RESUMO
Rationale: Epilepsy affects over 70 million people globally, with temporal lobe epilepsy with hippocampal sclerosis (TLE-HS) often progressing to a drug-resistant state. Recent research has highlighted the role of reactive astrocytes and glutamate dysregulation in epilepsy pathophysiology. This study aims to investigate the involvement of astrocytic xCT, a glutamate-cystine antiporter, and its regulation by the m6A reader protein YTHDC2 in TLE-HS. Methods: A pilocarpine-induced epilepsy model in mice was used to study the role of xCT in reactive astrocytes. The expression of xCT and its regulation by YTHDC2 were assessed through various molecular and cellular techniques. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were used to measure mRNA and protein levels of xCT and YTHDC2, respectively; immunofluorescence was utilized to visualize their localization and expression in astrocytes. In vivo glutamate measurements were conducted using microdialysis to monitor extracellular glutamate levels in the hippocampus. RNA immunoprecipitation-qPCR (RIP-qPCR) was performed to investigate the binding of YTHDC2 to SLC7A11 mRNA, while methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) was performed to quantify m6A modifications on SLC7A11 mRNA. A dual-luciferase reporter assay was conducted to assess the effect of m6A modifications on SLC7A11 mRNA translation, and polysome profiling was employed to evaluate the translational efficiency of SLC7A11 mRNA. Inhibition experiments involved shRNA-mediated knockdown of SLC7A11 (commonly known as xCT) and YTHDC2 expression in astrocytes. Video-electroencephalogram (EEG) recordings were used to monitor seizure activity in mice. Results: The xCT transporter in reactive astrocytes significantly contributes to elevated extracellular glutamate levels, enhancing neuronal excitability and seizure activity. Increased xCT expression is influenced by the m6A reader protein YTHDC2, which regulates its expression through m6A methylation. Inhibition of xCT or YTHDC2 in astrocytes reduces glutamate levels and effectively controls seizures in a mouse model. Specifically, mice with SLC7A11- or YTHDC2-knockdown astrocytes showed decreased glutamate concentration in the hippocampus and reduced frequency and duration of epileptic seizures. Conclusions: This study highlights the therapeutic potential of targeting YTHDC2 and xCT in reactive astrocytes to mitigate epilepsy. The findings provide a novel perspective on the mechanisms of glutamate dysregulation and their implications in seizure pathophysiology, suggesting that modulation of YTHDC2 and xCT could be a promising strategy for treating TLE.
Assuntos
Sistema y+ de Transporte de Aminoácidos , Astrócitos , Modelos Animais de Doenças , Epilepsia do Lobo Temporal , Ácido Glutâmico , Animais , Astrócitos/metabolismo , Epilepsia do Lobo Temporal/metabolismo , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/fisiopatologia , Camundongos , Sistema y+ de Transporte de Aminoácidos/metabolismo , Sistema y+ de Transporte de Aminoácidos/genética , Ácido Glutâmico/metabolismo , Masculino , Hipocampo/metabolismo , Pilocarpina , Camundongos Endogâmicos C57BL , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , HumanosRESUMO
The role of astroglial and microglial cells in the pathogenesis of epilepsy is currently under active investigation. It has been proposed that the activity of these cells may be regulated by the agonists of peroxisome proliferator-activated nuclear receptors (PPARs). This study investigated the effects of a seven-day treatment with the PPAR ß/δ agonist GW0742 (Fitorine, 5 mg/kg/day) on the behavior and gene expression of the astroglial and microglial proteins involved in the regulation of epileptogenesis in the rat brain within a lithium-pilocarpine model of temporal lobe epilepsy (TLE). TLE resulted in decreased social and increased locomotor activity in the rats, increased expression of astro- and microglial activation marker genes (Gfap, Aif1), pro- and anti-inflammatory cytokine genes (Tnfa, Il1b, Il1rn), and altered expression of other microglial (Nlrp3, Arg1) and astroglial (Lcn2, S100a10) genes in the dorsal hippocampus and cerebral cortex. GW0742 attenuated, but did not completely block, some of these impairments. Specifically, the treatment affected Gfap gene expression in the dorsal hippocampus and Aif1 gene expression in the cortex. The GW0742 injections attenuated the TLE-specific enhancement of Nlrp3 and Il1rn gene expression in the cortex. These results suggest that GW0742 may affect the expression of some genes involved in the regulation of epileptogenesis.
Assuntos
Astrócitos , Modelos Animais de Doenças , Epilepsia do Lobo Temporal , Microglia , PPAR delta , PPAR beta , Tiazóis , Animais , Microglia/efeitos dos fármacos , Microglia/metabolismo , Epilepsia do Lobo Temporal/tratamento farmacológico , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Ratos , PPAR delta/agonistas , PPAR delta/genética , PPAR delta/metabolismo , Masculino , Tiazóis/farmacologia , Tiazóis/uso terapêutico , PPAR beta/agonistas , PPAR beta/genética , PPAR beta/metabolismo , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Pilocarpina/farmacologia , Citocinas/metabolismo , Citocinas/genética , Fenóis , Compostos de SulfidrilaRESUMO
Temporal Lobe Epilepsy (TLE) is a chronic neurological disorder characterized by recurrent focal seizures originating in the temporal lobe. Despite the variety of antiseizure drugs currently available to treat TLE, about 30% of cases continue to have seizures. The etiology of TLE is complex and multifactorial. Increasing evidence indicates that Alzheimer's disease (AD) and drug-resistant TLE present common pathological features that may induce hyperexcitability, especially aberrant hyperphosphorylation of tau protein. Genetic polymorphic variants located in genes of the microtubule-associated protein tau (MAPT) and glycogen synthase kinase-3ß (GSK3B) have been associated with the risk of developing AD. The APOE ε4 allele is a major genetic risk factor for AD. Likewise, a gene-dose-dependent effect of ε4 seems to influence TLE. The present study aimed to investigate whether the APOE É4 allele and genetic variants located in the MAPT and GSK3B genes are associated with the risk of developing AD and drug-resistant TLE in a cohort of the Mexican population. A significant association with the APOE ε4 allele was observed in patients with AD and TLE. Additional genetic interactions were identified between this allele and variants of the MAPT and GSK3B genes.
Assuntos
Alelos , Doença de Alzheimer , Apolipoproteína E4 , Glicogênio Sintase Quinase 3 beta , Proteínas tau , Humanos , Proteínas tau/genética , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Masculino , Feminino , Pessoa de Meia-Idade , Apolipoproteína E4/genética , Adulto , Predisposição Genética para Doença , Idoso , Epilepsia Resistente a Medicamentos/genética , Epilepsia Resistente a Medicamentos/tratamento farmacológico , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/tratamento farmacológico , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BACKGROUND: The solute carrier (SLC) superfamily, which transports solutes across biological membranes, includes four members (SLC2A1, SLC6A1, SLC9A64, and SLC35A2) that have been linked to epilepsy. This study sought to examine the DNA methylation patterns near the promoters of these genes in temporal lobe epilepsy (TLE), as DNA methylation is a crucial epigenetic modification that can impact gene expression. METHODS: The study comprised 38 individuals with TLE and 38 healthy controls. Methylation experiments were performed using peripheral blood, while demethylation experiments were carried out using SH-SY5Y cells with the DNA methylation inhibitor decitabine. RESULTS: A significant difference was observed in the DNA methylation rate of SLC6A1 between TLE patients and controls, with TLE patients showing a lower rate (4.81% vs. 5.77%, p = 0.0000), which remained significant even after Bonferroni correction (p = 0.0000). Based on the hypomethylated SLC6A1 in TLE, a predictive model was established that showed promise in distinguishing and calibrating TLE. In the TLE group, there were differences in DNA methylation rates of SLC6A1 between the young patients and the older controls (4.42% vs. 5.22%, p = 0.0004). A similar trend (p = 0.0436) was noted after adjusting for sex, age at onset, and drug response. In addition, the study found that DNA methylation had a silencing impact on the expression of the SLC6A1 gene in SH-SY5Y cells, which were treated with decitabine at a set dose gradient. CONCLUSIONS: The evidence suggests that lower methylation of SLC6A1 may stimulate transcription in TLE, however, further investigation is necessary to confirm the exact mechanism.
Assuntos
Metilação de DNA , Epilepsia do Lobo Temporal , Regiões Promotoras Genéticas , Humanos , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/metabolismo , Feminino , Adulto , Masculino , Pessoa de Meia-Idade , Epigênese Genética , Sistema A de Transporte de Aminoácidos/genética , Sistema A de Transporte de Aminoácidos/metabolismo , Adulto Jovem , Decitabina/farmacologia , Transportador 1 de Aminoácido Excitatório/genética , Transportador 1 de Aminoácido Excitatório/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABARESUMO
Temporal lobe epilepsy (TLE) is a type of focal epilepsy characterized by spontaneous recurrent seizures originating from the hippocampus. The epigenetic reprogramming hypothesis of epileptogenesis suggests that the development of TLE is associated with alterations in gene transcription changes resulting in a hyperexcitable network in TLE. DNA 5-methylcytosine (5-mC) is an epigenetic mechanism that has been associated with chronic epilepsy. However, the contribution of 5-hydroxymethylcytosine (5-hmC), a product of 5-mC demethylation by the Ten-Eleven Translocation (TET) family proteins in chronic TLE is poorly understood. 5-hmC is abundant in the brain and acts as a stable epigenetic mark altering gene expression through several mechanisms. Here, we found that the levels of bulk DNA 5-hmC but not 5-mC were significantly reduced in the hippocampus of human TLE patients and in the kainic acid (KA) TLE rat model. Using 5-hmC hMeDIP-sequencing, we characterized 5-hmC distribution across the genome and found bidirectional regulation of 5-hmC at intergenic regions within gene bodies. We found that hypohydroxymethylated 5-hmC intergenic regions were associated with several epilepsy-related genes, including Gal, SV2, and Kcnj11 and hyperdroxymethylation 5-hmC intergenic regions were associated with Gad65, TLR4, and Bdnf gene expression. Mechanistically, Tet1 knockdown in the hippocampus was sufficient to decrease 5-hmC levels and increase seizure susceptibility following KA administration. In contrast, Tet1 overexpression in the hippocampus resulted in increased 5-hmC levels associated with improved seizure resiliency in response to KA. These findings suggest an important role for 5-hmC as an epigenetic regulator of epilepsy that can be manipulated to influence seizure outcomes.
Assuntos
5-Metilcitosina , Metilação de DNA , Modelos Animais de Doenças , Epilepsia do Lobo Temporal , Hipocampo , Animais , Hipocampo/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Masculino , Humanos , Epilepsia do Lobo Temporal/metabolismo , Epilepsia do Lobo Temporal/genética , Ratos , Ratos Sprague-Dawley , Feminino , Epigênese Genética , Adulto , Ácido CaínicoRESUMO
BACKGROUD: Temporal lobe epilepsy (TLE) is associated with abnormal dynamic functional connectivity patterns, but the dynamic changes in brain activity at each time point remain unclear, as does the potential molecular mechanisms associated with the dynamic temporal characteristics of TLE. METHODS: Resting-state functional magnetic resonance imaging (rs-fMRI) was acquired for 84 TLE patients and 35 healthy controls (HCs). The data was then used to conduct HMM analysis on rs-fMRI data from TLE patients and an HC group in order to explore the intricate temporal dynamics of brain activity in TLE patients with cognitive impairment (TLE-CI). Additionally, we aim to examine the gene expression profiles associated with the dynamic modular characteristics in TLE patients using the Allen Human Brain Atlas (AHBA) database. RESULTS: Five HMM states were identified in this study. Compared with HCs, TLE and TLE-CI patients exhibited distinct changes in dynamics, including fractional occupancy, lifetimes, mean dwell time and switch rate. Furthermore, transition probability across HMM states were significantly different between TLE and TLE-CI patients (p < 0.05). The temporal reconfiguration of states in TLE and TLE-CI patients was associated with several brain networks (including the high-order default mode network (DMN), subcortical network (SCN), and cerebellum network (CN). Furthermore, a total of 1580 genes were revealed to be significantly associated with dynamic brain states of TLE, mainly enriched in neuronal signaling and synaptic function. CONCLUSIONS: This study provides new insights into characterizing dynamic neural activity in TLE. The brain network dynamics defined by HMM analysis may deepen our understanding of the neurobiological underpinnings of TLE and TLE-CI, indicating a linkage between neural configuration and gene expression in TLE.
Assuntos
Epilepsia do Lobo Temporal , Imageamento por Ressonância Magnética , Cadeias de Markov , Humanos , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/fisiopatologia , Epilepsia do Lobo Temporal/diagnóstico por imagem , Masculino , Feminino , Adulto , Encéfalo/diagnóstico por imagem , Encéfalo/fisiopatologia , Regulação da Expressão Gênica , Estudos de Casos e Controles , Adulto Jovem , Pessoa de Meia-Idade , Descanso/fisiologia , Rede Nervosa/fisiopatologia , Rede Nervosa/diagnóstico por imagemRESUMO
BACKGROUND: Temporal lobe epilepsy (TLE), a prevalent neurological disorder, is associated with hippocampal oxidative stress and inflammation. A recent study reveals that the long noncoding RNA ILF3 divergent transcript (ILF3-AS1) level is elevated in the hippocampus of TLE patients; however, the functional roles of ILF3-AS1 in TLE and underlying mechanisms deserve further investigation. Hence, this study aimed to elucidate whether ILF3-AS1 is involved in the pathogenesis of TLE by regulating oxidative stress and inflammation and to explore its underlying mechanism in vitro. METHODS: Human hippocampal neurons were subjected to a magnesium-free (Mg2+-free) solution to establish an in vitro model of TLE. The potential binding sites between ILF3-AS1 and miRNA were predicted by TargetScan/Starbase and confirmed by dual luciferase reporter assay. Cell viability and damage were assessed by cell counting kit-8 and lactate dehydrogenase assay kits, respectively. Levels of reactive oxygen species, malondialdehyde, and superoxide dismutase were determined by commercial kits. Levels of Interleukin-6 (IL-6), IL-1ß, and tumor necrosis factor-alpha were quantified by enzyme-linked immunosorbent assay. The expressions of gene and protein were determined by quantitative real-time polymerase chain reaction and Western blot analysis. RESULTS: In Mg2+-free-treated hippocampal neurons, both ILF3-AS1 and HMGB1 were highly up-regulated, whereas miR-504-3p was down-regulated. ILF3-AS1 knockdown ameliorated Mg2+-free-induced cellular damage, oxidative stress, and inflammatory response. Bioinformatics analysis revealed that miR-504-3p was a target of ILF3-AS1 and was negatively regulated by ILF3-AS1. MiR-504-3p inhibitor blocked the protection of ILF3-AS1 knockdown against Mg2+-free-induced neuronal injury. Further analysis presented that ILF3-AS1 regulated HMGB1 expression by sponging miR-504-3p. Moreover, HMGB1 overexpression reversed the protective functions of ILF3-AS1 knockdown. CONCLUSION: Our findings indicate that ILF3-AS1 contributes to Mg2+-free-induced hippocampal neuron injuries, oxidative stress, and inflammation by targeting the miR-504-3p/HMGB1 axis. These results provide a novel mechanistic understanding of ILF3-AS1 in TLE and suggest potential therapeutic targets for the treatment of epilepsy.
Assuntos
Epilepsia do Lobo Temporal , Proteína HMGB1 , Hipocampo , Inflamação , MicroRNAs , Estresse Oxidativo , RNA Longo não Codificante , Humanos , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/metabolismo , Hipocampo/metabolismo , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Inflamação/metabolismo , Inflamação/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Neurônios/metabolismo , Proteínas do Fator Nuclear 90/metabolismo , Proteínas do Fator Nuclear 90/genética , Estresse Oxidativo/fisiologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
Although several adult rat models of medial temporal lobe epilepsy (mTLE) have been described in detail, our knowledge of mTLE epileptogenesis in infant rats is limited. Here, we present a novel infant rat model of mTLE (InfRPil-mTLE) based on a repetitive, triphasic injection regimen consisting of low-dose pilocarpine administrations (180 mg/kg. i.p.) on days 9, 11, and 15 post partum (pp). The model had a survival rate of >80% and exhibited characteristic spontaneous recurrent electrographic seizures (SRES) in both the hippocampus and cortex that persisted into adulthood. Using implantable video-EEG radiotelemetry, we quantified a complex set of seizure parameters that demonstrated the induction of chronic electroencephalographic seizure activity in our InfRPil-mTLE model, which predominated during the dark cycle. We further analyzed selected candidate genes potentially relevant to epileptogenesis using a RT-qPCR approach. Several candidates, such as the low-voltage-activated Ca2+ channel Cav3.2 and the auxiliary subunits ß 1 and ß 2, which were previously reported to be upregulated in the hippocampus of the adult pilocarpine mTLE model, were found to be downregulated (together with Cav2.1, Cav2.3, M1, and M3) in the hippocampus and cortex of our InfRPil-mTLE model. From a translational point of view, our model could serve as a blueprint for childhood epileptic disorders and further contribute to antiepileptic drug research and development in the future.
Assuntos
Modelos Animais de Doenças , Epilepsia do Lobo Temporal , Pilocarpina , Animais , Epilepsia do Lobo Temporal/fisiopatologia , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/metabolismo , Epilepsia do Lobo Temporal/induzido quimicamente , Ratos , Eletroencefalografia , Hipocampo/metabolismo , Animais Recém-Nascidos , Encéfalo/metabolismo , Ratos Sprague-Dawley , Masculino , FemininoRESUMO
OBJECTIVE: To expand the clinical phenotype and mutation spectrum of familial mesial temporal lobe epilepsy (FMTLE) and provide a new perspective for exploring the pathological mechanisms of epilepsy caused by leucine-rich glioma inactivated 1 (LGI1) variants. METHODS: We reported clinical data from two families with FMTLE and screened patients for variants in the LGI1 gene using Whole-exome sequencing and Sanger sequencing. The clinical features of FMTLE were analysed. The pathogenicity of the causative loci was assessed according to the American College of Medical Genetics and Genomics guidelines, and potential pathogenic mechanisms were predicted through multiple bioinformatics and molecular dynamics software. RESULTS: We identified two novel LGI1 truncating variants within two large families with FMTLE: LGI1 (c.1174C>T, p.Q392X) and LGI1 (c.703C>T, p.Q235X). Compared to previous reports, we found that focal to bilateral tonic-clonic seizures are a common type of seizure in FMTLE. The clinical phenotypes of patients with FMTLE caused by LGI1 variants were relatively mild, and all patients responded well to valproic acid. Bioinformatics analyses and molecular dynamics simulations showed that protein structure and interactions were considerably weakened or damaged as a result of both variants. CONCLUSION: This study presents the first report identifying LGI1 as a potential novel pathogenic gene within FMTLE families, thereby broadening the mutation spectrum associated with FMTLE. The findings of this study offer novel insights and avenues for understanding the intricate molecular mechanisms underlying LGI1 variants and their correlations with patient phenotypes. This study proposes the possibility of familial focal epilepsy syndromes overlapping.
Assuntos
Epilepsia do Lobo Temporal , Peptídeos e Proteínas de Sinalização Intracelular , Linhagem , Fenótipo , Adulto , Feminino , Humanos , Masculino , Adulto Jovem , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/fisiopatologia , Epilepsia do Lobo Temporal/congênito , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação , Criança , AdolescenteRESUMO
INTRODUCTION: Our previous work reveals a critical role of activation of neuronal Alox5 in exacerbating brain injury post seizures. However, whether neuronal Alox5 impacts the pathological process of epilepsy remains unknown. OBJECTIVES: To prove the feasibility of neuron-specific deletion of Alox5 via CRISPR-Cas9 in the blockade of seizure onset and epileptic progression. METHODS: Here, we employed a Clustered regularly interspaced short-palindromic repeat-associated proteins 9 system (CRISPR/Cas9) system delivered by adeno-associated virus (AAV) to specifically delete neuronal Alox5 gene in the hippocampus to explore its therapeutic potential in various epilepsy mouse models and possible mechanisms. RESULTS: Neuronal depletion of Alox5 was successfully achieved in the brain. AAV delivery of single guide RNA of Alox5 in hippocampus resulted in reducing seizure severity, delaying epileptic progression and improving epilepsy-associated neuropsychiatric comorbidities especially anxiety, cognitive deficit and autistic-like behaviors in pilocarpine- and kainic acid-induced temporal lobe epilepsy (TLE) models. In addition, neuronal Alox5 deletion also reversed neuron loss, neurodegeneration, astrogliosis and mossy fiber sprouting in TLE model. Moreover, a battery of tests including analysis of routine blood test, hepatic function, renal function, routine urine test and inflammatory factors demonstrated no noticeable toxic effect, suggesting that Alox5 deletion possesses the satisfactory biosafety. Mechanistically, the anti-epileptic effect of Alox5 deletion might be associated with reduction of glutamate level to restore excitatory/inhibitory balance by reducing CAMKII-mediated phosphorylation of Syn ISer603. CONCLUSION: Our findings showed the translational potential of AAV-mediated delivery of CRISPR-Cas9 system including neuronal Alox5 gene for an alternative promising therapeutic approach to treat epilepsy.
Assuntos
Araquidonato 5-Lipoxigenase , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Epilepsia , Hipocampo , Neurônios , Animais , Camundongos , Araquidonato 5-Lipoxigenase/metabolismo , Araquidonato 5-Lipoxigenase/genética , Neurônios/metabolismo , Hipocampo/metabolismo , Epilepsia/genética , Masculino , Camundongos Endogâmicos C57BL , Deleção de Genes , Epilepsia do Lobo Temporal/genética , Dependovirus/genética , PilocarpinaRESUMO
An epilepsy diagnosis reduces a patient's quality of life tremendously, and it is a fate shared by over 50 million people worldwide. Temporal lobe epilepsy (TLE) is largely considered a nongenetic or acquired form of epilepsy that develops in consequence of neuronal trauma by injury, malformations, inflammation, or a prolonged (febrile) seizure. Although extensive research has been conducted to understand the process of epileptogenesis, a therapeutic approach to stop its manifestation or to reliably cure the disease has yet to be developed. In this review, we briefly summarize the current literature predominately based on data from excitotoxic rodent models on the cellular events proposed to drive epileptogenesis and thoroughly discuss the major molecular pathways involved, with a focus on neurogenesis-related processes and transcription factors. Furthermore, recent investigations emphasized the role of the genetic background for the acquisition of epilepsy, including variants of neurodevelopmental genes. Mutations in associated transcription factors may have the potential to innately increase the vulnerability of the hippocampus to develop epilepsy following an injury-an emerging perspective on the epileptogenic process in acquired forms of epilepsy.
Assuntos
Epilepsia do Lobo Temporal , Humanos , Animais , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/metabolismo , Epilepsia do Lobo Temporal/patologia , Epilepsia do Lobo Temporal/terapia , Predisposição Genética para Doença , Astrócitos/metabolismo , Astrócitos/patologia , Fatores de Transcrição/metabolismo , Transdução de SinaisRESUMO
Over the past decades, the immune responses have been suspected of participating in the mechanisms for epilepsy. To assess the immune related pathway in temporal lobe epilepsy (TLE), we explored the altered immune pathways in TLE patients with and without hippocampal sclerosis (HS). We analyzed RNA-seq data from 3 TLE-HS and 3 TLE-nonHS patients, including identification of differentially expressed RNA, function pathway enrichment, the protein-protein interaction network and construction of ceRNA regulatory network. We illustrated the immune related landscape of molecules and pathways on human TLE-HS. Also, we identified several differential immune related genes like HSP90AA1 and SOD1 in TLE-HS patients. Further ceRNA regulatory network analysis found SOX2-OT connected to miR-671-5p and upregulated the target gene SPP1 in TLE-HS patients. Also, we identified both SOX2-OT and SPP1 were significantly upregulated in five different databases including TLE-HS patients and animal models. Our findings established the first immune related genes and possible regulatory pathways in TLE-HS patients and animal models, which provided a novel insight into disease pathogenesis in both patients and animal models. The immune related SOX2-OT/miR-671-5p/SPP1 axis may be the potential therapeutic target for TLE-HS.
Assuntos
Epilepsia do Lobo Temporal , Redes Reguladoras de Genes , Esclerose Hipocampal , MicroRNAs , Fatores de Transcrição SOXB1 , Adulto , Animais , Feminino , Humanos , Masculino , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/imunologia , Epilepsia do Lobo Temporal/fisiopatologia , Perfilação da Expressão Gênica , Esclerose Hipocampal/imunologia , Esclerose Hipocampal/fisiopatologia , MicroRNAs/genética , MicroRNAs/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , Mapas de Interação de Proteínas , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
The drug-resistant temporal lobe epilepsy (TLE) has recently been associated with single nucleotide variants (SNVs) in microRNA(miR)-146a (MIR-146A) (rs2910164) and Sodium Voltage-Gated Channel Alpha Subunit 1 (SCN1A) (rs2298771 and rs3812718) genes. Moreover, no studies have shown an association between these SNVs and susceptibility to drug-resistant and drug-responsive TLE in Brazil. Thus, deoxyribonucleic acid (DNA) samples from 120 patients with TLE (55 drug-responsive and 65 drug-resistant) were evaluated by real-time polymerase chain reaction (RT-PCR). A total of 1171 healthy blood donor individuals from the Online Archive of Brazilian Mutations (ABraOM, from Portuguese Arquivo Brasileiro On-line de Mutações), a repository containing genomic variants of the Brazilian population, were added as a control population for the studied SNVs. MIR-146A and SCN1A relative expression was performed by quantitative RT-PCR (qRT-PCR). The statistical analysis protocol was performed using an alpha error of 0.05. TLE patient samples and ABraOM control samples were in Hardy-Weinberg equilibrium for all studied SNVs. For rs2910164, the frequencies of the homozygous genotype (CC) (15.00% vs. 9.65%) and C allele (37.80% vs. 29.97%) were superior in patients with TLE compared to controls with a higher risk for TLE disease [odds ratio (OR) = 1.89 (95% confidence interval (95%CI) = 1.06-3.37); OR = 1.38 (95%CI = 1.04-1.82), respectively]. Drug-responsive patients also presented higher frequencies of the CC genotype [21.81% vs. 9.65%; OR = 2.58 (95%CI = 1.25-5.30)] and C allele [39.09% vs. 29.97%; OR = 1.50 (95%CI = 1.01-2.22)] compared to controls. For rs2298771, the frequency of the heterozygous genotype (AG) (51.67% vs. 40.40%) was superior in patients with TLE compared to controls with a higher risk for TLE disease [OR = 2.42 (95%CI = 1.08-5.41)]. Drug-resistant patients presented a higher AG frequency [56.92% vs. 40.40%; OR = 3.36 (95%CI = 1.04-17.30)] compared to the control group. For rs3812718, the prevalence of genotypes and alleles were similar in both studied groups. The MIR-146A relative expression level was lower in drug-resistant compared to drug-responsive patients for GC (1.6 vs. 0.1, p-value = 0.049) and CC (1.8 vs. 0.6, p-value = 0.039). Also, the SCN1A relative expression levels in samples from TLE patients were significantly higher in AG [2.09 vs. 1.10, p-value = 0.038] and GG (3.19 vs. 1.10, p-value < 0.001) compared to the AA genotype. In conclusion, the rs2910164-CC and rs2298771-AG genotypes are exerting significant risk influence, respectively, on responsive disease and resistant disease, probably due to an upregulated nuclear factor kappa B (NF-kB) and SCN1A loss of function.
Assuntos
Epilepsia do Lobo Temporal , MicroRNAs , Canal de Sódio Disparado por Voltagem NAV1.1 , Polimorfismo de Nucleotídeo Único , Humanos , Canal de Sódio Disparado por Voltagem NAV1.1/genética , MicroRNAs/genética , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/tratamento farmacológico , Feminino , Masculino , Brasil , Adulto , Predisposição Genética para Doença , Epilepsia Resistente a Medicamentos/genética , Epilepsia Resistente a Medicamentos/tratamento farmacológico , Pessoa de Meia-Idade , Adulto Jovem , Genótipo , Estudos de Coortes , Alelos , Frequência do Gene , Adolescente , Estudos de Casos e ControlesRESUMO
BACKGROUND AND OBJECTIVES: New-onset refractory status epilepticus (NORSE) occurs in previously healthy children or adults, often followed by refractory epilepsy and poor outcomes. The mechanisms that transform a normal brain into an epileptic one capable of seizing for prolonged periods despite treatment remain unclear. Nonetheless, several pieces of evidence suggest that immune dysregulation could contribute to hyperexcitability and modulate NORSE sequelae. METHODS: We used single-nucleus RNA sequencing to delineate the composition and phenotypic states of the CNS of 4 patients with NORSE, to better understand the relationship between hyperexcitability and immune disturbances. We compared them with 4 patients with chronic temporal lobe epilepsy (TLE) and 2 controls with no known neurologic disorder. RESULTS: Patients with NORSE and TLE exhibited a significantly higher proportion of excitatory neurons compared with controls, with no discernible difference in inhibitory GABAergic neurons. When examining the ratio between excitatory neurons and GABAergic neurons for each patient individually, we observed a higher ratio in patients with acute NORSE or TLE compared with controls. Furthermore, a negative correlation was found between the ratio of excitatory to GABAergic neurons and the proportion of GABAergic neurons. The ratio between excitatory neurons and GABAergic neurons correlated with the proportion of resident or infiltrating macrophages, suggesting the influence of microglial reactivity on neuronal excitability. Both patients with NORSE and TLE exhibited increased expression of genes associated with microglia activation, phagocytic activity, and NLRP3 inflammasome activation. However, patients with NORSE had decreased expression of genes related to the downregulation of the inflammatory response, potentially explaining the severity of their presentation. Microglial activation in patients with NORSE also correlated with astrocyte reactivity, possibly leading to higher degrees of demyelination. DISCUSSION: Our study sheds light on the complex cellular dynamics in NORSE, revealing the potential roles of microglia, infiltrating macrophages, and astrocytes in hyperexcitability and demyelination, offering potential avenues for future research targeting the identified pathways.
Assuntos
Encéfalo , Epilepsia Resistente a Medicamentos , Análise de Célula Única , Estado Epiléptico , Humanos , Estado Epiléptico/genética , Masculino , Feminino , Adulto , Epilepsia Resistente a Medicamentos/genética , Epilepsia Resistente a Medicamentos/imunologia , Encéfalo/metabolismo , Transcriptoma , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/fisiopatologia , Adulto Jovem , Criança , Pessoa de Meia-Idade , Adolescente , Neurônios GABAérgicos/metabolismo , Perfilação da Expressão Gênica , Microglia/metabolismoRESUMO
Familial epilepsy with auditory features (FEAF), previously known as autosomal-dominant lateral temporal lobe epilepsy (ADLTE) is a genetically heterogeneous syndrome, clinically characterized by focal seizures with prominent auditory symptoms. It is inherited with autosomal-dominant pattern with reduced penetrance (about 70%). Sporadic epilepsy with auditory features cases are more frequent and clinically indistinguishable from familial cases. One causal gene, MICAL-1, encodes MICAL-1, an intracellular multi-domain enzyme that is an important regulator of filamentous actin (F-actin) structures. Pathogenic variants in MICAL-1 account for approximately 7% of FEAF families. Here, we describe a de novo MICAL-1 pathogenic variant, p.Arg915Cys, in a sporadic case, an affected 21-year-old Italian man with no family history of epilepsy. Genetic testing was performed in the patient and his parents, using a next-generation sequencing panel. In cell-based assay, this variant significantly increased MICAL-1 oxidoreductase activity, which likely resulted in dysregulation of F-actin organization. This finding provides further support for a gain-of-function effect underlying MICAL-1-mediated epilepsy pathogenesis, as previously seen with other pathogenic variants. Furthermore, the case study provides evidence that de novo MICAL-1 pathogenic variants can occur in sporadic cases with epilepsy with auditory feature (EAF). PLAIN LANGUAGE SUMMARY: In this study, we report a new MICAL-1 pathogenic variant in a patient without family history for epilepsy, not inherited from his parents. MICAL-1 is a protein with enzymatic activity that reorganizes the structure of the cell. We proved the pathological effect of this variant by testing its enzymatic activity and found an increase of this activity. This result suggests that non-familial cases should be tested to find novel pathogenic variants in this gene.
Assuntos
Epilepsia do Lobo Temporal , Humanos , Masculino , Adulto Jovem , Epilepsia do Lobo Temporal/genética , Actinas/genéticaRESUMO
Although granule cell dispersion (GCD) in the hippocampus is known to be an important feature associated with epileptic seizures in temporal lobe epilepsy (TLE), the endogenous molecules that regulate GCD are largely unknown. In the present study, we have examined whether there is any change in AEG-1 expression in the hippocampus of a kainic acid (KA)-induced mouse model of TLE. In addition, we have investigated whether the modulation of astrocyte elevated gene-1 (AEG-1) expression in the dentate gyrus (DG) by intracranial injection of adeno-associated virus 1 (AAV1) influences pathological phenotypes such as GCD formation and seizure susceptibility in a KA-treated mouse. We have identified that the protein expression of AEG-1 is upregulated in the DG of a KA-induced mouse model of TLE. We further demonstrated that AEG-1 upregulation by AAV1 delivery in the DG-induced anticonvulsant activities such as the delay of seizure onset and inhibition of spontaneous recurrent seizures (SRS) through GCD suppression in the mouse model of TLE, while the inhibition of AEG-1 expression increased susceptibility to seizures. The present observations suggest that AEG-1 is a potent regulator of GCD formation and seizure development associated with TLE, and the significant induction of AEG-1 in the DG may have therapeutic potential against epilepsy.
Assuntos
Epilepsia do Lobo Temporal , Epilepsia , Animais , Camundongos , Astrócitos/metabolismo , Giro Denteado/metabolismo , Epilepsia/metabolismo , Epilepsia do Lobo Temporal/induzido quimicamente , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/tratamento farmacológico , Hipocampo/metabolismo , Ácido Caínico/efeitos adversos , Ácido Caínico/metabolismo , Convulsões/induzido quimicamente , Convulsões/genética , Convulsões/metabolismoRESUMO
OBJECTIVE: To investigate the effect of serum apolipoprotein E (APOE) levels on cognitive function in patients with temporal lobe epilepsy (TLE). METHODS: Clinical data were collected from 190 subjects including 110 TLE patients and 80 healthy people. Cognitive function was assessed using the Addenbrooke's Cognitive Examination Revised (ACE-R) scale. Serum levels of APOE were measured using ELISA kits. Genotyping of APOE in peripheral blood was detected by microarray hybridization. RESULTS: Patients with TLE had significantly lower ACE-R total score, memory and verbal fluency scores compared to the healthy group. Serum levels of APOE were significantly higher in TLE patients than in the healthy subjects. Serum APOE levels were significantly negatively correlated with ACE-R total score, memory and verbal fluency scores. The cognitive function score of TLE with APOE ε4 allele was lower than that of TLE without APOE ε4 allele. SIGNIFICANCE: Our study showed that serum APOE levels were higher in TLE patients than in the healthy population. And serum APOE levels were associated with cognitive dysfunction in TLE patients. APOE ε4 allele carriers have poor cognitive function in TLE patients.
Assuntos
Apolipoproteínas E , Epilepsia do Lobo Temporal , Testes Neuropsicológicos , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Apolipoproteínas E/genética , Apolipoproteínas E/sangue , Povo Asiático , China/epidemiologia , Cognição/fisiologia , Transtornos Cognitivos/sangue , Transtornos Cognitivos/etiologia , População do Leste Asiático , Epilepsia do Lobo Temporal/sangue , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/psicologia , GenótipoRESUMO
OBJECTIVE: The contribution of somatic variants to epilepsy has recently been demonstrated, particularly in the etiology of malformations of cortical development. The aim of this study was to determine the diagnostic yield of somatic variants in genes that have been previously associated with a somatic or germline epilepsy model, ascertained from resected brain tissue from patients with multidrug-resistant focal epilepsy. METHODS: Forty-two patients were recruited across three categories: (1) malformations of cortical development, (2) mesial temporal lobe epilepsy with hippocampal sclerosis, and (3) nonlesional focal epilepsy. Participants were subdivided based on histopathology of the resected brain. Paired blood- and brain-derived DNA samples were sequenced using high-coverage targeted next generation sequencing to high depth (585× and 1360×, respectively). Variants were identified using Genome Analysis ToolKit (GATK4) MuTect-2 and confirmed using high-coverage Amplicon-EZ sequencing. RESULTS: Sequence data on 41 patients passed quality control. Four somatic variants were validated following amplicon sequencing: within CBL, ALG13, MTOR, and FLNA. The diagnostic yield across 41 patients was 10%, 9% in mesial temporal lobe epilepsy with hippocampal sclerosis and 20% in malformations of cortical development. SIGNIFICANCE: This study provides novel insights into the etiology of mesial temporal lobe epilepsy with hippocampal sclerosis, highlighting a potential pathogenic role of somatic variants in CBL and ALG13. We also report candidate diagnostic somatic variants in FLNA in focal cortical dysplasia, while providing further insight into the importance of MTOR and related genes in focal cortical dysplasia. This work demonstrates the potential molecular diagnostic value of variants in both germline and somatic epilepsy genes.
Assuntos
Epilepsia Resistente a Medicamentos , Epilepsia do Lobo Temporal , Esclerose Hipocampal , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Epilepsia Resistente a Medicamentos/genética , Epilepsia Resistente a Medicamentos/etiologia , Epilepsia Resistente a Medicamentos/patologia , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/patologia , Filaminas/genética , Variação Genética , Esclerose Hipocampal/genética , Esclerose Hipocampal/patologia , Malformações do Desenvolvimento Cortical/genética , Malformações do Desenvolvimento Cortical/complicações , Malformações do Desenvolvimento Cortical/patologiaRESUMO
Epilepsy is a chronic and heterogenous disease characterized by recurrent unprovoked seizures, that are commonly resistant to antiseizure medications. This study applies a transcriptome network-based approach across epilepsies aiming to improve understanding of molecular disease pathobiology, recognize affected biological mechanisms and apply causal reasoning to identify therapeutic hypotheses. This study included the most common drug-resistant epilepsies (DREs), such as temporal lobe epilepsy with hippocampal sclerosis (TLE-HS), and mTOR pathway-related malformations of cortical development (mTORopathies). This systematic comparison characterized the global molecular signature of epilepsies, elucidating the key underlying mechanisms of disease pathology including neurotransmission and synaptic plasticity, brain extracellular matrix and energy metabolism. In addition, specific dysregulations in neuroinflammation and oligodendrocyte function were observed in TLE-HS and mTORopathies, respectively. The aforementioned mechanisms are proposed as molecular hallmarks of DRE with the identified upstream regulators offering opportunities for drug-target discovery and development.