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Epileptogenesis is the process by which a normal brain becomes hyperexcitable and capable of generating spontaneous recurrent seizures. The extensive dysregulation of gene expression associated with epileptogenesis is shaped, in part, by microRNAs (miRNAs) - short, non-coding RNAs that negatively regulate protein levels. Functional miRNA-mediated regulation can, however, be difficult to elucidate due to the complexity of miRNA-mRNA interactions. Here, we integrated miRNA and mRNA expression profiles sampled over multiple time-points during and after epileptogenesis in rats, and applied bi-clustering and Bayesian modelling to construct temporal miRNA-mRNA-mRNA interaction networks. Network analysis and enrichment of network inference with sequence- and human disease-specific information identified key regulatory miRNAs with the strongest influence on the mRNA landscape, and miRNA-mRNA interactions closely associated with epileptogenesis and subsequent epilepsy. Our findings underscore the complexity of miRNA-mRNA regulation, can be used to prioritise miRNA targets in specific systems, and offer insights into key regulatory processes in epileptogenesis with therapeutic potential for further investigation.
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Epilepsia , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , MicroARNs , ARN Mensajero , Convulsiones , Animales , MicroARNs/genética , MicroARNs/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Convulsiones/genética , Convulsiones/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Masculino , Regulación de la Expresión Génica , Teorema de Bayes , Modelos Animales de Enfermedad , TranscriptomaRESUMEN
Treating bone infections and ensuring bone repair is one of the greatest global challenges of modern orthopedics, made complex by antimicrobial resistance (AMR) risks due to long-term antibiotic treatment and debilitating large bone defects following infected tissue removal. An ideal multi-faceted solution would will eradicate bacterial infection without long-term antibiotic use, simultaneously stimulating osteogenesis and angiogenesis. Here, a multifunctional collagen-based scaffold that addresses these needs by leveraging the potential of antibiotic-free antimicrobial nanoparticles (copper-doped bioactive glass, CuBG) to combat infection without contributing to AMR in conjunction with microRNA-based gene therapy (utilizing an inhibitor of microRNA-138) to stimulate both osteogenesis and angiogenesis, is developed. CuBG scaffolds reduce the attachment of gram-positive bacteria by over 80%, showcasing antimicrobial functionality. The antagomiR-138 nanoparticles induce osteogenesis of human mesenchymal stem cells in vitro and heal a large load-bearing defect in a rat femur when delivered on the scaffold. Combining both promising technologies results in a multifunctional antagomiR-138-activated CuBG scaffold inducing hMSC-mediated osteogenesis and stimulating vasculogenesis in an in vivo chick chorioallantoic membrane model. Overall, this multifunctional scaffold catalyzes killing mechanisms in bacteria while inducing bone repair through osteogenic and angiogenic coupling, making this platform a promising multi-functional strategy for treating and repairing complex bone infections.
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MicroARNs , Nanopartículas , Humanos , Ratas , Animales , Andamios del Tejido , Regeneración Ósea , MicroARNs/genética , Antagomirs/farmacología , Osteogénesis , Antibacterianos/farmacología , Antibacterianos/uso terapéuticoRESUMEN
There remains an urgent need for new therapies for treatment-resistant epilepsy. Sodium channel blockers are effective for seizure control in common forms of epilepsy, but loss of sodium channel function underlies some genetic forms of epilepsy. Approaches that provide bidirectional control of sodium channel expression are needed. MicroRNAs (miRNA) are small noncoding RNAs which negatively regulate gene expression. Here we show that genome-wide miRNA screening of hippocampal tissue from a rat epilepsy model, mice treated with the antiseizure medicine cannabidiol, and plasma from patients with treatment-resistant epilepsy, converge on a single target-miR-335-5p. Pathway analysis on predicted and validated miR-335-5p targets identified multiple voltage-gated sodium channels (VGSCs). Intracerebroventricular injection of antisense oligonucleotides against miR-335-5p resulted in upregulation of Scn1a, Scn2a, and Scn3a in the mouse brain and an increased action potential rising phase and greater excitability of hippocampal pyramidal neurons in brain slice recordings, consistent with VGSCs as functional targets of miR-335-5p. Blocking miR-335-5p also increased voltage-gated sodium currents and SCN1A, SCN2A, and SCN3A expression in human induced pluripotent stem cell-derived neurons. Inhibition of miR-335-5p increased susceptibility to tonic-clonic seizures in the pentylenetetrazol seizure model, whereas adeno-associated virus 9-mediated overexpression of miR-335-5p reduced seizure severity and improved survival. These studies suggest modulation of miR-335-5p may be a means to regulate VGSCs and affect neuronal excitability and seizures. Changes to miR-335-5p may reflect compensatory mechanisms to control excitability and could provide biomarker or therapeutic strategies for different types of treatment-resistant epilepsy.
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Epilepsia , Células Madre Pluripotentes Inducidas , MicroARNs , Canales de Sodio Activados por Voltaje , Humanos , Ratones , Ratas , Animales , Células Madre Pluripotentes Inducidas/metabolismo , Convulsiones/inducido químicamente , Convulsiones/genética , Convulsiones/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Canales de Sodio Activados por Voltaje/genética , Canal de Sodio Activado por Voltaje NAV1.1/genética , Canal de Sodio Activado por Voltaje NAV1.1/metabolismo , Canal de Sodio Activado por Voltaje NAV1.3/genéticaRESUMEN
Assessment of cell cytotoxicity following transfection of cells with microRNA (miRNA) is an essential step in the evaluation of basic miRNA functional effects within cells in both 2D and 3D microenvironments. The lactate dehydrogenase (LDH) assay is a colorimetric assay that provides a basic, dependable method for determining cellular cytotoxicity through assessment of the level of plasma membrane damage in a cell population. Here, we describe the overexpression of miRNA in breast cancer cells when cultured in 3D collagen-based biomaterial scaffolds, achieved by Lipofectamine transfection, with subsequent examination of cell cytotoxicity using the LDH assay.
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Materiales Biocompatibles , MicroARNs , MicroARNs/genética , Transfección , Citotoxicidad Inmunológica , Bioensayo , L-Lactato Deshidrogenasa/genéticaRESUMEN
BACKGROUND: Inflammation, particularly cytokine release, contributes to epileptogenesis by influencing the cerebral tissue remodeling and neuronal excitability that occurs after a precipitating epileptogenic insult. While several cytokines have been explored in this process, release kinetics are less well investigated. Determining the time course of cytokine release in the epileptogenic zone is necessary for precisely timed preventive or therapeutic anti-inflammatory interventions. METHODS: Hippocampal extracellular levels of six cytokines and chemokines (IL-1ß, IL-6, IL-10, CCL2, CCL3, and CCL5) were quantified at various time points during epileptogenesis in a rat model of mesial temporal lobe epilepsy with hippocampal sclerosis (mTLE-HS) using microdialysis (MD). RESULTS: The analysis of microdialysates demonstrated consistent elevation at all time points during epileptogenesis for IL-1ß and IL-10. IL-10 release was maximal on day 1, IL-1ß release peaked at day 8. No correlation between local hippocampal IL-1ß concentrations and IL-1ß blood levels was found. CONCLUSION: The release kinetics of IL-1ß are consistent with its established pro-epileptogenic properties, while the kinetics of IL-10 suggest a counter-regulatory effect. This proof-of-concept study demonstrates the feasibility of intraindividual longitudinal monitoring of hippocampal molecular inflammatory processes via repetitive MD over several weeks and sheds light on the kinetics of hippocampal cytokine release during epileptogenesis.
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The majority of in vitro studies assessing cancer treatments are performed in two-dimensional (2D) monolayers and are subsequently validated in in vivo animal models. However, 2D models fail to accurately model the tumour microenvironment. Furthermore, animal models are not directly applicable to mimic the human scenario. Three-dimensional (3D) culture models may help to address the discrepancies of 2D and animal models. When cancer cells escape the primary tumour, they can invade at distant organs building secondary tumours, called metastasis. The development of metastasis leads to a dramatic decrease in the life expectancy of patients. Therefore, 3D systems to model the microenvironment of metastasis have also been developed. Several studies have demonstrated changes in cell behaviour and gene expression when cells are cultured in 3D compared to 2D and concluded a better comparability to cells in vivo. Of special importance is the effect seen in response to anti-cancer treatments as models are built primarily to serve as drug-testing platforms. This review highlights these changes between cancer cells grown in 2D and 3D models for some of the most common cancers including lung, breast and prostate tumours. In addition to models aiming to mimic the primary tumour site, the effects of 3D cell culturing in bone metastasis models are also described. STATEMENT OF SIGNIFICANCE: Most in vitro studies in cancer research are performed in 2D and are subsequently validated in in vivo animal models. However, both models possess numerous limitations: 2D models fail to accurately model the tumour microenvironment while animal models are expensive, time-consuming and can differ considerably from humans. It is accepted that the cancer microenvironment plays a critical role in the disease, thus, 3D models have been proposed as a potential solution to address the discrepancies of 2D and animal models. This review highlights changes in cell behaviour, including proliferation, gene expression and chemosensitivity, between cancer cells grown in 2D and 3D models for some of the most common cancers including lung, breast and prostate cancer as well as bone metastasis.
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Neoplasias de la Próstata , Microambiente Tumoral , Animales , Mama , Línea Celular Tumoral , Humanos , Pulmón , MasculinoRESUMEN
Nonviral vectors offer a safe alternative to viral vectors for gene therapy applications, albeit typically exhibiting lower transfection efficiencies. As a result, there remains a significant need for the development of a nonviral delivery system with low cytotoxicity and high transfection efficacy as a tool for safe and transient gene delivery. This study assesses MgAl-NO3 layered double hydroxide (LDH) as a nonviral vector to deliver nucleic acids (pDNA, miRNA and siRNA) to mesenchymal stromal cells (MSCs) in 2D culture and using a 3D tissue engineering scaffold approach. Nanoparticles were formulated by complexing LDH with pDNA, microRNA (miRNA) mimics and inhibitors, and siRNA at varying mass ratios of LDH:nucleic acid. In 2D monolayer, pDNA delivery demonstrated significant cytotoxicity issues, and low cellular transfection was deemed to be a result of the poor physicochemical properties of the LDH-pDNA nanoparticles. However, the lower mass ratios required to successfully complex with miRNA and siRNA cargo allowed for efficient delivery to MSCs. Furthermore, incorporation of LDH-miRNA nanoparticles into collagen-nanohydroxyapatite scaffolds resulted in successful overexpression of miRNA in MSCs, demonstrating the development of an efficacious miRNA delivery platform for gene therapy applications in regenerative medicine.
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Epilepsy diagnosis is complex, requires a team of specialists and relies on in-depth patient and family history, MRI-imaging and EEG monitoring. There is therefore an unmet clinical need for a non-invasive, molecular-based, biomarker to either predict the development of epilepsy or diagnose a patient with epilepsy who may not have had a witnessed seizure. Recent studies have demonstrated a role for microRNAs in the pathogenesis of epilepsy. MicroRNAs are short non-coding RNA molecules which negatively regulate gene expression, exerting profound influence on target pathways and cellular processes. The presence of microRNAs in biofluids, ease of detection, resistance to degradation and functional role in epilepsy render them excellent candidate biomarkers. Here we performed the first multi-model, genome-wide profiling of plasma microRNAs during epileptogenesis and in chronic temporal lobe epilepsy animals. From video-EEG monitored rats and mice we serially sampled blood samples and identified a set of dysregulated microRNAs comprising increased miR-93-5p, miR-142-5p, miR-182-5p, miR-199a-3p and decreased miR-574-3p during one or both phases. Validation studies found miR-93-5p, miR-199a-3p and miR-574-3p were also dysregulated in plasma from patients with intractable temporal lobe epilepsy. Treatment of mice with common anti-epileptic drugs did not alter the expression levels of any of the five miRNAs identified, however administration of an anti-epileptogenic microRNA treatment prevented dysregulation of several of these miRNAs. The miRNAs were detected within the Argonuate2-RISC complex from both neurons and microglia indicating these miRNA biomarker candidates can likely be traced back to specific brain cell types. The current studies identify additional circulating microRNA biomarkers of experimental and human epilepsy which may support diagnosis of temporal lobe epilepsy via a quick, cost-effective rapid molecular-based test.
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MicroARN Circulante/genética , Epilepsia del Lóbulo Temporal/genética , Animales , Anticonvulsivantes/farmacología , Barrera Hematoencefálica/metabolismo , MicroARN Circulante/efectos de los fármacos , Modelos Animales de Enfermedad , Estimulación Eléctrica , Epilepsia del Lóbulo Temporal/sangre , Epilepsia del Lóbulo Temporal/inducido químicamente , Agonistas de Aminoácidos Excitadores/toxicidad , Ácido Kaínico/toxicidad , Masculino , Ratones , Agonistas Muscarínicos/toxicidad , Vía Perforante , Pilocarpina/toxicidad , RatasRESUMEN
Temporal lobe epilepsy is the most common drug-resistant form of epilepsy in adults. The reorganization of neural networks and the gene expression landscape underlying pathophysiologic network behavior in brain structures such as the hippocampus has been suggested to be controlled, in part, by microRNAs. To systematically assess their significance, we sequenced Argonaute-loaded microRNAs to define functionally engaged microRNAs in the hippocampus of three different animal models in two species and at six time points between the initial precipitating insult through to the establishment of chronic epilepsy. We then selected commonly up-regulated microRNAs for a functional in vivo therapeutic screen using oligonucleotide inhibitors. Argonaute sequencing generated 1.44 billion small RNA reads of which up to 82% were microRNAs, with over 400 unique microRNAs detected per model. Approximately half of the detected microRNAs were dysregulated in each epilepsy model. We prioritized commonly up-regulated microRNAs that were fully conserved in humans and designed custom antisense oligonucleotides for these candidate targets. Antiseizure phenotypes were observed upon knockdown of miR-10a-5p, miR-21a-5p, and miR-142a-5p and electrophysiological analyses indicated broad safety of this approach. Combined inhibition of these three microRNAs reduced spontaneous seizures in epileptic mice. Proteomic data, RNA sequencing, and pathway analysis on predicted and validated targets of these microRNAs implicated derepressed TGF-ß signaling as a shared seizure-modifying mechanism. Correspondingly, inhibition of TGF-ß signaling occluded the antiseizure effects of the antagomirs. Together, these results identify shared, dysregulated, and functionally active microRNAs during the pathogenesis of epilepsy which represent therapeutic antiseizure targets.
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Epilepsia del Lóbulo Temporal/tratamiento farmacológico , Epilepsia del Lóbulo Temporal/metabolismo , MicroARNs/efectos de los fármacos , MicroARNs/metabolismo , Oligonucleótidos Antisentido/farmacología , Convulsiones/tratamiento farmacológico , Convulsiones/metabolismo , Animales , Antagomirs/farmacología , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Biomarcadores , Modelos Animales de Enfermedad , Epilepsia , Femenino , Hipocampo/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Proteómica , Ratas , Ratas Sprague-Dawley , Convulsiones/genética , Análisis de Sistemas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: Up to 80% of mesial temporal lobe epilepsy patients with hippocampal sclerosis (mTLE-HS) are resistant to pharmacological treatment, often necessitating surgical resection. Deep brain stimulation (DBS) has emerged as an alternative treatment for patients who do not qualify for resective brain surgery. Brain stimulation may also exert disease-modifying effects, and noncoding microRNAs have recently been proposed to shape the gene expression landscape in epilepsy. OBJECTIVE: We compared the effect of DBS of 4 different hippocampal target regions on epileptogenesis and manifest epilepsy in a rat model of mTLE-HS. To explore mechanisms, we profiled the effect of the most effective DBS paradigm on hippocampal microRNA levels. METHODS: MTLE-HS was induced by electrical stimulation of the perforant pathway (PP) in rats. This paradigm leads to spontaneous seizures within 4 weeks. We investigated DBS of 4 targets: PP, fimbria fornix (FF) formation, dentate gyrus (DG) and ventral hippocampal commissure (VHC). We applied both high- (130â¯Hz) and low-frequency (5â¯Hz or 1â¯Hz) stimulation. Functional microRNAs were identified in the hippocampus immediately after VHC-DBS and after a 97-day recording period by sequencing small RNAs bound to Argonaute-2, a component of the miRNA silencing complex. RESULTS: Low frequency DBS of the VHC significantly delayed the occurrence of the first spontaneous recurrent seizure in the PPS model by â¼300%, from 19 to 56 days. No other stimulation regime altered the latency phase. Upregulation of 5 microRNAs during epileptogenesis was suppressed by VHC-stimulation. CONCLUSION: We conclude that DBS of the VHC delays epilepsy in the PPS model in rats and is associated with differential regulation of several miRNAs. Additional studies are required to determine whether VHC-regulated miRNAs serve causal roles in the anti-epileptogenic effects of this DBS model.
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Estimulación Encefálica Profunda/métodos , Epilepsia del Lóbulo Temporal/metabolismo , Epilepsia del Lóbulo Temporal/terapia , Fórnix/metabolismo , MicroARNs/biosíntesis , Animales , Epilepsia del Lóbulo Temporal/genética , Expresión Génica , Masculino , MicroARNs/genética , Ratas , Ratas Sprague-Dawley , Convulsiones/genética , Convulsiones/metabolismo , Convulsiones/terapiaRESUMEN
Current anti-epileptic drugs (AEDs) act on a limited set of neuronal targets, are ineffective in a third of patients with epilepsy, and do not show disease-modifying properties. MicroRNAs are small noncoding RNAs that regulate levels of proteins by post-transcriptional control of mRNA stability and translation. MicroRNA-134 is involved in controlling neuronal microstructure and brain excitability and previous studies showed that intracerebroventricular injections of locked nucleic acid (LNA), cholesterol-tagged antagomirs targeting microRNA-134 (Ant-134) reduced evoked and spontaneous seizures in mouse models of status epilepticus. Translation of these findings would benefit from evidence of efficacy in non-status epilepticus models and validation in another species. Here, we report that electrographic seizures and convulsive behavior are strongly reduced in adult mice pre-treated with Ant-134 in the pentylenetetrazol model. Pre-treatment with Ant-134 did not affect the severity of status epilepticus induced by perforant pathway stimulation in adult rats, a toxin-free model of acquired epilepsy. Nevertheless, Ant-134 post-treatment reduced the number of rats developing spontaneous seizures by 86% in the perforant pathway stimulation model and Ant-134 delayed epileptiform activity in a rat ex vivo hippocampal slice model. The potent anticonvulsant effects of Ant-134 in multiple models may encourage pre-clinical development of this approach to epilepsy therapy.
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OBJECTIVE: Kainic acid (KA) is a potent glutamate analog that is used to induce neurodegeneration and model temporal lobe epilepsy (TLE) in rodents. KA reliably induces severe, prolonged seizures, that is, convulsive status epilepticus (cSE), which is typically fatal without pharmacologic intervention. Although the use of KA to model human epilepsy has proven unquestionably valuable for >30 years, significant variability and mortality continue to confound results. These issues are probably the consequence of cSE, an all-or-nothing response that is inherently capricious and uncontrollable. The relevance of cSE to the human condition is dubious, however, as most patients with epilepsy never experienced it. We sought to develop a simple, KA-based animal model of TLE that avoids cSE and its confounds. METHODS: Adult, male Sprague-Dawley rats received coincident subcutaneous injections of KA (5 mg) and lorazepam (0.25 mg), approximately 15.0 and 0.75 mg/kg, respectively. Continuous video-electroencephalography (EEG) was used to monitor acute seizure activity and detect spontaneous seizures. Immunocytochemistry, Fluoro-Jade B staining, and Timm staining were used to characterize both acute and chronic neuropathology. RESULTS: Acutely, focal hippocampal seizures were induced, which began after about 30 min and were self-terminating after a few hours. Widespread hippocampal neurodegeneration was detected after 4 days. Spontaneous, focal hippocampal seizures began after an average of 12 days in all animals. Classic hippocampal sclerosis and mossy fiber sprouting characterized the long-term neuropathology. Morbidity and mortality rates were both 0%. SIGNIFICANCE: We show here that the effects of systemic KA can be limited to the hippocampus simply with coadministration of a benzodiazepine at a low dose. This means that lorazepam can block convulsive seizures without truly stopping seizure activity. This novel, cSE-free animal model reliably mimics the defining characteristics of acquired mesial TLE: hippocampal sclerosis and spontaneous hippocampal-onset seizures after a prolonged seizure-free period, without significant morbidity, mortality, or nonresponders.