RESUMO
The role of hydrophobicity of phenylalanine-glycine nucleoporins (FG-Nups) in determining the transport of receptor-bound cargo across the nuclear pore complex (NPC) is investigated using Langevin dynamics simulations. A coarse-grained, minimal model of the NPC, comprising a cylindrical pore and hydrophobic-hydrophilic random copolymers for FG-Nups was employed. Karyopherin-bound receptor-cargo complexes (Kaps) were modeled as rigid, coarse-grained spheres without (inert) and with (patchy) FG-binding hydrophobic domains. With a sequence-agnostic description of FG-Nups and the absence of any anisotropies associated with either NPC or cargo, the model described tracer transport only as a function of FG-Nup hydrophobicity, f. The simulations showed the emergence of two important features of cargo transport, namely, NPC selectivity and specificity. NPC selectivity to patchy tracers emerged due to hydrophobic Kap-FG interactions and despite the sequence-agnostic description of FG-Nups. Furthermore, NPC selectivity was observed only in a specific range of FG-hydrophobic fraction, 0.05 ≤ f ≤ 0.20, resulting in specificity of NPC transport with respect to f. Significantly, this range corresponded to the number fraction of FG-repeats observed in both S. cerevisiae and H. sapiens NPCs. This established the central role of the FG-hydrophobic fraction in determining NPC transport, and provided a biophysical basis for conservation of the FG-Nup hydrophobic fraction across evolutionarily distant NPCs. Specificity in NPC transport emerged from the formation of a hydrogel-like network inside the pore with a characteristic mesh size dependent on f. This network rejected cargo for f > 0.2 based on size exclusion, which resulted in enhanced translocation probability for 0.05 ≤ f ≤ 0.20. Extended brush configurations outside the pore resulted in entropic repulsion and exclusion of inert cargo in this range. Thus, our minimal NPC model exhibited a hybrid cargo translocation mechanism, with aspects of both virtual gate and selective-phase models, in this range of FG-hydrophobic fraction.
Assuntos
Poro Nuclear , Saccharomyces cerevisiae , Poro Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/análise , Complexo de Proteínas Formadoras de Poros Nucleares/química , Glicina/química , Fenilalanina/químicaRESUMO
Epileptogenesis is characterized by intrinsic changes in neuronal firing, resulting in hyperactive neurons and the subsequent generation of seizure activity. These alterations are accompanied by changes in gene transcription networks, first with the activation of early-immediate genes and later with the long-term activation of genes involved in memory. Our objective was to engineer a promoter containing binding sites for activity-dependent transcription factors upregulated in chronic epilepsy (EpiPro) and validate it in multiple rodent models of epilepsy. First, we assessed the activity dependence of EpiPro: initial electrophysiology studies found that EpiPro-driven GFP expression was associated with increased firing rates when compared with unlabeled neurons, and the assessment of EpiPro-driven GFP expression revealed that GFP expression was increased ~150× after status epilepticus. Following this, we compared EpiPro-driven GFP expression in two rodent models of epilepsy, rat lithium/pilocarpine and mouse electrical kindling. In rodents with chronic epilepsy, GFP expression was increased in most neurons, but particularly in dentate granule cells, providing in vivo evidence to support the "breakdown of the dentate gate" hypothesis of limbic epileptogenesis. Finally, we assessed the time course of EpiPro activation and found that it was rapidly induced after seizures, with inactivation following over weeks, confirming EpiPro's potential utility as a gene therapy driver for epilepsy.
Assuntos
Epilepsia , Estado Epiléptico , Ratos , Camundongos , Animais , Epilepsia/genética , Epilepsia/terapia , Epilepsia/metabolismo , Convulsões/genética , Convulsões/terapia , Convulsões/metabolismo , Neurônios/metabolismo , Estado Epiléptico/genética , Estado Epiléptico/terapia , Estado Epiléptico/metabolismo , Pilocarpina , Terapia Genética , Modelos Animais de Doenças , Hipocampo/metabolismoRESUMO
SCN8A epileptic encephalopathy is a devastating epilepsy syndrome caused by mutant SCN8A, which encodes the voltage-gated sodium channel NaV1.6. To date, it is unclear if and how inhibitory interneurons, which express NaV1.6, influence disease pathology. Using both sexes of a transgenic mouse model of SCN8A epileptic encephalopathy, we found that selective expression of the R1872W SCN8A mutation in somatostatin (SST) interneurons was sufficient to convey susceptibility to audiogenic seizures. Patch-clamp electrophysiology experiments revealed that SST interneurons from mutant mice were hyperexcitable but hypersensitive to action potential failure via depolarization block under normal and seizure-like conditions. Remarkably, GqDREADD-mediated activation of WT SST interneurons resulted in prolonged electrographic seizures and was accompanied by SST hyperexcitability and depolarization block. Aberrantly large persistent sodium currents, a hallmark of SCN8A mutations, were observed and were found to contribute directly to aberrant SST physiology in computational modeling and pharmacological experiments. These novel findings demonstrate a critical and previously unidentified contribution of SST interneurons to seizure generation not only in SCN8A epileptic encephalopathy, but epilepsy in general.SIGNIFICANCE STATEMENTSCN8A epileptic encephalopathy is a devastating neurological disorder that results from de novo mutations in the sodium channel isoform Nav1.6. Inhibitory neurons express NaV1.6, yet their contribution to seizure generation in SCN8A epileptic encephalopathy has not been determined. We show that mice expressing a human-derived SCN8A variant (R1872W) selectively in somatostatin (SST) interneurons have audiogenic seizures. Physiological recordings from SST interneurons show that SCN8A mutations lead to an elevated persistent sodium current which drives initial hyperexcitability, followed by premature action potential failure because of depolarization block. Furthermore, chemogenetic activation of WT SST interneurons leads to audiogenic seizure activity. These findings provide new insight into the importance of SST inhibitory interneurons in seizure initiation, not only in SCN8A epileptic encephalopathy, but for epilepsy broadly.
Assuntos
Interneurônios/fisiologia , Convulsões/fisiopatologia , Somatostatina/metabolismo , Potenciais de Ação , Animais , Ondas Encefálicas , Interneurônios/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Convulsões/genética , Convulsões/metabolismo , Somatostatina/genéticaRESUMO
OBJECTIVE: Sudden unexpected death in epilepsy (SUDEP) is an unpredictable and devastating comorbidity of epilepsy that is believed to be due to cardiorespiratory failure immediately after generalized convulsive seizures. METHODS: We performed cardiorespiratory monitoring of seizure-induced death in mice carrying either a p.Arg1872Trp or p.Asn1768Asp mutation in a single Scn8a allele-mutations identified from patients who died from SUDEP-and of seizure-induced death in pentylenetetrazole-treated wild-type mice. RESULTS: The primary cause of seizure-induced death for all mice was apnea, as (1) apnea began during a seizure and continued for tens of minutes until terminal asystole, and (2) death was prevented by mechanical ventilation. Fatal seizures always included a tonic phase that was coincident with apnea. This tonic phase apnea was not sufficient to produce death, as it also occurred during many nonfatal seizures; however, all seizures that were fatal had tonic phase apnea. We also made the novel observation that continuous tonic diaphragm contraction occurred during tonic phase apnea, which likely contributes to apnea by preventing exhalation, and this was only fatal when breathing did not resume after the tonic phase ended. Finally, recorded seizures from a patient with developmental epileptic encephalopathy with a previously undocumented SCN8A likely pathogenic variant (p.Leu257Val) revealed similarities to those of the mice, namely, an extended tonic phase that was accompanied by apnea. INTERPRETATION: We conclude that apnea coincident with the tonic phase of a seizure, and subsequent failure to resume breathing, are the determining events that cause seizure-induced death in Scn8a mutant mice. ANN NEUROL 2021;89:1023-1035.
Assuntos
Apneia/complicações , Epilepsia/complicações , Morte Súbita Inesperada na Epilepsia , Animais , Convulsivantes , Diafragma/fisiopatologia , Eletroencefalografia , Eletromiografia , Feminino , Humanos , Lactente , Masculino , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Pentilenotetrazol , Gravidez , Respiração Artificial , Mecânica RespiratóriaRESUMO
AIM: The study aimed to profile the volatile phytocomposition of snow mountain garlic (SMG) compared to normal garlic and investigate the anti-Candida efficacy against clinically relevant multi-drug resistant isolates of Candida species. METHODS AND RESULTS: Herein, SMG has shown significantly superior fungicidal power at 2x-MIC dose against C. albicans and C. glabrata in killing kinetic evaluation unlike the fungistatic effect of normal garlic. GC-MS headspace-based profiling of SMG showed 5 unique volatile compounds and a 5-fold higher content of saponins than normal garlic. In an in-silico analysis, cholesta-4,6-dien-3-ol,(3-beta) was uniquely identified in SMG as a potential inhibitor with high binding affinity to the active site of exo-1,3-betaglucan synthase, an established anti-candida drug target crucial for the biofilm matrix formation, thus suggesting a plausible anti-Candida mechanism. CONCLUSION: The in-vitro and in-silico studies have demonstrated the Candida-cidal and anti-biofilm activities of SMG, distinguishing it from the Candida-static efficacy of normal garlic. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that identifies several phytochemical signatures of SMG along with a potential anti-Candida compound, that is cholesta-4,6-dien-3-ol,(3-beta)-, which appears worthy of detailed studies in the future to explore the utility of SMG as a fungal phytotherapy agent, especially against drug-resistant Candida sp.
Assuntos
Alho , Antifúngicos/metabolismo , Candida , Candida albicans , Candida glabrata , Alho/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Testes de Sensibilidade MicrobianaRESUMO
De novo mutations of the sodium channel gene SCN8A result in an epileptic encephalopathy with refractory seizures, developmental delay, and elevated risk of sudden death. p.Arg1872Trp is a recurrent de novo SCN8A mutation reported in 14 unrelated individuals with epileptic encephalopathy that included seizure onset in the prenatal or infantile period and severe verbal and ambulatory comorbidities. The major biophysical effect of the mutation was previously shown to be impaired channel inactivation accompanied by increased current density. We have generated a conditional mouse mutation in which expression of this severe gain-of-function mutation is dependent upon Cre recombinase. Global activation of p.Arg1872Trp by EIIa-Cre resulted in convulsive seizures and lethality at 2 weeks of age. Neural activation of the p.Arg1872Trp mutation by Nestin-Cre also resulted in early onset seizures and death. Restriction of p.Arg1872Trp expression to excitatory neurons using Emx1-Cre recapitulated seizures and juvenile lethality between 1 and 2 months of age. In contrast, activation of p.Arg1872Trp in inhibitory neurons by Gad2-Cre or Dlx5/6-Cre did not induce seizures or overt neurological dysfunction. The sodium channel modulator GS967/Prax330 prolonged survival of mice with global expression of R1872W and also modulated the activity of the mutant channel in transfected cells. Activation of the p.Arg1872Trp mutation in adult mice was sufficient to generate seizures and death, indicating that successful therapy will require lifelong treatment. These findings provide insight into the pathogenic mechanism of this gain-of-function mutation of SCN8A and identify excitatory neurons as critical targets for therapeutic intervention.
Assuntos
Encefalopatias/genética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Integrases/genética , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Neurônios/fisiologia , Prosencéfalo/fisiologia , Animais , Encefalopatias/patologia , Células Cultivadas , Feminino , Mutação com Ganho de Função/genética , Integrases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/patologia , Técnicas de Cultura de Órgãos , Prosencéfalo/patologiaRESUMO
The putative cache (Ca2+ channel and chemotaxis receptor) domain containing 1 (CACHD1) protein has predicted structural similarities to members of the α2δ voltage-gated Ca2+ channel auxiliary subunit family. CACHD1 mRNA and protein were highly expressed in the male mammalian CNS, in particular in the thalamus, hippocampus, and cerebellum, with a broadly similar tissue distribution to CaV3 subunits, in particular CaV3.1. In expression studies, CACHD1 increased cell-surface localization of CaV3.1, and these proteins were in close proximity at the cell surface, consistent with the formation of CACHD1-CaV3.1 complexes. In functional electrophysiological studies, coexpression of human CACHD1 with CaV3.1, CaV3.2, and CaV3.3 caused a significant increase in peak current density and corresponding increases in maximal conductance. By contrast, α2δ-1 had no effect on peak current density or maximal conductance in CaV3.1, CaV3.2, or CaV3.3. A comparison of CACHD1-mediated increases in CaV3.1 current density and gating currents revealed an increase in channel open probability. In hippocampal neurons from male and female embryonic day 19 rats, CACHD1 overexpression increased CaV3-mediated action potential firing frequency and neuronal excitability. These data suggest that CACHD1 is structurally an α2δ-like protein that functionally modulates CaV3 voltage-gated calcium channel activity.SIGNIFICANCE STATEMENT This is the first study to characterize the Ca2+ channel and chemotaxis receptor domain containing 1 (CACHD1) protein. CACHD1 is widely expressed in the CNS, in particular in the thalamus, hippocampus, and cerebellum. CACHD1 distribution is similar to that of low voltage-activated (CaV3, T-type) calcium channels, in particular to CaV3.1, a protein that regulates neuronal excitability and is a potential therapeutic target in conditions such as epilepsy and pain. CACHD1 is structurally an α2δ-like protein that functionally increases CaV3 calcium current. CACHD1 increases the presence of CaV3.1 at the cell surface, forms complexes with CaV3.1 at the cell surface, and causes an increase in channel open probability. In hippocampal neurons, CACHD1 causes increases in neuronal firing. Thus, CACHD1 represents a novel protein that modulates CaV3 activity.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo T/biossíntese , Hipocampo/metabolismo , Proteínas de Membrana/metabolismo , Animais , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo T/química , Canais de Cálcio Tipo T/genética , Feminino , Células HEK293 , Humanos , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Ratos , Ratos WistarRESUMO
OBJECTIVE: Monoallelic de novo gain-of-function variants in the voltage-gated sodium channel SCN8A are one of the recurrent causes of severe developmental and epileptic encephalopathy (DEE). In addition, a small number of de novo or inherited monoallelic loss-of-function variants have been found in patients with intellectual disability, autism spectrum disorder, or movement disorders. Inherited monoallelic variants causing either gain or loss-of-function are also associated with less severe conditions such as benign familial infantile seizures and isolated movement disorders. In all three categories, the affected individuals are heterozygous for a SCN8A variant in combination with a wild-type allele. In the present study, we describe two unusual families with severely affected individuals who inherited biallelic variants of SCN8A. METHODS: We identified two families with biallelic SCN8A variants by diagnostic gene panel sequencing. Functional analysis of the variants was performed using voltage clamp recordings from transfected ND7/23 cells. RESULTS: We identified three probands from two unrelated families with DEE due to biallelic SCN8A variants. Each parent of an affected individual carried a single heterozygous SCN8A variant and exhibited mild cognitive impairment without seizures. In both families, functional analysis demonstrated segregation of one allele with complete loss-of-function, and one allele with altered biophysical properties consistent with partial loss-of-function. SIGNIFICANCE: These studies demonstrate that SCN8A DEE may, in rare cases, result from inheritance of two variants, both of which exhibit reduced channel activity. In these families, heterozygosity for the dominant variants results in less severe disease than biallelic inheritance of two variant alleles. The clinical consequences of variants with partial and complete loss of SCN8A function are variable and likely to be influenced by genetic background.
Assuntos
Encefalopatias/genética , Deficiências do Desenvolvimento/genética , Epilepsia/genética , Frequência do Gene/genética , Variação Genética/genética , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Adulto , Encefalopatias/complicações , Encefalopatias/diagnóstico , Pré-Escolar , Deficiências do Desenvolvimento/complicações , Deficiências do Desenvolvimento/diagnóstico , Epilepsia/complicações , Epilepsia/diagnóstico , Feminino , Humanos , Masculino , LinhagemRESUMO
SCN8A encephalopathy, or early infantile epileptic encephalopathy 13 (EIEE13), is caused predominantly by de novo gain-of-function mutations in the voltage-gated Na channel Nav1.6. Affected individuals suffer from refractory seizures, developmental delay, cognitive disability, and elevated risk of sudden unexpected death in epilepsy (SUDEP). A knock-in mouse model carrying the patient mutation p.Asn1768Asp (N1768D) reproduces many features of the disorder, including spontaneous seizures and SUDEP. We used the mouse model to examine the effects of the mutation on layer II stellate neurons of the medial entorhinal cortex (mEC), which transmit excitatory input to the hippocampus. Heterozygous (Scn8aD/+), homozygous (Scn8aD/D)), and WT (Scn8a+/+) littermates were compared at 3 weeks of age, the time of seizure onset for homozygous mice. Heterozygotes remain seizure free for another month. mEC layer II neurons of heterozygous and homozygous mice were hyperexcitable and generated long-lasting depolarizing potentials with bursts of action potentials after synaptic stimulation. Recording of Na currents revealed proexcitatory increases in persistent and resurgent currents and rightward shifts in inactivation parameters, leading to significant increases in the magnitude of window currents. The proexcitatory changes were more pronounced in homozygous mice than in heterozygotes, consistent with the earlier age of seizure onset in homozygotes. These studies demonstrate that the N1768D mutation increases the excitability of mEC layer II neurons by increasing persistent and resurgent Na currents and disrupting channel inactivation. The aberrant activities of mEC layer II neurons would provide excessive excitatory input to the hippocampus and contribute to hyperexcitability of hippocampal neurons in this model of SCN8A encephalopathy.SIGNIFICANCE STATEMENTSCN8A encephalopathy is a devastating neurological disorder that results from de novo mutations in the Na channel Nav1.6. In addition to seizures, patients suffer from cognitive and developmental delays and are at high risk for sudden unexpected death in epilepsy (SUDEP). A mouse knock-in model expressing the patient mutation N1768D reproduces several pathological phenotypes, including spontaneous seizures and sudden death. We demonstrate that medial entorhinal cortex (mEC) neurons from the mouse model exhibit proexcitatory alterations in Na channel activity, some of which were not seen in hippocampal or cortical neurons, and resulting in neuronal hyperexcitability. Because mEC neurons regulate the activity of the hippocampus, which plays an important role in seizure onset, we propose that these profound changes in mEC neuron excitability associated with the gain-of-function mutation of Nav1.6 may increase excitatory drive into the hippocampus, culminating in seizure activity and SUDEP.
Assuntos
Encefalopatias/genética , Encefalopatias/fisiopatologia , Modelos Animais de Doenças , Córtex Entorrinal/fisiopatologia , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Neurônios/fisiologia , Potenciais de Ação/fisiologia , Animais , Síndrome de Brugada/genética , Síndrome de Brugada/fisiopatologia , Epilepsia/genética , Epilepsia/fisiopatologia , Feminino , Técnicas de Introdução de Genes/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Técnicas de Cultura de Órgãos , Canais de Sódio/genéticaRESUMO
Variants in the neuronal sodium channel gene SCN8A have been implicated in several neurological disorders. Early infantile epileptic encephalopathy type 13 results from de novo gain-of-function mutations that alter the biophysical properties of the channel. Complete loss-of-function variants of SCN8A have been identified in cases of isolated intellectual disability. We now report a novel heterozygous SCN8A variant, p.Pro1719Arg, in a small pedigree with five family members affected with autosomal dominant upper limb isolated myoclonus without seizures or cognitive impairment. Functional analysis of the p.Pro1719Arg variant in transfected neuron-derived cells demonstrated greatly reduced Nav 1.6 channel activity without altered gating properties. Hypomorphic alleles of Scn8a in the mouse are known to result in similar movement disorders. This study expands the phenotypic and functional spectrum of SCN8A variants to include inherited nonepileptic isolated myoclonus. SCN8A can be considered as a candidate gene for isolated movement disorders without seizures.
Assuntos
Epilepsia/genética , Deficiência Intelectual/genética , Mioclonia/genética , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Criança , Epilepsia/fisiopatologia , Feminino , Heterozigoto , Humanos , Deficiência Intelectual/fisiopatologia , Mutação com Perda de Função/genética , Masculino , Pessoa de Meia-Idade , Mutação , Mioclonia/fisiopatologia , Linhagem , Convulsões/genética , Convulsões/fisiopatologiaRESUMO
We present here the correlation of picomolar affinities between surface-plasmon and electrochemical immunoassays for the binding of serum glutamic acid decarboxylase 65 autoantibody (GADA), a biomarker of type 1 diabetes (T1D), to its antigen GAD-65. Carboxylated (â¼5.0%)-graphene-modified immunoassembly on a gold surface-plasmon chip or on an electrochemical array provided significantly larger binding affinity, higher sensitivity, and lower detection limits than a self-assembled monolayer surface of mercaptopropionic acid (MPA). Estimation of the relative surface -COOH groups by covalent tagging of an electroactive aminoferrocene showed that the graphenyl surface displayed a greater number of -COOH groups (9-fold) than the MPA surface. X-ray-photoelectron-spectroscopy analysis showed more C-O and CâO functionalities on the graphene-COOH surface than on the MPA surface. The graphene-COOH coating on gold exhibited â¼5.5-fold enhancement of plasmon signals compared with a similar coating on a plain glass surface. In summary, this article provides a quantitative comparison of carboxylated graphene with a mercapto-monolayer immunoassembly. Additionally, we propose that the binding-constant value can be useful as a quality-control checkpoint for reproducible and reliable production of large-scale biosensors for clinical bioassays.
Assuntos
Ácido 3-Mercaptopropiônico/química , Autoanticorpos/sangue , Técnicas Eletroquímicas , Glutamato Descarboxilase/sangue , Imunoensaio , Fragmentos de Peptídeos/sangue , Ressonância de Plasmônio de Superfície , Autoanticorpos/metabolismo , Sítios de Ligação , Técnicas Biossensoriais , Glutamato Descarboxilase/metabolismo , Humanos , Fragmentos de Peptídeos/metabolismo , Propriedades de SuperfícieRESUMO
OBJECTIVE: De novo mutations of SCN8A, encoding the voltage-gated sodium channel NaV 1.6, have been associated with a severe infant onset epileptic encephalopathy. Individuals with SCN8A encephalopathy have a mean age of seizure onset of 4-5 months, with multiple seizure types that are often refractory to treatment with available drugs. Anecdotal reports suggest that high-dose phenytoin is effective for some patients, but there are associated adverse effects and potential for toxicity. Functional characterization of several SCN8A encephalopathy variants has shown that elevated persistent sodium current is one of several common biophysical defects. Therefore, specifically targeting elevated persistent current may be a useful therapeutic strategy in some cases. METHODS: The novel sodium channel modulator GS967 has greater preference for persistent as opposed to peak current and nearly 10-fold greater potency than phenytoin. We evaluated the therapeutic effect of GS967 in the Scn8aN1768D/+ mouse model carrying an SCN8A patient mutation that results in elevated persistent sodium current. We also performed patch clamp recordings to assess the effect of GS967 on peak and persistent sodium current and excitability in hippocampal neurons from Scn8aN1768D/+ mice. RESULTS: GS967 potently blocked persistent sodium current without affecting peak current, normalized action potential morphology, and attenuated excitability in neurons from heterozygous Scn8aN1768D/+ mice. Acute treatment with GS967 provided dose-dependent protection against maximal electroshock-induced seizures in Scn8aN1768D/+ and wild-type mice. Chronic treatment of Scn8aN1768D/+ mice with GS967 resulted in lower seizure burden and complete protection from seizure-associated lethality observed in untreated Scn8aN1768D/+ mice. Protection was achieved at a chronic dose that did not cause overt behavioral toxicity or sedation. SIGNIFICANCE: Persistent sodium current modulators like GS967 may be an effective precision targeting strategy for SCN8A encephalopathy and other functionally similar channelopathies when elevated persistent sodium current is the primary dysfunction.
Assuntos
Anticonvulsivantes/uso terapêutico , Epilepsia/tratamento farmacológico , Mutação/genética , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Piridinas/uso terapêutico , Triazóis/uso terapêutico , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Animais , Anticonvulsivantes/farmacologia , Encefalopatias/complicações , Encefalopatias/genética , Modelos Animais de Doenças , Esquema de Medicação , Eletrochoque/efeitos adversos , Epilepsia/etiologia , Epilepsia/genética , Epilepsia/patologia , Feminino , Hipocampo/patologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Uso Off-Label , Fenitoína/farmacologia , Fenitoína/uso terapêutico , Piridinas/farmacologia , Triazóis/farmacologiaRESUMO
Temporal lobe epilepsy (TLE) is a common form of adult epilepsy involving the limbic structures of the temporal lobe. Subiculum neurons act to provide a major output from the hippocampus and consist of a large population of endogenously bursting excitatory neurons. In TLE, subiculum neurons are largely spared, become hyperexcitable and show spontaneous epileptiform activity. The basis for this hyperexcitability is unclear, but is likely to involve alterations in the expression levels and function of various ion channels. In this study, we sought to determine the importance of sodium channel currents in facilitating neuronal hyperexcitability of subiculum neurons in the continuous hippocampal stimulation (CHS) rat model of TLE. Subiculum neurons from TLE rats were hyperexcitable, firing a higher frequency of action potentials after somatic current injection and action potential (AP) bursts after synaptic stimulation. Voltage clamp recordings revealed increases in resurgent (INaR) and persistent (INaP) sodium channel currents and pro-excitatory shifts in sodium channel activation and inactivation parameters that would facilitate increases in AP generation. Attenuation of INaR and INaP currents with 4,9-anhydro-tetrodotoxin (4,9-ah TTX; 100nM), a toxin with increased potency against Nav1.6 channels, suppressed neuronal firing frequency and inhibited AP bursting induced by synaptic stimulation in TLE neurons. These findings support an important role of sodium channels, particularly Nav1.6, in facilitating subiculum neuron hyperexcitability in TLE and provide further support for the importance of INaR and INaP currents in establishing epileptiform activity of subiculum neurons.
Assuntos
Epilepsia do Lobo Temporal/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Canais de Sódio/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Anticonvulsivantes/farmacologia , Modelos Animais de Doenças , Estimulação Elétrica , Eletrodos Implantados , Epilepsia do Lobo Temporal/tratamento farmacológico , Epilepsia do Lobo Temporal/patologia , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Imuno-Histoquímica , Masculino , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurotransmissores/farmacologia , Técnicas de Patch-Clamp , Ratos Sprague-Dawley , Estado Epiléptico , Técnicas de Cultura de TecidosRESUMO
OBJECTIVE: SCN8A encephalopathy (early infantile epileptic encephalopathy; EIEE13) is caused by gain-of-function mutations resulting in hyperactivity of the voltage-gated sodium channel Nav 1.6. The channel is concentrated at the axon initial segment (AIS) and is involved in establishing neuronal excitability. Clinical features of SCN8A encephalopathy include seizure onset between 0 and 18 months of age, intellectual disability, and developmental delay. Seizures are often refractory to treatment with standard antiepileptic drugs, and sudden unexpected death in epilepsy (SUDEP) has been reported in approximately 10% of patients. In a recent study, high doses of phenytoin were effective in four patients with SCN8A encephalopathy. In view of this observation, we have investigated the relationship between the functional effect of the SCN8A mutation p.Ile1327Val and its response to phenytoin. METHODS: The mutation was introduced into the Scn8a cDNA by site-directed mutagenesis. Channel activity was characterized in transfected ND7/23 cells. The effects of phenytoin (100 µm) on mutant and wild-type (WT) channels were compared. RESULTS: Channel activation parameters were shifted in a hyperpolarizing direction in the mutant channel, whereas inactivation parameters were shifted in a depolarizing direction, increasing Na channel window current. Macroscopic current decay was slowed in I1327V channels, indicating an impairment in the transition from open state to inactivated state. Channel deactivation was also delayed, allowing more channels to remain in the open state. Phenytoin (100 µm) resulted in hyperpolarized activation and inactivation curves as well as greater tonic block and use-dependent block of I1327V mutant channels relative to WT. SIGNIFICANCE: SCN8A - I1327V is a gain-of-function mutation with altered features that are predicted to increase neuronal excitability and seizure susceptibility. Phenytoin is an effective inhibitor of the mutant channel and may be of use in treating patients with gain-of-function mutations of SCN8A.
Assuntos
Anticonvulsivantes/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Mutação/genética , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Fenitoína/farmacologia , Linhagem Celular Transformada , Estimulação Elétrica , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Isoleucina/genética , Masculino , Modelos Moleculares , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia , Transfecção , Valina/genéticaRESUMO
SCN8A epileptic encephalopathy (EE) is a severe epilepsy syndrome resulting from de novo mutations in the voltage-gated sodium channel Na v 1.6, encoded by the gene SCN8A . Na v 1.6 is expressed in both excitatory and inhibitory neurons, yet previous studies have primarily focused on the impact SCN8A mutations have on excitatory neuron function, with limited studies on the importance of inhibitory interneurons to seizure onset and progression. Inhibitory interneurons are critical in balancing network excitability and are known to contribute to the pathophysiology of other epilepsies. Parvalbumin (PV) interneurons are the most prominent inhibitory neuron subtype in the brain, making up about 40% of inhibitory interneurons. Notably, PV interneurons express high levels of Na v 1.6. To assess the role of PV interneurons within SCN8A EE, we used two mouse models harboring patient-derived SCN8A gain-of-function mutations, Scn8a D/+ , where the SCN8A mutation N1768D is expressed globally, and Scn8a W/+ -PV, where the SCN8A mutation R1872W is selectively expressed in PV interneurons. Expression of the R1872W SCN8A mutation selectively in PV interneurons led to the development of spontaneous seizures in Scn8a W/+ -PV mice and seizure-induced death, decreasing survival compared to wild-type. Electrophysiology studies showed that PV interneurons in Scn8a D/+ and Scn8a W/+ -PV mice were susceptible to depolarization block, a state of action potential failure. Scn8a D/+ and Scn8a W/+ -PV interneurons also exhibited increased persistent sodium current, a hallmark of SCN8A gain-of-function mutations that contributes to depolarization block. Evaluation of synaptic connections between PV interneurons and pyramidal cells showed an increase in synaptic transmission failure at high frequencies (80-120Hz) as well as an increase in synaptic latency in Scn8a D/+ and Scn8a W/+ -PV interneurons. These data indicate a distinct impairment of synaptic transmission in SCN8A EE, potentially decreasing overall cortical network inhibition. Together, our novel findings indicate that failure of PV interneuron spiking via depolarization block along with frequency-dependent inhibitory synaptic impairment likely elicits an overall reduction in the inhibitory drive in SCN8A EE, leading to unchecked excitation and ultimately resulting in seizures and seizure-induced death.
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Background Glioblastoma (GBM) is the most common primary malignant brain tumor. The aim of this study was to elucidate the role of microenvironment and intrinsic T-type calcium channels (Cav3) in regulating tumor growth and progression. Methods We grafted syngeneic GBM cells into Cav3.2 knockout mice to assess the role of microenvironment T-Type calcium channels on GBM tumor growth. We performed single-cell RNA-seq (scRNA-seq) of tumors from WT and Cav3.2 KO mice to elucidate the regulation of tumors by the microenvironment. We used neurons from WT and Cav3.2 KO mice in co-culture with GBM stem cells (GSC) to assess the effects of Cav3.2 on neuron/GSC synaptic connections and tumor cell growth. Results Cav3.2 KO in the microenvironment led to significant reduction of GBM growth and prolongation of animal survival. scRNA-seq showed that microenvironment Cav3.2 regulates neuronal and glial biological processes. Microenvironment Cav3.2 downregulated numerous genes associated with regulating the OPC cell state in GBM tumors such as SOX10 and Olig2. Neuronal Cav3.2 promoted neuron/GSC synaptic connections and GSC growth. Treatment of GSCs with the Cav3 blocker mibefradil downregulated genes associated with neuronal processes. The Cav3 blocker drug mibefradil synergized with temozolomide (TMZ) and radiation to reduce in vivo tumor growth and prolong animal survival. Conclusions Together these data reveal a role for microenvironment Cav3 in promoting GBM tumor progression through regulating neuronal and glial processes particularly associated with the OPC-cell state. Targeting both intrinsic and microenvironment Cav3 with the inhibitor mibefradil significantly enhanced the anti-GBM effects of TMZ and radiation.
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During epileptogenesis a series of molecular and cellular events occur, culminating in an increase in neuronal excitability, leading to seizure initiation. The entorhinal cortex has been implicated in the generation of epileptic seizures in both humans and animal models of temporal lobe epilepsy. This hyperexcitability is due, in part, to proexcitatory changes in ion channel activity. Sodium channels play an important role in controlling neuronal excitability, and alterations in their activity could facilitate seizure initiation. We sought to investigate whether medial entorhinal cortex (mEC) layer II neurons become hyperexcitable and display proexcitatory behavior of Na channels during epileptogenesis. Experiments were conducted 7 days after electrical induction of status epilepticus (SE), a time point during the latent period of epileptogenesis and before the onset of seizures. mEC layer II stellate neurons from post-SE animals were hyperexcitable, eliciting action potentials at higher frequencies compared with control neurons. Na channel currents recorded from post-SE neurons revealed increases in Na current amplitudes, particularly persistent and resurgent currents, as well as depolarized shifts in inactivation parameters. Immunocytochemical studies revealed increases in voltage-gated Na (Nav) 1.6 isoform levels. The toxin 4,9-anhydro-tetrodotoxin, which has greater selectivity for Nav1.6 over other Na channel isoforms, suppressed neuronal hyperexcitability, reduced macroscopic Na currents, persistent and resurgent Na current densities, and abolished depolarized shifts in inactivation parameters in post-SE neurons. These studies support a potential role for Nav1.6 in facilitating the hyperexcitability of mEC layer II neurons during epileptogenesis.
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Córtex Entorrinal/fisiopatologia , Epilepsia/fisiopatologia , Canal de Sódio Disparado por Voltagem NAV1.6/fisiologia , Neurônios/fisiologia , Animais , Técnicas In Vitro , Masculino , Canal de Sódio Disparado por Voltagem NAV1.6/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.6/metabolismo , Ratos , Ratos Sprague-Dawley , Sódio/fisiologia , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/análogos & derivados , Tetrodotoxina/farmacologia , Fatores de TempoRESUMO
We report the application of nano crystalline tin oxide quantum dots (SnO2-QDs) for electrochemical detection of Vibrio cholerae based on DNA hybridization technique. SnO2-QDs (- 1-5 nm) have been synthesized by laser ablation technique in liquid (LAL) and electrophoretically deposited onto hydrolyzed surface of indium tin oxide (ITO) coated glass electrode. A single stranded oligonucleotide probe (23 bases) have been designed form the virulent gene sequence of V. cholerae and has been immobilized onto SnO2-QDs/ITO surface for the fabrication of ssDNA/SnO2-QDs/ITO bioelectrode and these bioelectrode have been further used for DNA hybridization (dsDNA/SnO2-QDs/ITO). The electrochemical response studies have been carried out with different concentration genomic DNA (100-500 ng/microL), which indicated that SnO2 provides an effective surface to bind with the phosphate group of DNA, thus resulting in an enhanced electron transport. The hybridized electrode exhibits linear response with regression coefficient (R) 0.974, high sensitivity 35.20 nA/ng/cm2, low detection limit (31.5 ng/microL), faster response time (3 s) and high stability of 0-120 days when stored under refrigerated conditions.
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DNA Bacteriano/análise , Pontos Quânticos , Compostos de Estanho/química , Vibrio cholerae/isolamento & purificação , Sequência de Bases , Sondas de DNA , Microscopia de Força Atômica , Espectroscopia de Infravermelho com Transformada de Fourier , Vibrio cholerae/genética , Vibrio cholerae/patogenicidade , VirulênciaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Snow Mountain Garlic (SMG) (Allium ampeloprasum L.) is a wild trans-Himalayan member of the genus Allium, valued for its anti-inflammatory and anti-arthritic properties in the mountain folk medicinal system (Sowa-Rigpa). Despite its age-old medicinal usage by traditional therapists and the native population for various ailments including rheumatism, there is no scientific validation of its phyto-pharmaceutical merits. AIM OF THE STUDY: The present pre-clinical study compared the in-vivo anti-arthritic effects of SMG with reported efficacy doses of normal garlic (Allium sativum L.) extract and dexamethasone in a complete Freund's adjuvant (CFA)-induced arthritis rat model. MATERIALS AND METHODS: The female Wistar rats were immunized by the subplannter injection of CFA into the right hind footpad. Aqueous extracts of SMG and normal garlic were administered orally at a dose of 250 mg/kg and 500 mg/kg for 28 days. Dexamethasone was used as positive control drug. Behavioral parameters including paw markers, arthritis index, joint stiffness, body weight change, etc. were measured. Also, the changes in histopathological indices, hematological profile, inflammatory mediators, and serum cytokines level was determined. RESULTS: Treatment of rats with SMG extracts significantly (p < 0.001) prevented the reduction in body weight and hematological changes as well as ameliorated clinical symptoms such as arthritic index, joint stiffness, arthritis score, edema, hyperalgesia, and histopathological indices. This was associated with a significant reduction in the serum levels of RF, CRP, anti-CCP, and proinflammatory cytokines exhibiting strong anti-arthritic potential. SMG extracts could also significantly down regulate the NF-κB, COX-2, and iNOS expression in the ankle joint tissues. CONCLUSIONS: The present study is the first attempt to validate the phyto-pharmaceutical efficacy of this folk garlic variety from the trans-Himalayan region. Overall, SMG extract showed remarkable preventive anti-inflammatory and anti-arthritic activities which were closely comparable to therapeutic effects of dexamethasone and at par or even better than normal garlic w.r.t. several study parameters.
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Artrite Experimental , Produtos Biológicos , Alho , Animais , Feminino , Ratos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Antioxidantes/uso terapêutico , Artrite Experimental/induzido quimicamente , Artrite Experimental/tratamento farmacológico , Produtos Biológicos/uso terapêutico , Peso Corporal , Citocinas/metabolismo , Dexametasona/uso terapêutico , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Ratos WistarRESUMO
PURPOSE: The adenosinergic system is known to exert an inhibitory affect in the brain, and as such adenosine has been considered an endogenous anticonvulsant. Entorhinal cortex (EC) layer II neurons, which serve as the primary input to the hippocampus, are spared in temporal lobe epilepsy (TLE) and become hyperexcitable. Because these neurons also express adenosine receptors, the activity of these neurons may be controlled by adenosine, specifically during seizure activity when adenosine levels are thought to rise. In light of this, we determined if the actions of adenosine on medial EC (mEC) layer II stellate neurons are augmented in TLE and by which receptor subtype. METHODS: Horizontal brain slices were prepared from rats exhibiting spontaneous seizures (TLE) induced by electrical stimulation and compared with age-matched control rats. mEC layer II stellate neurons were visually identified, and action potentials (APs) were evoked either by a series of depolarizing current injection steps or via presynaptic stimulation of mEC deep layers. The effects of adenosine were compared with actions of adenosine A(1) and A(2A) receptor-specific agonists (CPA and CGS-21680) and antagonists (DPCPX and ZM-241385), respectively. Immunohistochemical and qPCR techniques were also employed to assess relative adenosine A(1)-receptor message and expression. KEY FINDINGS: mEC layer II stellate neurons were hyperexcitable in TLE, evoking a higher frequency of APs when depolarized and generating bursts of APs when synaptically stimulated. Adenosine reduced AP frequency and synaptically evoked APs in a dose-dependent manner (500 nM-100 µM); however, in TLE, the inhibitory actions of adenosine occurred at concentrations that were without affect in control neurons. In both cases, the inhibitory actions of adenosine were mediated via activation of the A(1)- and not the A(2A)-receptor subtype. Quantitative polymerase chain reaction (qPCR) and immunohistochemical experiments revealed an upregulation of the adenosine A(1) mRNA and an increase in A(1)-receptor staining in TLE neurons compared to control. SIGNIFICANCE: Our data indicate that the actions of adenosine on mEC layer II stellate neurons is accentuated in TLE due to an upregulation of adenosine A(1)-receptors. Because adenosine levels are thought to rise during seizure activity, activation of adenosine A(1)-receptors could provide a possible endogenous mechanism to suppress seizure activity and spread within the temporal lobe.