as those acting at the excitatory synapse, at the inhibitory synapse, on the extrasynaptic neuronal membrane, or with multiple or miscellaneous mechanisms of action. We herein briefly summarize and illustrate the action of AEDs on neurons and related findings that are pertinent to the clinical aspect of epileptology.
Assuntos
Anticonvulsivantes/farmacologia , Neurônios/efeitos dos fármacos , Animais , Humanos , Sinapses/efeitos dos fármacosRESUMO
PURPOSE: This study was performed to clarify the clinical features of Japanese patients with PRRT2 mutations. METHODS: The PRRT2 gene was analyzed in 135 patients with benign infantile epilepsy (BIE) or paroxysmal kinesigenic dyskinesia (PKD) using a direct sequencing method: 92 patients had BIE alone, 25 had both BIE and PKD, and 18 had PKD alone. Of the cases, 105 were familial, and 30 were sporadic. Clinical information was collected using a structured questionnaire. RESULTS: PRRT2 mutations were identified in 104 patients. Among the familial cases, PRRT2 mutations were found in at least one individual in 21 of 28 families with BIE alone, in 26 of 27 families with infantile convulsions and choreoathetosis, and in 2 of 3 families with PKD alone. Among the sporadic cases, PRRT2 mutations were observed in 7 of 25 patients with BIE alone, in 1 of 1 patient with BIE and PKD, and in 3 of 4 patients with PKD alone. The c.649dupC mutation was the most frequent, followed by the c.981C > G mutation. Among the patients with epilepsy, the median age at BIE onset was 5 months, the median age at the last seizure was 6 months, and the median number of seizures was 5. CONCLUSION: PRRT2 mutations were found in 68% of Japanese probands with BIE or PKD. The phenotypes of BIE associated with PRRT2 mutations were consistent with those of BIE diagnosed clinically.
Assuntos
Distonia/genética , Epilepsia Neonatal Benigna/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Humanos , Lactente , Japão , Mutação , LinhagemRESUMO
We report a female patient with Dravet syndrome (DS) with erratic segmental myoclonus, the origin of which was first identified in the cerebral cortex by the detection of myoclonus-associated cortical discharges. The discharges were disclosed through jerk-locked back-averaging of electroencephalogram (EEG) data using the muscle activity of myoclonus as triggers. The detected spikes on the contralateral parieto-central region preceded myoclonic muscle activity in the forearms by 28-46ms. The patient was six months old at the time of examination, and was developing normally before seizure onset at two months of age. She suffered from recurrent afebrile or febrile generalized tonic-clonic seizures that often developed into status epilepticus. Interictal EEG and brain magnetic resonance imaging (MRI) showed no significant findings. The amplitudes of the somatosensory-evoked potentials were not extremely large. She has a chromosomal microdeletion involving SCN1A and adjacent genes.
Assuntos
Córtex Cerebral/diagnóstico por imagem , Eletroencefalografia/métodos , Epilepsias Mioclônicas/diagnóstico por imagem , Mioclonia/diagnóstico por imagem , Córtex Cerebral/fisiopatologia , Diagnóstico Diferencial , Epilepsias Mioclônicas/tratamento farmacológico , Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/fisiopatologia , Feminino , Humanos , Lactente , Mioclonia/tratamento farmacológico , Mioclonia/genética , Mioclonia/fisiopatologia , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Deleção de Sequência , Estado Epiléptico/diagnóstico por imagem , Estado Epiléptico/tratamento farmacológico , Estado Epiléptico/genética , Estado Epiléptico/fisiopatologiaRESUMO
OBJECTIVE: The importance of epileptic high-frequency oscillations (HFOs) in electroencephalogram (EEG) is growing. Action potentials generating some HFOs are observed in the vicinity of neurons in experimental animals. However electrodes that are remote from neurons, as in case of clinical situations, should not record action potentials. We propose to resolve this question by a realistic simulation of epileptic neuronal network. METHODS: The rat dentate gyrus with sclerosis was simulated in silico. We computed the current dipole moment generated by each granule cell and the field potentials in a measurement area far from neurons. RESULTS: The dentate gyrus was stimulated through synaptic input to evoke discharges resembling interictal epileptiform discharges, which had superimposed HFOs⩽295Hz that were recordable with remote electrodes and represented bursts of action potentials of granule cells. The increase in power of HFOs was associated with the progression of sclerosis, the reduction of GABAergic inhibition, and the increase in cell connectivity. Spectral frequency of HFOs had similar tendencies. CONCLUSIONS: HFOs recorded with electrodes remote from neurons could actually be generated by clusters of action potentials. SIGNIFICANCE: The phenomenon of action potentials recorded with remote electrodes can possibly extend the clinical meaning of EEG.
Assuntos
Potenciais de Ação/fisiologia , Giro Denteado/fisiologia , Eletroencefalografia/métodos , Epilepsia/fisiopatologia , Neurônios/fisiologia , Animais , Giro Denteado/citologia , Eletroencefalografia/instrumentação , Masculino , Microeletrodos , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: Developmental disorders including cognitive deficit, hyperkinetic disorder, and autistic behaviors are frequently comorbid in epileptic patients with SCN1A mutations. However, the mechanisms underlying these developmental disorders are poorly understood and treatments are currently unavailable. Using a rodent model with an Scn1a mutation, we aimed to elucidate the pathophysiologic basis and potential therapeutic treatments for developmental disorders stemming from Scn1a mutations. METHODS: We conducted behavioral analyses on rats with the N1417H-Scn1a mutation. With high-performance liquid chromatography, we measured dopamine and its metabolites in the frontal cortex, striatum, nucleus accumbens, and midbrain. Methylphenidate was administered intraperitoneally to examine its effects on developmental disorder-like behaviors and hyperthermia-induced seizures. RESULTS: Behavioral studies revealed that Scn1a-mutant rats had repetitive behavior, hyperactivity, anxiety-like behavior, spatial learning impairments, and motor imbalance. Dopamine levels in the striatum and nucleus accumbens in Scn1a-mutant rats were significantly lower than those in wild-type rats. In Scn1a-mutant rats, methylphenidate, by increasing dopamine levels in the synaptic cleft, improved hyperactivity, anxiety-like behavior, and spatial learning impairments. Surprisingly, methylphenidate also strongly suppressed hyperthermia-induced seizures. SIGNIFICANCE: Dysfunction of the mesolimbic dopamine reward pathway may contribute to the hyperactivity and learning impairments in Scn1a-mutant rats. Methylphenidate was effective for treating hyperactivity, learning impairments, and hyperthermia-induced seizures. We propose that methylphenidate treatment may ameliorate not only developmental disorders but also epileptic seizures in patients with SCN1A mutations.
Assuntos
Estimulantes do Sistema Nervoso Central/uso terapêutico , Febre/complicações , Aprendizagem/efeitos dos fármacos , Metilfenidato/uso terapêutico , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Convulsões/tratamento farmacológico , Animais , Comportamento Animal/efeitos dos fármacos , Corpo Estriado/química , Dopamina/análise , Hipercinese/tratamento farmacológico , Hipercinese/genética , Masculino , Mutação de Sentido Incorreto/genética , Núcleo Accumbens/química , Ratos , Ratos Mutantes , Convulsões/etiologia , Convulsões/genética , Comportamento Estereotipado/efeitos dos fármacosRESUMO
Topical therapy is the most favored form of treatment for whitening against hyper-pigmentation and sunburn because it lends itself to self-administration, patient compliance and an absence of systemic adverse effects. However, high-molecular-weight, hydrophilic chemicals are difficult to use as transdermal delivery drugs and the use of topical drugs has been highly limited. There are now many potent tyrosinase inhibitors, for example, sulfite or kojic acid, but the efficacy of their skin transduction remains a big problem. Furthermore, melanogenesis inhibitors from natural sources have great potential, as they are considered to be safe and largely free from adverse side effects. We applied 11-arginine (11R), a cell-membrane-permeable peptide, as a transdermal delivery system with a skin delivery enhancer, pyrenbutyrate. We performed intracellular screening for melanogenesis inhibitors with 11R fused with several kinds of tyrosinase inhibitory peptides from natural sources. Of 28 tyrosinase peptides, 13 melanin synthesis inhibitory peptides were selected. Peptide No. 10 found in gliadin protein, a wheat component, most strongly inhibited melanin production. This No. 10 peptide, of only 8 amino acids, fused to 11R showed no cytotoxicity and inhibited melanin synthesis as determined through melanin content measured using an absorption spectrometer and observation with a transmission electron microscope. Next, we transduced this 11R-No. 10 into skin with an 11R transdermal delivery system after previous treatment with pyrenbutyrate and performed daily repetitive topical application for two weeks against a UV-induced sun-tanning guinea pig model. We observed a whitening effect in a model skin sample by Masson-Fontana staining and the 11R-No. 10 peptide-applied area showed significant melanogenesis inhibition. These results show that 11R using a transdermal drug delivery system with melanogenesis inhibitory peptide is a very safe and promising method for applications from cosmetics to the pharmaceutical industry.
Assuntos
Melaninas/metabolismo , Monofenol Mono-Oxigenase/antagonistas & inibidores , Peptídeos/química , Peptídeos/farmacologia , Administração Cutânea , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Sistemas de Liberação de Medicamentos , Feminino , Cobaias , Humanos , Melanoma Experimental , Peptídeos/administração & dosagem , Peptídeos/farmacocinética , Pele/efeitos dos fármacos , Pele/metabolismo , Pigmentação da Pele/efeitos dos fármacosRESUMO
New anti-cancer therapy with boron neutron capture therapy (BNCT) is based on the nuclear reaction of boron-10 with neutron irradiation. The median survival of BNCT patients with glioblastoma was almost twice as long as those receiving standard therapy in a Japanese BNCT clinical trial. In this clinical trial, two boron compounds, BPA (boronophenylalanine) and BSH (sodium borocaptate), were used for BNCT. BPA is taken up into cells through amino acid transporters that are expressed highly in almost all malignant cells, but BSH cannot pass through the cell membrane and remains outside the cell. We simulated the energy transfer against the nucleus at different locations of boron from outside the cell to the nuclear region with neutron irradiation and concluded that there was a marked difference between inside and outside the cell in boron localization. To overcome this disadvantage of BSH in BNCT, we used a cell-penetrating peptide system for transduction of BSH. CPP (cell-membrane penetrating peptide) is very common peptide domains that transduce many physiologically active substances into cells in vitro and in vivo. BSH-fused CPPs can penetrate the cell membrane and localize inside a cell. To increase the boron ratio in one BSH-peptide molecule, 8BSH fused to 11R with a dendritic lysine structure was synthesized and administrated to malignant glioma cells and a brain tumor mouse model. 8BSH-11R localized at the cell nucleus and showed a very high boron value in ICP results. With neutron irradiation, the 8BSH-11R administrated group showed a significant cancer killing effect compared to the 100 times higher concentration of BSH-administrated group. We concluded that BSH-fused CPPs were one of the most improved and potential boron compounds in the next-stage BNCT trial and 8BSH-11R may be applied in the clinical setting.
Assuntos
Boroidretos/uso terapêutico , Terapia por Captura de Nêutron de Boro , Peptídeos/uso terapêutico , Compostos de Sulfidrila/uso terapêutico , Neoplasias Encefálicas/radioterapia , Glioblastoma/radioterapia , HumanosRESUMO
The aim of this study was to assess the anticonvulsant effect of carbon dioxide (CO2) on Scn1a mutation-related febrile seizures. We examined physiological changes in the blood gas levels after the induction of hyperthermia-induced seizures (HISs), which were associated with the Scn1a missense mutation. We determined the efficacy of inhalation of 5% or 10% CO2 to treat HISs. HISs were evoked in Scn1a mutant and wild-type (WT) rats by hot water baths. To determine the anticonvulsant effect of CO2 inhalation, rats were placed in a chamber filled with air or mixed gas containing 5% CO2 or 10% CO2 for 3 min, immediately after the induction of HISs. We also analyzed the blood gas levels at the end of inhalation of CO2. Hot water bathing induced a significant reduction in the partial pressure of CO2 (pCO2) and respiratory alkalosis in the WT and Scn1a mutant rats. HISs were evoked in 100% of the Scn1a mutant rats within 5 min, but in none of the WT rats. The Scn1a mutant rats demonstrated a higher HISs susceptibility associated with respiratory alkalosis than the WT rats. Inhalation of 10% CO2 shortened the seizure duration from 62.6±12.1 s to 15.5±1.0 s. Blood gas analysis after the inhalation of 10% CO2 demonstrated an elevated pCO2 level and respiratory acidosis. Inhalation of 10% CO2 demonstrated a potent and fast-acting anticonvulsant effect against HISs.
Assuntos
Dióxido de Carbono/administração & dosagem , Febre/genética , Mutação de Sentido Incorreto/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Convulsões/genética , Animais , Febre/tratamento farmacológico , Febre/metabolismo , Inalação/fisiologia , Masculino , Ratos , Ratos Endogâmicos F344 , Ratos Mutantes , Convulsões/tratamento farmacológico , Convulsões/metabolismo , Fatores de TempoRESUMO
We examined high-frequency oscillations (HFOs) in the ictal cortical EEGs of hyperthermia-induced seizures in a rat model of febrile seizures with an SCN1A mutation as a means of investigating the pathophysiological mechanisms underlying the generation of febrile seizures. We used 13 male homozygous Scn1a-N1417H mutant rats (F344/NSlc-Scn1a(Kyo811)) and 10 wild-type control rats. Generalized tonic-clonic seizures were induced in all mutant rats, and HFOs with frequencies ranging from 200 to 400 Hz were found to precede spikes during the clonic phases of these seizures in the ictal EEGs. The proportion of all spikes in each seizure that were associated with HFOs increased with age. In time-frequency spectra of the EEG data, the HFOs had a mean peak frequency of 301.1 ± 65.4 Hz (range: 156.3-468.8Hz) and a mean peak power of 24.6 ± 3.8 dB (range: 11.4-33.4 dB); the peak power increased with age. Regarding the wild-type rats, a brief seizure without unmistakable HFOs was exceptionally induced in only one rat. The generation mechanism of febrile seizures is still an unanswered question. The detection of HFOs from the ictal EEGs of hyperthermia-induced seizures may provide a cue to answering this open question, although in this research we were unable to provide sufficient evidence to prove that the generation of HFOs depended on the mutation.
Assuntos
Eletroencefalografia/métodos , Febre/genética , Febre/fisiopatologia , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Convulsões Febris/genética , Convulsões Febris/fisiopatologia , Animais , Masculino , Ratos , Ratos MutantesRESUMO
Dravet syndrome is an intractable epileptic syndrome beginning in the first year of life. De novo mutations of SCN1A, which encode the Na(v)1.1 neuronal voltage-gated sodium channel, are considered the major cause of Dravet syndrome. In this study, we investigated genetic modifiers of this syndrome. We performed a mutational analysis of all coding exons of CACNA1A in 48 subjects with Dravet syndrome. To assess the effects of CACNA1A variants on the epileptic phenotypes of Dravet syndrome, we compared clinical features in two genotype groups: 1) subjects harboring SCN1A mutations but no CACNA1A variants (n=20) and 2) subjects with SCN1A mutations plus CACNA1A variants (n=20). CACNA1A variants detected in patients were studied using heterologous expression of recombinant human Ca(v)2.1 in HEK 293 cells and whole-cell patch-clamp recording. Nine CACNA1A variants, including six novel ones, were detected in 21 of the 48 subjects (43.8%). Based on the incidence of variants in healthy controls, most of the variants seemed to be common polymorphisms. However, the subjects harboring SCN1A mutations and CACNA1A variants had absence seizures more frequently than the patients with only SCN1A mutations (8/20 vs. 0/20, p=0.002). Moreover, the former group of subjects exhibited earlier onset of seizures and more frequent prolonged seizures before one year of age, compared to the latter group of subjects. The electrophysiological properties of four of the five novel Ca(v)2.1 variants exhibited biophysical changes consistent with gain-of-function. We conclude that CACNA1A variants in some persons with Dravet syndrome may modify the epileptic phenotypes.
Assuntos
Canais de Cálcio/genética , Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/fisiopatologia , Adulto , Sequência de Bases , Criança , Análise Mutacional de DNA , Eletroencefalografia , Genótipo , Humanos , Dados de Sequência Molecular , Técnicas de Patch-Clamp , FenótipoRESUMO
Topical therapy is the most favored form of treatment for whitening against hyperpigmentation and sunburn because it lends itself to self-administration, patient compliance, and absence of systemic adverse effects. However, transdermal delivery of hydrophilic chemicals is difficult. The main purpose of this study is to develop a delivering system of hydrophilic drugs and proteins across the skin. Hydroquinone (HQ), a well-known tyrosinase inhibitor and antimelanogenesis compound, and enhanced green fluorescent protein (EGFP) were fused with eleven poly-arginine (11R). Both HQ-11R and EGFP-11R were efficiently delivered in B16 cells, a mouse melanoma cell line. HQ-11R was as effective as HQ alone at inhibiting melanin synthesis in B16 cells. EGFP-11R was efficiently delivered into cells of the epidermis with 4-(1-pyrenyl)-butyric acid (PB), a counteranion bearing an aromatic hydrophobic moiety, in vivo, but EGFP alone or EGFP-11R without PB was not. Finally, topical application of HQ-11R with PB significantly inhibited UV irradiation-induced pigmentation in guinea pigs compared with HQ alone. These results suggest that topical therapy using poly-arginine in combination with PB is useful for the delivery of hydrophilic drugs and proteins by the transdermal route.
Assuntos
Ácido Butírico/química , Sistemas de Liberação de Medicamentos/métodos , Proteínas de Fluorescência Verde/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Peptídeos/química , Pirenos/química , Administração Cutânea , Animais , Ânions , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Cobaias , Hidroquinonas/administração & dosagem , Hidroquinonas/farmacologia , Hidroquinonas/uso terapêutico , Melaninas/biossíntese , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Camundongos , Peptídeos/síntese química , Pigmentação da Pele/efeitos dos fármacos , Pigmentação da Pele/efeitos da radiação , Raios UltravioletaRESUMO
Mutations of the neuronal voltage-gated sodium channel alpha subunit type II (SCN2A) cause various epileptic syndromes, but have never been reported in association with acute encephalopathy. To validate the involvement of SCN2A mutations in acute encephalopathy, we screened 25 patients and found a novel missense mutation (Met1128Thr) in a patient with acute encephalitis with refractory, repetitive partial seizures (AERRPS). This finding suggests that SCN2A mutation is a predisposing factor for acute encephalopathy.
Assuntos
Encefalopatias/genética , Epilepsias Parciais/genética , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Mutação Puntual/genética , Estado Epiléptico/genética , Doença Aguda , Adulto , Sequência de Aminoácidos , Humanos , Masculino , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.2/química , Estrutura Terciária de ProteínaRESUMO
Protein transduction with cell-penetrating peptides such as poly-arginine and HIV TAT peptides is widely used to deliver proteins, peptides, siRNA and biologically active compounds. It has been thought that poly-arginine peptides transduce proteins in a manner dependent on the number of arginine residues and oligo-peptides such as three arginines (3R) are ineffective. Here we showed that 3R-fused proteins were effectively delivered and functioned in cells co-treated with pyrenebutyrate, a counteranion bearing an aromatic hydrophobic moiety. Little 3R was transduced in glioma cells without pyrenebutyrate whereas the oligo-arginine was effectively delivered with pyrenebutyrate. Enhanced green fluorescence protein (eGFP) fused with 3R was effectively delivered into various kinds of cells including primary cultured cells and suspended cells in the presence of pyrenebutyrate. p53 fused with 3R (3R-p53) was delivered into glioma cells without pyrenebutyrate but could not be translocated into the nucleus. In contrast, 3R-p53 was observed in nuclei of glioma cells when co-applied with pyrenebutyrate. Although 3R-p53 was delivered less effectively than 11R-p53 with pyrenebutyrate, its transcriptional activity was higher than that of 11R-p53. Moreover, a single administration of 3R-p53 with pyrenebutyrate significantly inhibited the growth of cancer cells. These results suggest protein transduction using an oligo-arginine (3R) with pyrenebutyrate to be a good tool for the delivery of functional transcription factors and a promising method of treating cancer.
Assuntos
Núcleo Celular/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Vetores Genéticos/química , Peptídeos/química , Fatores de Transcrição/metabolismo , Transdução Genética/métodos , Linhagem Celular , Proteínas de Fluorescência Verde/química , Células HeLa , Humanos , Proteínas , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The neuropeptide oxytocin (OT) has been shown to exert multiple functions in both males and females, and to play a key role in the regulation of emotionality in the central nervous system (CNS). OT has an anxiolytic effect in the CNS of rodents and humans. However, the molecular mechanisms of this effect are unclear. Here we show that OT induced the expression of regulator of G-protein signaling 2 (RGS2), a regulatory factor for anxiety, in the central amygdala (CeA) of female mice. Bath application of OT increased RGS2 levels in slices of the amygdala of virgin mice. RGS2 levels in the CeA were higher in lactating mice than in virgin mice. In contrast, RGS2 levels in mice that had given birth did not increase when the pups were removed. Acute restraint stress for 4h induced RGS2 expression within the CeA, and local administration of an OT receptor antagonist inhibited this expression. Behavioral experiments revealed that transient restraint stress had an anxiolytic effect in wild-type females, and RGS2 levels in the CeA correlated with the anxiolytic behavior. By contrast, in the OT receptor-deficient mice, restraint stress neither increased RGS2 levels in the CeA nor had an anxiolytic effect. These results suggest that OT displays an anxiolytic effect through the induction of RGS2 expression in the CNS.