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1.
Nature ; 591(7848): 157-161, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33597751

RESUMO

Citrate is best known as an intermediate in the tricarboxylic acid cycle of the cell. In addition to this essential role in energy metabolism, the tricarboxylate anion also acts as both a precursor and a regulator of fatty acid synthesis1-3. Thus, the rate of fatty acid synthesis correlates directly with the cytosolic concentration of citrate4,5. Liver cells import citrate through the sodium-dependent citrate transporter NaCT (encoded by SLC13A5) and, as a consequence, this protein is a potential target for anti-obesity drugs. Here, to understand the structural basis of its inhibition mechanism, we determined cryo-electron microscopy structures of human NaCT in complexes with citrate or a small-molecule inhibitor. These structures reveal how the inhibitor-which binds to the same site as citrate-arrests the transport cycle of NaCT. The NaCT-inhibitor structure also explains why the compound selectively inhibits NaCT over two homologous human dicarboxylate transporters, and suggests ways to further improve the affinity and selectivity. Finally, the NaCT structures provide a framework for understanding how various mutations abolish the transport activity of NaCT in the brain and thereby cause epilepsy associated with mutations in SLC13A5 in newborns (which is known as SLC13A5-epilepsy)6-8.


Assuntos
Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/química , Ácido Cítrico/metabolismo , Microscopia Crioeletrônica , Malatos/farmacologia , Fenilbutiratos/farmacologia , Simportadores/antagonistas & inibidores , Simportadores/química , Sítios de Ligação , Encéfalo/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/ultraestrutura , Ácido Cítrico/química , Transportadores de Ácidos Dicarboxílicos/química , Transportadores de Ácidos Dicarboxílicos/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Humanos , Malatos/química , Modelos Moleculares , Mutação , Fenilbutiratos/química , Multimerização Proteica , Sódio/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/genética , Simportadores/genética , Simportadores/ultraestrutura
2.
Arch Biochem Biophys ; 699: 108763, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33460581

RESUMO

MicroRNAs (miRNAs) are small non-coding highly conserved RNA molecules that can act as master regulators of gene expression in a sequence-specific manner either by translation repression or mRNA degradation, influencing a wide range of biologic processes that are essential for the maintenance of cellular homeostasis. Chronic pediatric diseases are the leading cause of death worldwide among children and the recent evidence indicates that aberrant miRNA expression significantly contributes to the development of chronic pediatric diseases. This review focuses on the role of miRNAs in five major chronic pediatric diseases including bronchial asthma, congenital heart diseases, cystic fibrosis, type 1 diabetes mellitus, and epilepsy, and their potential use as novel biomarkers for the diagnosis and prognosis of these disorders.


Assuntos
Asma/fisiopatologia , Fibrose Cística/fisiopatologia , Diabetes Mellitus Tipo 1/fisiopatologia , Epilepsia/fisiopatologia , Cardiopatias Congênitas/fisiopatologia , MicroRNAs/fisiologia , Asma/diagnóstico , Asma/metabolismo , Biomarcadores/sangue , Biomarcadores/metabolismo , Doença Crônica , Fibrose Cística/diagnóstico , Fibrose Cística/metabolismo , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/metabolismo , Epilepsia/diagnóstico , Epilepsia/metabolismo , Cardiopatias Congênitas/diagnóstico , Cardiopatias Congênitas/metabolismo , Humanos , MicroRNAs/sangue , MicroRNAs/metabolismo , Pediatria , Prognóstico
3.
Anal Chem ; 93(4): 2490-2499, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33433198

RESUMO

Epilepsy is a chronic neurodegenerative disease that has seriously threatened human health. Accumulating evidence reveals that the pathological progression of epilepsy is closely related to peroxynitrite (ONOO-). Unfortunately, understanding the physiological roles of ONOO- in epilepsy is still challenging due to the lack of powerful imaging probes for the determination of the level of fluctuations of ONOO- in the epileptic brain. Herein, a near-infrared (NIR) two-photon (TP) fluorescent probe [dicyanomethylene-4H-pyran (DCM)-ONOO] is presented to trace ONOO- in living cells and in kainate (KA)-induced rat epilepsy models with satisfactory sensitivity and selectivity. The probe is composed of a NIR TP DCM fluorophore and a recognition moiety diphenylphosphinamide. The phosphoramide bond of the probe is interrupted after reacting with ONOO- for 10 min, and then, the released amino groups emit strong fluorescence due to the restoration of the intramolecular charge transfer process. The probe can effectively detect the changes of endogenous ONOO- with excellent temporal and spatial resolution in living cells and in rat epileptic brain. The imaging results demonstrate that the increasing level of ONOO- is closely associated with epilepsy and severe neuronal damage in the brain under KA stimulation. In addition, the low-dose resveratrol can effectively inhibit ONOO- overexpression and further relieve neuronal damage. With the assistance of TP fluorescence imaging in the epileptic brain tissue, we hypothesize that the abnormal levels of ONOO- may serve as a potential indicator for the diagnosis of epilepsy. The TP fluorescence imaging based on DCM-ONOO provides a great potential approach for understanding the epilepsy pathology and diagnosis.


Assuntos
Epilepsia/induzido quimicamente , Epilepsia/metabolismo , Corantes Fluorescentes/química , Ácido Peroxinitroso/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ácido Caínico/toxicidade , Camundongos , Estrutura Molecular , Ratos , Ratos Sprague-Dawley
4.
Am J Hum Genet ; 108(2): 357-367, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33508234

RESUMO

Focal segmental glomerulosclerosis (FSGS) is the main pathology underlying steroid-resistant nephrotic syndrome (SRNS) and a leading cause of chronic kidney disease. Monogenic forms of pediatric SRNS are predominantly caused by recessive mutations, while the contribution of de novo variants (DNVs) to this trait is poorly understood. Using exome sequencing (ES) in a proband with FSGS/SRNS, developmental delay, and epilepsy, we discovered a nonsense DNV in TRIM8, which encodes the E3 ubiquitin ligase tripartite motif containing 8. To establish whether TRIM8 variants represent a cause of FSGS, we aggregated exome/genome-sequencing data for 2,501 pediatric FSGS/SRNS-affected individuals and 48,556 control subjects, detecting eight heterozygous TRIM8 truncating variants in affected subjects but none in control subjects (p = 3.28 × 10-11). In all six cases with available parental DNA, we demonstrated de novo inheritance (p = 2.21 × 10-15). Reverse phenotyping revealed neurodevelopmental disease in all eight families. We next analyzed ES from 9,067 individuals with epilepsy, yielding three additional families with truncating TRIM8 variants. Clinical review revealed FSGS in all. All TRIM8 variants cause protein truncation clustering within the last exon between residues 390 and 487 of the 551 amino acid protein, indicating a correlation between this syndrome and loss of the TRIM8 C-terminal region. Wild-type TRIM8 overexpressed in immortalized human podocytes and neuronal cells localized to nuclear bodies, while constructs harboring patient-specific variants mislocalized diffusely to the nucleoplasm. Co-localization studies demonstrated that Gemini and Cajal bodies frequently abut a TRIM8 nuclear body. Truncating TRIM8 DNVs cause a neuro-renal syndrome via aberrant TRIM8 localization, implicating nuclear bodies in FSGS and developmental brain disease.


Assuntos
Proteínas de Transporte/genética , Deficiências do Desenvolvimento/genética , Epilepsia/genética , Glomerulosclerose Segmentar e Focal/genética , Espaço Intranuclear/metabolismo , Síndrome Nefrótica/genética , Síndrome Nefrótica/metabolismo , Proteínas do Tecido Nervoso/genética , Adulto , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Linhagem Celular , Criança , Pré-Escolar , Códon sem Sentido , Deficiências do Desenvolvimento/metabolismo , Epilepsia/metabolismo , Feminino , Glomerulosclerose Segmentar e Focal/metabolismo , Humanos , Rim/metabolismo , Masculino , Camundongos , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Podócitos/metabolismo , Sequenciamento Completo do Exoma
5.
Int J Mol Sci ; 22(2)2021 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-33466734

RESUMO

Cannabis sativa L. turned out to be a valuable source of chemical compounds of various structures, showing pharmacological activity. The most important groups of compounds include phytocannabinoids and terpenes. The pharmacological activity of Cannabis (in epilepsy, sclerosis multiplex (SM), vomiting and nausea, pain, appetite loss, inflammatory bowel diseases (IBDs), Parkinson's disease, Tourette's syndrome, schizophrenia, glaucoma, and coronavirus disease 2019 (COVID-19)), which has been proven so far, results from the affinity of these compounds predominantly for the receptors of the endocannabinoid system (the cannabinoid receptor type 1 (CB1), type two (CB2), and the G protein-coupled receptor 55 (GPR55)) but, also, for peroxisome proliferator-activated receptor (PPAR), glycine receptors, serotonin receptors (5-HT), transient receptor potential channels (TRP), and GPR, opioid receptors. The synergism of action of phytochemicals present in Cannabis sp. raw material is also expressed in their increased bioavailability and penetration through the blood-brain barrier. This review provides an overview of phytochemistry and pharmacology of compounds present in Cannabis extracts in the context of the current knowledge about their synergistic actions and the implications of clinical use in the treatment of selected diseases.


Assuntos
Canabinoides/farmacologia , Cannabis/química , Descoberta de Drogas , Compostos Fitoquímicos/farmacologia , Terpenos/farmacologia , Animais , Canabinoides/química , Canabinoides/uso terapêutico , Sinergismo Farmacológico , Endocanabinoides/metabolismo , Epilepsia/tratamento farmacológico , Epilepsia/metabolismo , Humanos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Compostos Fitoquímicos/química , Compostos Fitoquímicos/uso terapêutico , Receptores de Canabinoides/metabolismo , Esquizofrenia/tratamento farmacológico , Esquizofrenia/metabolismo , Terpenos/química , Terpenos/uso terapêutico , Síndrome de Tourette/tratamento farmacológico , Síndrome de Tourette/metabolismo
6.
Methods Mol Biol ; 2188: 133-155, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33119850

RESUMO

Genetic mutations have long been implicated in epilepsy, particularly in genes that encode ion channels and neurotransmitter receptors. Among some of those identified are voltage-gated sodium, potassium and calcium channels, and ligand-gated gamma-aminobutyric acid (GABA), neuronal nicotinic acetylcholine (CHRN), and glutamate receptors, making them key therapeutic targets. In this chapter we discuss the use of automated electrophysiological technologies to examine the impact of gene defects in two potassium channels associated with different epilepsy syndromes. The hKCNC1 gene encodes the voltage-gated potassium channel hKV3.1, and mutations in this gene cause progressive myoclonus epilepsy (PME) and ataxia due to a potassium channel mutation (MEAK). The hKCNT1 gene encodes the weakly voltage-dependent sodium-activated potassium channel hKCNT1, and mutations in this gene cause a wide spectrum of seizure disorders, including severe autosomal dominant sleep-related hypermotor epilepsy (ADSHE) and epilepsy of infancy with migrating focal seizures (EIMFS), both conditions associated with drug-resistance. Importantly, both of these potassium channels play vital roles in regulating neuronal excitability. Since its discovery in the late nineteen seventies, the patch-clamp technique has been regarded as the bench-mark technology for exploring ion channel characteristics. In more recent times, innovations in automated patch-clamp technologies, of which there are many, are enabling the study of ion channels with much greater productivity that manual systems are capable of. Here we describe aspects of Nanion NPC-16 Patchliner, examining the effects of temperature on stably and transiently transfected mammalian cells, the latter of which for most automated systems on the market is quite challenging. Remarkable breakthroughs in the development of other automated electrophysiological technologies, such as multielectrode arrays that support extracellular signal recordings, provide additional features to examine network activity in the area of ion channel research, particularly epilepsy. Both of these automated technologies enable the acquisition of consistent, robust, and reproducible data. Numerous systems have been developed with very similar capabilities, however, not all the systems on the market are adapted to work with primary cells, particularly neurons that can be problematic. This chapter also showcases methods that demonstrate the versatility of Nanion NPC-16 Patchliner and the Multi Channel Systems (MCS) multielectrode array (MEA) assay for acutely dissociated murine primary cortical neurons, enabling the study of potassium channel mutations implicated in severe refractory epilepsies.


Assuntos
Epilepsia/patologia , Neurônios/patologia , Técnicas de Patch-Clamp/métodos , Animais , Células Cultivadas , Fenômenos Eletrofisiológicos , Epilepsia/genética , Epilepsia/metabolismo , Desenho de Equipamento , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Técnicas de Patch-Clamp/instrumentação , Canais de Potássio Ativados por Sódio/genética , Canais de Potássio Ativados por Sódio/metabolismo , Canais de Potássio Shaw/genética , Canais de Potássio Shaw/metabolismo , Transfecção/instrumentação , Transfecção/métodos
7.
Life Sci ; 267: 118935, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33359246

RESUMO

PURPOSE: Long non-coding RNAs (lncRNAs) have been reported to be involved in regulating epilepsy. The purpose of this study is to investigate the possibly regulatory mechanism of small nucleolar RNA host gene 1 (SNHG1) on epilepsy. METHODS: Quantitative real-time PCR was utilized to detect the expression of SNHG1, microRNA (miR)-181a, and B-cell lymphoma-2 (BCL-2). Through an enzyme-linked immunosorbent assay, the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, and cyclooxygenase-2 (COX-2) were determined. The viability and apoptosis of CTX-TNA2 cells were measured using MTT assay and flow cytometry analysis, respectively. Western blot assay was performed to analyze the protein levels of Bcl-2, BCL2-associated X, and Caspase-3. The relationships between miR-181a and SNHG1/BCL-2 were confirmed by the dual-luciferase reporter assay. RESULTS: SNHG1 expression was down-regulated in EP tissues and kainic acid (KA)-induced CTX-TNA2 cells. The apoptosis and release of inflammatory factors (TNF-α, IL-1ß, IL-6, and COX-2) in KA-induced CTX-TNA2 cells were suppressed by SNHG1 overexpression and promoted by miR-181a up-regulation. In addition, we confirmed that SNHG1 targeted miR-181a, whereas BCL-2 was a target gene of miR-181a. Negative correlations between SNHG1 and miR-181a, as well as miR-181a and BCL-2 were exhibited. Both the up-regulation of miR-181a and down-regulation of BCL-2 reversed the inhibiting effects of SNHG1 on apoptosis and inflammatory response of KA-induced CTX-TNA2 cells, and the promoting effect upon cell viability. CONCLUSIONS: SNHG1 alleviated the progression of EP by modulating the miR-181a/BCL-2 axis in vitro, thus SNHG1 could act as a possible therapeutic target for treating EP.


Assuntos
Epilepsia/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Animais , Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Progressão da Doença , Epilepsia/metabolismo , Interleucina-1beta/metabolismo , Masculino , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Longo não Codificante/metabolismo , RNA Nucleolar Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
8.
Int J Mol Sci ; 21(24)2020 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-33348808

RESUMO

Both α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR) have been reported as targets for treatment of epilepsy. To investigate the roles and interactions of AMPAR and NMDAR in ictogenesis of epileptic hippocampus, we analyzed AMPAR antagonists (perampanel and GYKI 52466)-mediated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) regulation and glutamate ionotropic receptor NMDA type subunit 2B (GluN2B) tyrosine (Y) 1472 phosphorylation in epilepsy rats. Both perampanel and GYKI 52466 increased PTEN expression and its activity (reduced phosphorylation), concomitant with decreased activities (phosphorylations) of Src family-casein kinase 2 (CK2) signaling pathway. Compatible with these, they also restored the upregulated GluN2B Y1472 and Ca2+/cAMP response element-binding protein (CREB) serine (S) 133 phosphorylations and surface expression of glutamate ionotropic receptor AMPA type subunit 1 (GRIA1) to basal level in the epileptic hippocampus. These effects of perampanel and GYKI 52466 are observed in responders (whose seizure activities are responsive to AMPAR antagonists), but not non-responders (whose seizure activities were uncontrolled by AMPAR antagonists). Therefore, our findings suggest that Src/CK2/PTEN-mediated GluN2B Y1472 and CREB S133 regulations may be one of the responsible signaling pathways for the generation of refractory seizures in non-responders to AMPAR antagonists.


Assuntos
Benzodiazepinas/farmacologia , Epilepsia/tratamento farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de AMPA/antagonistas & inibidores , Animais , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Epilepsia/induzido quimicamente , Epilepsia/metabolismo , Epilepsia/patologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Masculino , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais , Quinases da Família src/genética , Quinases da Família src/metabolismo
9.
PLoS One ; 15(12): e0242773, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33338084

RESUMO

Autism Spectrum Disorder (ASD) is a heterogeneous disorder that is often accompanied with many co-morbidities. Recent genetic studies have identified various pathways from hundreds of candidate risk genes with varying levels of association to ASD. However, it is unknown which pathways are specific to the core symptoms or which are shared by the co-morbidities. We hypothesised that critical ASD candidates should appear widely across different scoring systems, and that comorbidity pathways should be constituted by genes expressed in the relevant tissues. We analysed the Simons Foundation for Autism Research Initiative (SFARI) database and four independently published scoring systems and identified 292 overlapping genes. We examined their mRNA expression using the Genotype-Tissue Expression (GTEx) database and validated protein expression levels using the human protein atlas (HPA) dataset. This led to clustering of the overlapping ASD genes into 2 groups; one with 91 genes primarily expressed in the central nervous system (CNS geneset) and another with 201 genes expressed in both CNS and peripheral tissues (CNS+PT geneset). Bioinformatic analyses showed a high enrichment of CNS development and synaptic transmission in the CNS geneset, and an enrichment of synapse, chromatin remodelling, gene regulation and endocrine signalling in the CNS+PT geneset. Calcium signalling and the glutamatergic synapse were found to be highly interconnected among pathways in the combined geneset. Our analyses demonstrate that 2/3 of ASD genes are expressed beyond the brain, which may impact peripheral function and involve in ASD co-morbidities, and relevant pathways may be explored for the treatment of ASD co-morbidities.


Assuntos
Transtorno do Espectro Autista/genética , Sinalização do Cálcio/genética , Epilepsia/genética , Redes Reguladoras de Genes , Proteínas do Tecido Nervoso/genética , Transtornos da Personalidade/genética , Comportamento Autodestrutivo/genética , Adulto , Idoso , Atlas como Assunto , Transtorno do Espectro Autista/diagnóstico , Transtorno do Espectro Autista/metabolismo , Transtorno do Espectro Autista/patologia , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Cromatina/metabolismo , Cromatina/ultraestrutura , Comorbidade , Bases de Dados Genéticas , Conjuntos de Dados como Assunto , Epilepsia/diagnóstico , Epilepsia/metabolismo , Epilepsia/patologia , Feminino , Regulação da Expressão Gênica , Ontologia Genética , Humanos , Masculino , Pessoa de Meia-Idade , Anotação de Sequência Molecular , Proteínas do Tecido Nervoso/classificação , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Sistema Nervoso Periférico/metabolismo , Sistema Nervoso Periférico/patologia , Transtornos da Personalidade/diagnóstico , Transtornos da Personalidade/metabolismo , Transtornos da Personalidade/patologia , Comportamento Autodestrutivo/diagnóstico , Comportamento Autodestrutivo/metabolismo , Comportamento Autodestrutivo/patologia , Sinapses/metabolismo , Sinapses/patologia , Sinapses/ultraestrutura , Transmissão Sináptica , Transcrição Genética
10.
Cell Prolif ; 53(8): e12856, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32648622

RESUMO

OBJECTIVES: Glial cell activation contributes to the inflammatory response and occurrence of epilepsy. Our preliminary study demonstrated that the long non-coding RNA, H19, promotes hippocampal glial cell activation during epileptogenesis. However, the precise mechanisms underlying this effect remain unclear. MATERIALS AND METHODS: H19 and let-7b were overexpressed or silenced using an adeno-associated viral vector in vivo. Their expression in a kainic acid-induced epilepsy model was evaluated by real-time quantitative PCR, fluorescence in situ hybridization, and cytoplasmic and nuclear RNA isolation. A dual-luciferase reporter assay was used to evaluate the direct binding of let-7b to its target genes and H19. Western blot, video camera monitoring and Morris water maze were performed to confirm the role of H19 and let7b on epileptogenesis. RESULTS: H19 was increased in rat hippocampus neurons after status epilepticus, which might be due to epileptic seizure-induced hypoxia. Increased H19 aggravated the epileptic seizures, memory impairment and mossy fibre sprouting of the epileptic rats. H19 could competitively bind to let-7b to suppress its expression. Overexpression of let-7b inhibited hippocampal glial cell activation, inflammatory response and epileptic seizures by targeting Stat3. Moreover, overexpressed H19 reversed the inhibitory effect of let-7b on glial cell activation. CONCLUSIONS: LncRNA H19 could competitively bind to let-7b to promote hippocampal glial cell activation and epileptic seizures by targeting Stat3 in a rat model of temporal lobe epilepsy.


Assuntos
Epilepsia do Lobo Temporal/genética , Hipocampo/metabolismo , MicroRNAs/genética , RNA Longo não Codificante/genética , Fator de Transcrição STAT3/metabolismo , Animais , Modelos Animais de Doenças , Epilepsia/genética , Epilepsia/metabolismo , Epilepsia do Lobo Temporal/metabolismo , Genes Supressores de Tumor/fisiologia , Masculino , Ratos Sprague-Dawley , Convulsões/genética , Convulsões/metabolismo
11.
Epilepsia ; 61(7): e79-e84, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32562438

RESUMO

The assay of saliva samples provides a valuable alternative to the use of blood samples for therapeutic drug monitoring (TDM), at least for certain categories of patients. To determine the feasibility of using saliva sampling for the TDM of rufinamide, we compared rufinamide concentrations in paired samples of saliva and plasma collected from 26 patients with epilepsy at steady state. Within-patient relationships between plasma rufinamide concentrations and dose, and the influence of comedication were also investigated. Assay results in the two tested fluids showed a good correlation (r2  = .78, P < .0001), but concentrations in saliva were moderately lower than those in plasma (mean saliva to plasma ratio = 0.7 ± 0.2). In eight patients evaluated at three different dose levels, plasma rufinamide concentrations increased linearly with increasing dose. Patients receiving valproic acid comedication had higher dose-normalized plasma rufinamide levels than patients comedicated with drugs devoid of strong enzyme-inducing or enzyme-inhibiting activity. Overall, these findings indicate that use of saliva represents a feasible option for the application of TDM in patients treated with rufinamide. Because rufinamide concentrations are lower in saliva than in plasma, a correction factor is needed if measurements made in saliva are used as a surrogate for plasma concentrations.


Assuntos
Anticonvulsivantes/metabolismo , Monitoramento de Medicamentos/métodos , Epilepsia/tratamento farmacológico , Epilepsia/metabolismo , Saliva/metabolismo , Triazóis/metabolismo , Adolescente , Adulto , Anticonvulsivantes/sangue , Anticonvulsivantes/uso terapêutico , Criança , Epilepsia/sangue , Feminino , Humanos , Masculino , Triazóis/sangue , Triazóis/uso terapêutico , Adulto Jovem
12.
Pharmacol Rev ; 72(3): 606-638, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32540959

RESUMO

Epilepsy is a chronic neurologic disorder that affects over 70 million people worldwide. Despite the availability of over 20 antiseizure drugs (ASDs) for symptomatic treatment of epileptic seizures, about one-third of patients with epilepsy have seizures refractory to pharmacotherapy. Patients with such drug-resistant epilepsy (DRE) have increased risks of premature death, injuries, psychosocial dysfunction, and a reduced quality of life, so development of more effective therapies is an urgent clinical need. However, the various types of epilepsy and seizures and the complex temporal patterns of refractoriness complicate the issue. Furthermore, the underlying mechanisms of DRE are not fully understood, though recent work has begun to shape our understanding more clearly. Experimental models of DRE offer opportunities to discover, characterize, and challenge putative mechanisms of drug resistance. Furthermore, such preclinical models are important in developing therapies that may overcome drug resistance. Here, we will review the current understanding of the molecular, genetic, and structural mechanisms of ASD resistance and discuss how to overcome this problem. Encouragingly, better elucidation of the pathophysiological mechanisms underpinning epilepsies and drug resistance by concerted preclinical and clinical efforts have recently enabled a revised approach to the development of more promising therapies, including numerous potential etiology-specific drugs ("precision medicine") for severe pediatric (monogenetic) epilepsies and novel multitargeted ASDs for acquired partial epilepsies, suggesting that the long hoped-for breakthrough in therapy for as-yet ASD-resistant patients is a feasible goal. SIGNIFICANCE STATEMENT: Drug resistance provides a major challenge in epilepsy management. Here, we will review the current understanding of the molecular, genetic, and structural mechanisms of drug resistance in epilepsy and discuss how the problem might be overcome.


Assuntos
Anticonvulsivantes/farmacologia , Epilepsia/tratamento farmacológico , Animais , Anticonvulsivantes/farmacocinética , Anticonvulsivantes/uso terapêutico , Resistência a Medicamentos , Epilepsia/genética , Epilepsia/metabolismo , Humanos , Ensaios Clínicos Controlados Aleatórios como Assunto
13.
Epilepsia ; 61(6): 1120-1128, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32378757

RESUMO

OBJECTIVE: Therapeutic drug monitoring (TDM) of antiepileptic drugs (AEDs) helps optimize drug management for patients with epilepsy. Salivary testing is both noninvasive and easy, and has several other advantages. Due to technical advances, salivary TDM has become feasible for several drugs, including AEDs, and its value has been investigated. Until recently, saliva TDM of perampanel (PER) had not been reported. The purpose of our study was to confirm whether saliva is a biological substitute for plasma in PER TDM. METHODS: Adult patients diagnosed with epilepsy who received PER from August 2018 to March 2019 at Seoul National University Hospital were enrolled. Total and free PER were measured in simultaneously obtained plasma and saliva samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high-performance liquid chromatographic (HPLC). We examined the correlations between saliva and plasma PER concentrations and whether the use of concomitant medications classified as cytochrome P450 (CYP)3A4 inducers affected the correlations. RESULTS: Thirty patients were enrolled, aged 16 to 60; 10 (33%) were women. Patients received 2 to 12 mg (mean, 6 mg) of PER. The average total and free concentrations of PER were 343.02 (46.6-818.0) and 1.53 (0.51-2.92) ng/mL in plasma and 9.74 (2.21-33.0) and 2.83 (1.01-6.8) ng/mL in saliva, respectively. A linear relationship was observed between the total PER concentrations in saliva and the total and free PER concentrations in plasma (both P < .001; r = .678 and r = .619, respectively). The change in the PER concentration caused by the CYP3A4 inducer did not affect the correlation between saliva and plasma concentrations (all P < .001). SIGNIFICANCE: The PER concentration in saliva was correlated with that in plasma. This correlation was not affected by CYP3A4 inducers. Our results demonstrate for the first time that PER is measurable in saliva and suggest the potential for the clinical application of the saliva PER TDM matrix.


Assuntos
Anticonvulsivantes/metabolismo , Monitoramento de Medicamentos/métodos , Epilepsia/tratamento farmacológico , Epilepsia/metabolismo , Piridonas/metabolismo , Saliva/metabolismo , Adolescente , Adulto , Anticonvulsivantes/sangue , Anticonvulsivantes/uso terapêutico , Cromatografia Líquida/métodos , Epilepsia/sangue , Feminino , Humanos , Masculino , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Piridonas/sangue , Piridonas/uso terapêutico , Adulto Jovem
14.
Nat Commun ; 11(1): 2510, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32427860

RESUMO

In mammals, a subset of arginine tRNA isoacceptors are methylated in the anticodon loop by the METTL2 methyltransferase to form the 3-methylcytosine (m3C) modification. However, the mechanism by which METTL2 identifies specific tRNA arginine species for m3C formation as well as the biological role of m3C in mammals is unknown. Here, we show that human METTL2 forms a complex with DALR anticodon binding domain containing 3 (DALRD3) protein to recognize particular arginine tRNAs destined for m3C modification. DALRD3-deficient human cells exhibit nearly complete loss of the m3C modification in tRNA-Arg species. Notably, we identify a homozygous nonsense mutation in the DALRD3 gene that impairs m3C formation in human patients exhibiting developmental delay and early-onset epileptic encephalopathy. These findings uncover an unexpected function for the DALRD3 protein in the targeting of distinct arginine tRNAs for m3C modification and suggest a crucial biological role for DALRD3-dependent tRNA modification in proper neurological development.


Assuntos
Citosina/análogos & derivados , Epilepsia/metabolismo , RNA de Transferência de Arginina/metabolismo , tRNA Metiltransferases/metabolismo , Idade de Início , Linhagem Celular , Citosina/metabolismo , Epilepsia/genética , Humanos , Conformação de Ácido Nucleico , Ligação Proteica , RNA de Transferência de Arginina/química , RNA de Transferência de Arginina/genética , tRNA Metiltransferases/genética
15.
J Neurosci ; 40(23): 4586-4595, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32341095

RESUMO

The epilepsy-linked gene SV2A, has a number of potential roles in the synaptic vesicle (SV) life cycle. However, how loss of SV2A function translates into presynaptic dysfunction and ultimately seizure activity is still undetermined. In this study, we examined whether the first SV2A mutation identified in human disease (R383Q) could provide information regarding which SV2A-dependent events are critical in the translation to epilepsy. We utilized a molecular replacement strategy in which exogenous SV2A was expressed in mouse neuronal cultures of either sex, which had been depleted of endogenous SV2A to mimic the homozygous human condition. We found that the R383Q mutation resulted in a mislocalization of SV2A from SVs to the plasma membrane, but had no effect on its activity-dependent trafficking. This SV2A mutant displayed reduced mobility when stranded on the plasma membrane and reduced binding to its interaction partner synaptotagmin-1 (Syt1). Furthermore, the R383Q mutant failed to rescue reduced expression and dysfunctional activity-dependent trafficking of Syt1 in the absence of endogenous SV2A. This suggests that the inability to control Syt1 expression and trafficking at the presynapse may be key in the transition from loss of SV2A function to seizure activity.SIGNIFICANCE STATEMENT SV2A is a synaptic vesicle (SV) protein, the absence or dysfunction of which is linked to epilepsy. However, the series of molecular events that result in this neurological disorder is still undetermined. We demonstrate here that the first human mutation in SV2A identified in an individual with epilepsy displays reduced binding to synaptotagmin-1 (Syt1), an SV protein essential for synchronous neurotransmitter release. Furthermore, this mutant cannot correct alterations in both Syt1 expression and trafficking when expressed in the absence of endogenous SV2A (to mimic the homozygous human condition). This suggests that the inability to control Syt1 expression and trafficking may be key in the transition from loss of SV2A function to seizure activity.


Assuntos
Epilepsia/genética , Glicoproteínas de Membrana/genética , Mutação de Sentido Incorreto/fisiologia , Proteínas do Tecido Nervoso/genética , Transporte Proteico/fisiologia , Sinaptotagmina I/biossíntese , Sinaptotagmina I/genética , Animais , Células Cultivadas , Epilepsia/metabolismo , Feminino , Expressão Gênica , Células HEK293 , Humanos , Masculino , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/deficiência
16.
Proc Natl Acad Sci U S A ; 117(19): 10155-10164, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32327603

RESUMO

Myeloperoxidase (MPO)-mediated oxidative stress has been suggested to play an important role in the pathological dysfunction of epileptic brains. However, there is currently no robust brain-imaging tool to detect real-time endogenous hypochlorite (HClO) generation by MPO or a fluorescent probe for rapid high-throughput screening of antiepileptic agents that control the MPO-mediated chlorination stress. Herein, we report an efficient two-photon fluorescence probe (named HCP) for the real-time detection of endogenous HClO signals generated by MPO in the brain of kainic acid (KA)-induced epileptic mice, where HClO-dependent chlorination of quinolone fluorophore gives the enhanced fluorescence response. With this probe, we visualized directly the endogenous HClO fluxes generated by the overexpression of MPO activity in vivo and ex vivo in mouse brains with epileptic behaviors. Notably, by using HCP, we have also constructed a high-throughput screening approach to rapidly screen the potential antiepileptic agents to control MPO-mediated oxidative stress. Moreover, from this screen, we identified that the flavonoid compound apigenin can relieve the MPO-mediated oxidative stress and inhibit the ferroptosis of neuronal cells. Overall, this work provides a versatile fluorescence tool for elucidating the role of HClO generation by MPO in the pathology of epileptic seizures and for rapidly discovering additional antiepileptic agents to prevent and treat epilepsy.


Assuntos
Apigenina/farmacologia , Encéfalo/efeitos dos fármacos , Epilepsia/tratamento farmacológico , Ferroptose , Ácido Hipocloroso/metabolismo , Estresse Oxidativo , Peroxidase/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Mapeamento Encefálico/métodos , Epilepsia/metabolismo , Epilepsia/patologia , Corantes Fluorescentes/química , Camundongos , Neuroimagem/métodos , Fármacos Neuroprotetores/farmacologia
17.
PLoS One ; 15(4): e0230787, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32275724

RESUMO

The mechanisms of epileptic discharge generation and spread are not yet fully known. A recently proposed simple biophysical model of interictal and ictal discharges, Epileptor-2, reproduces well the main features of neuronal excitation and ionic dynamics during discharge generation. In order to distinguish between two hypothesized mechanisms of discharge propagation, we extend the model to the case of two-dimensional propagation along the cortical neural tissue. The first mechanism is based on extracellular potassium diffusion, and the second is the propagation of spikes and postsynaptic signals along axons and dendrites. Our simulations show that potassium diffusion is too slow to reproduce an experimentally observed speed of ictal wavefront propagation (tenths of mm/s). By contrast, the synaptic mechanism predicts well the speed and synchronization of the pre-ictal bursts before the ictal front and the afterdischarges in the ictal core. Though this fact diminishes the role of diffusion and electrodiffusion, the model nevertheless highlights the role of potassium extrusion during neuronal excitation, which provides a positive feedback that changes at the ictal wavefront the balance of excitation versus inhibition in favor of excitation. This finding may help to find a target for a treatment to prevent seizure propagation.


Assuntos
Dendritos/metabolismo , Epilepsia/metabolismo , Epilepsia/fisiopatologia , Potássio/metabolismo , Convulsões/metabolismo , Convulsões/fisiopatologia , Potenciais de Ação/fisiologia , Axônios/metabolismo , Córtex Cerebral/metabolismo , Córtex Cerebral/fisiopatologia , Pareamento Cromossômico/fisiologia , Modelos Neurológicos , Neurônios/metabolismo , Potenciais Sinápticos/fisiologia
18.
J Integr Neurosci ; 19(1): 77-87, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-32259888

RESUMO

There is a need to investigate the role of nuclear factor kappa B in the regulation of cyclooxygenase-2 expression in the epileptic rat brain and cultured hippocampal neurons. Immunofluorescence and polymerase chain reaction was used to detect the expression of nuclear factor kappa B and cyclooxygenase-2. In cultured hippocampal neurons and rat brain: the control group compared with the normal group, nuclear factor kappa B expression in the hippocampal dentate gyrus, cerebral cortex, the piriform cortex brain regions were significantly increased (P < 0.01). This is accompanied by a significant increase in cyclooxygenase-2 protein and mRNA expressions in the hippocampus (P < 0.01). In the experimental group compared to the control group, the nuclear factor-kappa B expression in the hippocampal dentate gyrus, cerebral cortex, piriform cortex, and other brain regions was significantly lower (P < 0.01), with the accompanying decrease in cyclooxygenase-2 protein and mRNA expression (P < 0.01) in the hippocampus. In conclusion, κB-decoy can inhibit nuclear factor kappa B activation in epileptic rat brain and cyclooxygenase-2 overexpression.


Assuntos
Encéfalo/metabolismo , Ciclo-Oxigenase 2/metabolismo , Epilepsia/metabolismo , Hipocampo/metabolismo , NF-kappa B/metabolismo , Neurônios/metabolismo , Convulsões/metabolismo , Animais , Células Cultivadas , Masculino , Ratos Sprague-Dawley
19.
Sci Rep ; 10(1): 3760, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111960

RESUMO

Epilepsy is a widespread neurological disease characterized by abnormal neuronal activity resulting in recurrent seizures. There is mounting evidence that a circadian system disruption, involving clock genes and their downstream transcriptional regulators, is associated with epilepsy. In this study, we characterized the hippocampal expression of clock genes and PAR bZIP transcription factors (TFs) in a mouse model of temporal lobe epilepsy induced by intrahippocampal injection of kainic acid (KA). The expression of PAR bZIP TFs was significantly altered following KA injection as well as in other rodent models of acquired epilepsy. Although the PAR bZIP TFs are regulated by proinflammatory cytokines in peripheral tissues, we discovered that the regulation of their expression is inflammation-independent in hippocampal tissue and rather mediated by clock genes and hyperexcitability. Furthermore, we report that hepatic leukemia factor (Hlf), a member of PAR bZIP TFs family, is invariably downregulated in animal models of acquired epilepsy, regulates neuronal activity in vitro and its overexpression in dentate gyrus neurons in vivo leads to altered expression of genes associated with seizures and epilepsy. Overall, our study provides further evidence of PAR bZIP TFs involvement in epileptogenesis and points to Hlf as the key player.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Giro Denteado/metabolismo , Epilepsia/metabolismo , Regulação da Expressão Gênica , Animais , Giro Denteado/patologia , Modelos Animais de Doenças , Epilepsia/induzido quimicamente , Ácido Caínico/efeitos adversos , Ácido Caínico/farmacologia , Masculino , Camundongos
20.
Chem Commun (Camb) ; 56(27): 3871-3874, 2020 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-32134089

RESUMO

A two-photon (TP) fluorescence probe has been developed for imaging endogenous FA fluxes during metabolic and epigenetic processes in animal models, especially in live brains.


Assuntos
Encéfalo/metabolismo , Epilepsia/metabolismo , Corantes Fluorescentes/farmacologia , Formaldeído/metabolismo , Animais , Caenorhabditis elegans , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Camundongos , Imagem Óptica , Fótons
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