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1.
Proc Natl Acad Sci U S A ; 117(27): 15977-15988, 2020 07 07.
Article in English | MEDLINE | ID: mdl-32581127

ABSTRACT

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.


Subject(s)
Epilepsy, Temporal Lobe/drug therapy , Epilepsy, Temporal Lobe/metabolism , MicroRNAs/drug effects , MicroRNAs/metabolism , Oligonucleotides, Antisense/pharmacology , Seizures/drug therapy , Seizures/metabolism , Animals , Antagomirs/pharmacology , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Biomarkers , Disease Models, Animal , Epilepsy , Female , Hippocampus/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , Proteomics , Rats , Rats, Sprague-Dawley , Seizures/genetics , Systems Analysis , Up-Regulation/drug effects
2.
Brain ; 143(7): 2139-2153, 2020 07 01.
Article in English | MEDLINE | ID: mdl-32594159

ABSTRACT

Temporal lobe epilepsy is the most common and refractory form of epilepsy in adults. Gene expression within affected structures such as the hippocampus displays extensive dysregulation and is implicated as a central pathomechanism. Post-transcriptional mechanisms are increasingly recognized as determinants of the gene expression landscape, but key mechanisms remain unexplored. Here we show, for first time, that cytoplasmic mRNA polyadenylation, one of the post-transcriptional mechanisms regulating gene expression, undergoes widespread reorganization in temporal lobe epilepsy. In the hippocampus of mice subjected to status epilepticus and epilepsy, we report >25% of the transcriptome displays changes in their poly(A) tail length, with deadenylation disproportionately affecting genes previously associated with epilepsy. Suggesting cytoplasmic polyadenylation element binding proteins (CPEBs) being one of the main contributors to mRNA polyadenylation changes, transcripts targeted by CPEBs were particularly enriched among the gene pool undergoing poly(A) tail alterations during epilepsy. Transcripts bound by CPEB4 were over-represented among transcripts with poly(A) tail alterations and epilepsy-related genes and CPEB4 expression was found to be increased in mouse models of seizures and resected hippocampi from patients with drug-refractory temporal lobe epilepsy. Finally, supporting an adaptive function for CPEB4, deletion of Cpeb4 exacerbated seizure severity and neurodegeneration during status epilepticus and the development of epilepsy in mice. Together, these findings reveal an additional layer of gene expression regulation during epilepsy and point to novel targets for seizure control and disease-modification in epilepsy.


Subject(s)
Epilepsy, Temporal Lobe/metabolism , Gene Expression Regulation/physiology , Polyadenylation/physiology , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Animals , Epilepsy, Temporal Lobe/genetics , Female , Humans , Male , Mice , Mice, Inbred C57BL
3.
J Neurosci ; 39(27): 5377-5392, 2019 07 03.
Article in English | MEDLINE | ID: mdl-31048325

ABSTRACT

Extracellular ATP activates inflammatory responses to tissue injury. It is also implicated in establishing lasting network hyperexcitability in the brain by acting upon independent receptor systems. Whereas the fast-acting P2X channels have well-established roles driving neuroinflammation and increasing hyperexcitability, the slower-acting metabotropic P2Y receptors have received much less attention. Recent studies of P2Y1 receptor function in seizures and epilepsy have produced contradictory results, suggesting that the role of this receptor during seizure pathology may be highly sensitive to context. Here, by using male mice, we demonstrate that the metabotropic P2Y1 receptor mediates either proconvulsive or anticonvulsive responses, dependent on the time point of activation in relation to the induction of status epilepticus. P2Y1 deficiency or a P2Y1 antagonist (MRS2500) administered before a chemoconvulsant, exacerbates epileptiform activity, whereas a P2Y1 agonist (MRS2365) administered at this time point is anticonvulsant. When these drugs are administered after the onset of status epilepticus, however, their effect on seizure severity is reversed, with the antagonist now anticonvulsant and the agonist proconvulsant. This result was consistent across two different mouse models of status epilepticus (intra-amygdala kainic acid and intraperitoneal pilocarpine). Pharmacologic P2Y1 blockade during status epilepticus reduces also associated brain damage, delays the development of epilepsy and, when applied during epilepsy, suppresses spontaneous seizures, in mice. Our data show a context-specific role for P2Y1 during seizure pathology and demonstrate that blocking P2Y1 after status epilepticus and during epilepsy has potent anticonvulsive effects, suggesting that P2Y1 may be a novel candidate for the treatment of drug-refractory status epilepticus and epilepsy.SIGNIFICANCE STATEMENT This is the first study to fully characterize the contribution of a metabotropic purinergic P2Y receptor during acute seizures and epilepsy. The findings suggest that targeting P2Y1 may offer a potential novel treatment strategy for drug-refractory status epilepticus and epilepsy. Our data demonstrate a context-specific role of P2Y1 activation during seizures, switching from a proconvulsive to an anticonvulsive role depending on physiopathological context. Thus, our study provides a possible explanation for seemingly conflicting results obtained between studies of different brain diseases where P2Y1 targeting has been proposed as a potential treatment strategy and highlights that the timing of pharmacological interventions is of critical importance to the understanding of how receptors contribute to the generation of seizures and the development of epilepsy.


Subject(s)
Brain/physiopathology , Epilepsy/physiopathology , Receptors, Purinergic P2Y1/physiology , Status Epilepticus/physiopathology , Adenosine Diphosphate/administration & dosage , Adenosine Diphosphate/analogs & derivatives , Animals , Brain/drug effects , Deoxyadenine Nucleotides/administration & dosage , Disease Models, Animal , Electroencephalography , Male , Mice, Inbred C57BL , Mice, Knockout , Purinergic P2Y Receptor Agonists/administration & dosage , Purinergic P2Y Receptor Antagonists/administration & dosage , Receptors, Purinergic P2Y1/genetics
4.
Neurobiol Dis ; 144: 105048, 2020 10.
Article in English | MEDLINE | ID: mdl-32800995

ABSTRACT

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.


Subject(s)
Circulating MicroRNA/genetics , Epilepsy, Temporal Lobe/genetics , Animals , Anticonvulsants/pharmacology , Blood-Brain Barrier/metabolism , Circulating MicroRNA/drug effects , Disease Models, Animal , Electric Stimulation , Epilepsy, Temporal Lobe/blood , Epilepsy, Temporal Lobe/chemically induced , Excitatory Amino Acid Agonists/toxicity , Kainic Acid/toxicity , Male , Mice , Muscarinic Agonists/toxicity , Perforant Pathway , Pilocarpine/toxicity , Rats
5.
Biochim Biophys Acta Mol Cell Res ; 1864(2): 255-266, 2017 Feb.
Article in English | MEDLINE | ID: mdl-27840225

ABSTRACT

Cells have developed complex transcriptional regulatory mechanisms to maintain intracellular homeostasis and withstand pathophysiological stressors. Feed-forward loops comprising transcription factors that drive expression of both target gene and a microRNA as negative regulator, are gaining increasing recognition as key regulatory elements of cellular homeostasis. The ATP-gated purinergic P2X7 receptor (P2X7R) is an important driver of inflammation and has been implicated in the pathogenesis of numerous brain diseases including epilepsy. Changes in P2X7R expression have been reported in both experimental models and in epilepsy patients but the mechanism(s) controlling P2X7R levels remain incompletely understood. The specificity protein 1 (Sp1) has been shown to induce P2X7R transcription in vitro and recent data has identified microRNA-22 as a post-transcriptional repressor of P2X7R expression after seizures. In the present study we show that Sp1 can induce the transcription of both microRNA-22 and P2X7R in vitro during increased neuronal activity and in vivo in a mouse model of status epilepticus. We further show that Sp1-driven microRNA-22 transcription is calcium-sensitive and Sp1 occupancy of the microRNA-22 promoter region is blocked under conditions of seizure activity sufficient to elicit neuronal death. Taken together, our results suggest a neuronal activity-dependent P2X7R expression which is induced by the transcription factor Sp1 and repressed in a calcium-dependent manner by microRNA-22.


Subject(s)
Adenosine Triphosphate/metabolism , Calcium/metabolism , MicroRNAs/metabolism , Receptors, Purinergic P2X7/physiology , Sp1 Transcription Factor/physiology , Animals , Male , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , Promoter Regions, Genetic , Receptors, Purinergic P2X7/genetics , Transcription, Genetic/physiology
6.
Neurochem Res ; 42(7): 2033-2054, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28397067

ABSTRACT

Brief, non-harmful seizures (preconditioning) can temporarily protect the brain against prolonged, otherwise injurious seizures. Following focal-onset status epilepticus (SE) in preconditioned (tolerance) and sham-preconditioned (injury) mice, we screened for protein changes using a proteomic approach and identified several putative candidates of epileptic tolerance. Among SE-induced changes to both proteomic screens, proteins clustered in key regulatory pathways, including protein trafficking and cytoskeletal regulation. Downregulation of one such protein, ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), was unique to injury and not evident in tolerance. UCHL1 inhibition decreased hippocampal ubiquitin, disrupted UPS function, interfered with seizure termination and exacerbated seizure-induced cell death. Though UCHL1 transcription was maintained after SE, we observed downregulation of the pro-translational antisense Uchl1 (AsUchl1) and confirmed that both AsUchl1 and rapamycin can increase UCHL1 expression in vivo. These data indicate that the post-transcriptional loss of UCHL1 following SE is deleterious to neuronal survival and may contribute to hyperexcitability, and are suggestive of a novel modality of rapamycin therapy.


Subject(s)
Hippocampus/injuries , Hippocampus/metabolism , Proteomics/methods , Status Epilepticus/metabolism , Ubiquitin Thiolesterase/antagonists & inhibitors , Ubiquitin Thiolesterase/metabolism , Animals , Cell Survival/drug effects , Cell Survival/physiology , Hippocampus/drug effects , Indoles/administration & dosage , Injections, Intraventricular , Male , Mice , Mice, Inbred C57BL , Neurons/drug effects , Neurons/metabolism , Oximes/administration & dosage , Random Allocation , Status Epilepticus/drug therapy , Status Epilepticus/genetics , Ubiquitin Thiolesterase/genetics
7.
Neurobiol Dis ; 83: 100-14, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26341542

ABSTRACT

Seizures are common during the neonatal period, often due to hypoxic-ischemic encephalopathy and may contribute to acute brain injury and the subsequent development of cognitive deficits and childhood epilepsy. Here we explored short- and long-term consequences of neonatal hypoxia-induced seizures in 7 day old C57BL/6J mice. Seizure activity, molecular markers of hypoxia and histological injury were investigated acutely after hypoxia and response to chemoconvulsants and animal behaviour was explored at adulthood. Hypoxia was induced by exposing pups to 5% oxygen for 15 min (global hypoxia). Electrographically defined seizures with behavioral correlates occurred in 95% of these animals and seizures persisted for many minutes after restitution of normoxia. There was minimal morbidity or mortality. Pre- or post-hypoxia injection of phenobarbital (50mg/kg) had limited efficacy at suppressing seizures. The hippocampus from neonatal hypoxia-seizure mice displayed increased expression of vascular endothelial growth factor and the immediate early gene c-fos, minimal histological evidence of cell injury and activation of caspase-3 in scattered neurons. Behavioral analysis of mice five weeks after hypoxia-induced seizures detected novel anxiety-related and other behaviors, while performance in a spatial memory test was similar to controls. Seizure threshold tests with kainic acid at six weeks revealed that mice previously subject to neonatal hypoxia-induced seizures developed earlier, more frequent and longer-duration seizures. This study defines a set of electro-clinical, molecular, pharmacological and behavioral consequences of hypoxia-induced seizures that indicate short- and long-term deleterious outcomes and may be a useful model to investigate the pathophysiology and treatment of neonatal seizures in humans.


Subject(s)
Anxiety/etiology , Cerebral Cortex/physiopathology , Hippocampus/metabolism , Hypoxia/complications , Seizures/etiology , Seizures/physiopathology , Animals , Animals, Newborn , Anticonvulsants/administration & dosage , Anxiety/physiopathology , Behavior, Animal/physiology , Cerebral Cortex/drug effects , Cerebral Cortex/pathology , Electroencephalography , Female , Hippocampus/pathology , Hypoxia/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Male , Mice , Mice, Inbred C57BL , Motor Activity , Neurons/metabolism , Neurons/pathology , Phenobarbital/administration & dosage , Seizures/metabolism , Time Factors , Vascular Endothelial Growth Factor A/metabolism
8.
Brain ; 136(Pt 2): 577-92, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23361066

ABSTRACT

Hippocampal sclerosis is a frequent pathological finding in patients with temporal lobe epilepsy and can be caused by prolonged single or repeated brief seizures. Both DNA damage and endoplasmic reticulum stress have been implicated as underlying molecular mechanisms in seizure-induced brain injury. The CCAAT/enhancer-binding protein homologous protein (CHOP) is a transcriptional regulator induced downstream of DNA damage and endoplasmic reticulum stress, which can promote or inhibit apoptosis according to context. Recent work has proposed inhibition of CHOP as a suitable neuroprotective strategy. Here, we show that transcript and protein levels of CHOP increase in surviving subfields of the hippocampus after prolonged seizures (status epilepticus) in mouse models. CHOP was also elevated in the hippocampus from epileptic mice and patients with pharmacoresistant epilepsy. The hippocampus of CHOP-deficient mice was much more vulnerable to damage in mouse models of status epilepticus. Moreover, compared with wild-type animals, CHOP-deficient mice subject to status epilepticus developed more spontaneous seizures, displayed protracted hippocampal neurodegeneration and a deficit in a hippocampus-dependent object-place recognition task. The absence of CHOP was associated with a supra-maximal induction of p53 after status epilepticus, and inhibition of p53 abolished the cell death-promoting consequences of CHOP deficiency. The protective effect of CHOP could be partly explained by activating transcription of murine double minute 2 that targets p53 for degradation. These data demonstrate that CHOP is required for neuronal survival after seizures and caution against inhibition of CHOP as a neuroprotective strategy where excitotoxicity is an underlying pathomechanism.


Subject(s)
Neurons/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Seizures/metabolism , Transcription Factor CHOP/physiology , Tumor Suppressor Protein p53/metabolism , Animals , Cell Survival/physiology , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurons/pathology , Proto-Oncogene Proteins c-mdm2/physiology , Seizures/genetics , Seizures/pathology , Tumor Suppressor Protein p53/physiology
9.
Neuropharmacology ; 253: 109968, 2024 Aug 01.
Article in English | MEDLINE | ID: mdl-38692453

ABSTRACT

Microglia are described as the immune cells of the brain, their immune properties have been extensively studied since first described, however, their neural functions have only been explored over the last decade. Microglia have an important role in maintaining homeostasis in the central nervous system by surveying their surroundings to detect pathogens or damage cells. While these are the classical functions described for microglia, more recently their neural functions have been defined; they are critical to the maturation of neurons during embryonic and postnatal development, phagocytic microglia remove excess synapses during development, a process called synaptic pruning, which is important to overall neural maturation. Furthermore, microglia can respond to neuronal activity and, together with astrocytes, can regulate neural activity, contributing to the equilibrium between excitation and inhibition through a feedback loop. Hypoxia at birth is a serious neurological condition that disrupts normal brain function resulting in seizures and epilepsy later in life. Evidence has shown that microglia may contribute to this hyperexcitability after neonatal hypoxia. This review will summarize the existing data on the role of microglia in the pathogenesis of neonatal hypoxia and the plausible mechanisms that contribute to the development of hyperexcitability after hypoxia in neonates. This article is part of the Special Issue on "Microglia".


Subject(s)
Epilepsy , Microglia , Microglia/physiology , Microglia/pathology , Humans , Animals , Epilepsy/physiopathology , Epilepsy/pathology , Infant, Newborn , Hypoxia/physiopathology , Brain/pathology , Brain/physiopathology
10.
J Neurosci ; 32(5): 1577-88, 2012 Feb 01.
Article in English | MEDLINE | ID: mdl-22302800

ABSTRACT

Prolonged seizures (status epilepticus) produce pathophysiological changes in the hippocampus that are associated with large-scale, wide-ranging changes in gene expression. Epileptic tolerance is an endogenous program of cell protection that can be activated in the brain by previous exposure to a non-harmful seizure episode before status epilepticus. A major transcriptional feature of tolerance is gene downregulation. Here, through methylation analysis of 34,143 discrete loci representing all annotated CpG islands and promoter regions in the mouse genome, we report the genome-wide DNA methylation changes in the hippocampus after status epilepticus and epileptic tolerance in adult mice. A total of 321 genes showed altered DNA methylation after status epilepticus alone or status epilepticus that followed seizure preconditioning, with >90% of the promoters of these genes undergoing hypomethylation. These profiles included genes not previously associated with epilepsy, such as the polycomb gene Phc2. Differential methylation events generally occurred throughout the genome without bias for a particular chromosomal region, with the exception of a small region of chromosome 4, which was significantly overrepresented with genes hypomethylated after status epilepticus. Surprisingly, only few genes displayed differential hypermethylation in epileptic tolerance. Nevertheless, gene ontology analysis emphasized the majority of differential methylation events between the groups occurred in genes associated with nuclear functions, such as DNA binding and transcriptional regulation. The present study reports select, genome-wide DNA methylation changes after status epilepticus and in epileptic tolerance, which may contribute to regulating the gene expression environment of the seizure-damaged hippocampus.


Subject(s)
CA3 Region, Hippocampal/metabolism , DNA Methylation/genetics , Status Epilepticus/genetics , Status Epilepticus/metabolism , Animals , Down-Regulation/genetics , Genome-Wide Association Study/methods , Male , Mice , Mice, Inbred C57BL , Status Epilepticus/prevention & control
11.
J Neurochem ; 124(6): 749-56, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23278239

ABSTRACT

FOXO3a is member of the Forkhead box class O transcription factors, which functions in diverse pathways to regulate cellular metabolism, differentiation, and apoptosis. FOXO3a shuttles between the cytoplasm and nucleus and may be activated in neurons by stressors, including seizures. A subset of nuclear transcription factors may localize to mitochondria, but whether FOXO3a is present within brain mitochondria is unknown. Here, we report that purified mitochondrial fractions from rat, mouse, and human hippocampus, as well as HT22 hippocampal cells, contain FOXO3a protein. Immunogold electron microscopy supported the presence of FOXO3a within brain mitochondria, and chromatin immunoprecipitation analysis suggested FOXO3a was associated with mitochondrial DNA. Over-expression of a mitochondrially targeted FOXO3a fusion protein in HT22 cells, but not primary hippocampal neurons, conferred superior protection against glutamate toxicity than FOXO3a alone. Mitochondrial FOXO3a levels were reduced in the damaged region of the mouse hippocampus after status epilepticus, while mitochondrial fractions from the hippocampus of patients with temporal lobe epilepsy displayed higher levels of FOXO3a than controls. These results support mitochondria as a site of FOXO3a localization, which may contribute to the overall physiological and pathophysiological functions of this transcription factor.


Subject(s)
Forkhead Transcription Factors/metabolism , Hippocampus/chemistry , Mitochondria/chemistry , Animals , Brain/metabolism , Cell Line , Cell Survival/physiology , Forkhead Box Protein O3 , Hippocampus/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , Rats , Rats, Sprague-Dawley
12.
Methods Mol Biol ; 2595: 65-73, 2023.
Article in English | MEDLINE | ID: mdl-36441454

ABSTRACT

MicroRNAs are small molecules of non-coding RNAs involved in the regulation of mRNA expression, generally through inhibition of translation. Major efforts have been made to understand their role in health and disease, and more recently, microRNAs have been extensively studied as potential disease biomarkers. While the profiling and analysis of microRNAs from large quantities of biofluids are well established, difficulties still remain in the use of small volume of samples for this purpose. These include tissue samples for neurodevelopmental conditions, which may require the analysis of specific areas of the brain, e.g., hippocampus, or serum or plasma samples with a volume of fewer than 100 microliters. This chapter will give an overview of the preparation, profiling, and analysis of microRNAs from brain tissue with a starting mass of fewer than 100 micrograms.


Subject(s)
MicroRNAs , Neurodevelopmental Disorders , Humans , Brain , Hippocampus , MicroRNAs/genetics , Real-Time Polymerase Chain Reaction
13.
Methods Mol Biol ; 2595: 93-100, 2023.
Article in English | MEDLINE | ID: mdl-36441456

ABSTRACT

MicroRNAs are key posttranscriptional regulators of protein levels in cells. The brain is particularly enriched in microRNAs, and important roles have been demonstrated for these noncoding RNAs in various neurological disorders. To this end, visualization of microRNAs in specific cell types and subcellular compartments within tissue sections provides researchers with essential insights that support understanding of the cell and molecular mechanisms of microRNAs in brain diseases. In this chapter we describe an in situ hybridization protocol for the detection of microRNAs in mouse brain sections, which provides cellular resolution of the expression of microRNAs in the brain.


Subject(s)
Brain Diseases , MicroRNAs , Animals , Mice , Humans , MicroRNAs/genetics , In Situ Hybridization , Brain , Research Personnel
14.
Br J Pharmacol ; 180(13): 1710-1729, 2023 07.
Article in English | MEDLINE | ID: mdl-36637008

ABSTRACT

BACKGROUND AND PURPOSE: Neonatal seizures represent a clinical emergency. However, current anti-seizure medications fail to resolve seizures in ~50% of infants. The P2X7 receptor (P2X7R) is an important driver of inflammation, and evidence suggests that P2X7R contributes to seizures and epilepsy in adults. However, no genetic proof has yet been provided to determine what contribution P2X7R makes to neonatal seizures, its effects on inflammatory signalling during neonatal seizures, and the therapeutic potential of P2X7R-based treatments on long-lasting brain excitability. EXPERIMENTAL APPROACH: Neonatal seizures were induced by global hypoxia in 7-day-old mouse pups (P7). The role of P2X7Rs during seizures was analysed in P2X7R-overexpressing and knockout mice. Treatment of wild-type mice after hypoxia with the P2X7R antagonist JNJ-47965567 was used to determine the effects of the P2X7R on long-lasting brain hyperexcitability. Cell type-specific P2X7R expression was analysed in P2X7R-EGFP reporter mice. RNA sequencing was used to monitor P2X7R-dependent hippocampal downstream signalling. KEY RESULTS: P2X7R deletion reduced seizure severity, whereas P2X7R overexpression exacerbated seizure severity and reduced responsiveness to anti-seizure medication. P2X7R deficiency led to an anti-inflammatory phenotype in microglia, and treatment of mice with a P2X7R antagonist reduced long-lasting brain hyperexcitability. RNA sequencing identified several pathways altered in P2X7R knockout mice after neonatal hypoxia, including a down-regulation of genes implicated in inflammation and glutamatergic signalling. CONCLUSION AND IMPLICATIONS: Treatments based on targeting the P2X7R may represent a novel therapeutic strategy for neonatal seizures with P2X7Rs contributing to the generation of neonatal seizures, driving inflammatory processes and long-term hyperexcitability states.


Subject(s)
Receptors, Purinergic P2X7 , Seizures , Animals , Mice , Animals, Newborn , Brain/metabolism , Epilepsy/chemically induced , Epilepsy/metabolism , Hypoxia/complications , Inflammation/drug therapy , Mice, Knockout , Receptors, Purinergic P2X7/genetics , Receptors, Purinergic P2X7/metabolism , Seizures/metabolism
15.
Am J Pathol ; 179(5): 2519-32, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21945804

ABSTRACT

When an otherwise harmful insult to the brain is preceded by a brief, noninjurious stimulus, the brain becomes tolerant, and the resulting damage is reduced. Epileptic tolerance develops when brief seizures precede an episode of prolonged seizures (status epilepticus). MicroRNAs (miRNAs) are small, noncoding RNAs that function as post-transcriptional regulators of gene expression. We investigated how prior seizure preconditioning affects the miRNA response to status epilepticus evoked by intra-amygdalar kainic acid in mice. The miRNA was extracted from the ipsilateral CA3 subfield 24 hours after focal-onset status epilepticus in animals that had previously received either seizure preconditioning (tolerance) or no preconditioning (injury), and mature miRNA levels were measured using TaqMan low-density arrays. Expression of 21 miRNAs was increased, relative to control, after status epilepticus alone, and expression of 12 miRNAs was decreased. Increased miR-132 levels were matched with increased binding to Argonaute-2, a constituent of the RNA-induced silencing complex. In tolerant animals, expression responses of >40% of the injury-group-detected miRNAs differed, being either unchanged relative to control or down-regulated, and this included miR-132. In vivo microinjection of locked nucleic acid-modified oligonucleotides (antagomirs) against miR-132 depleted hippocampal miR-132 levels and reduced seizure-induced neuronal death. Thus, our data strongly suggest that miRNAs are important regulators of seizure-induced neuronal death.


Subject(s)
Hippocampus/metabolism , MicroRNAs/metabolism , Status Epilepticus/prevention & control , Amygdala/metabolism , Animals , Antagomirs , Argonaute Proteins/metabolism , Down-Regulation , Excitatory Amino Acid Agonists/toxicity , Injections, Intralesional , Kainic Acid/toxicity , Male , Mice , Mice, Inbred C57BL , MicroRNAs/antagonists & inhibitors , MicroRNAs/pharmacology , Oligonucleotides/pharmacology , Up-Regulation
16.
Biomedicines ; 10(11)2022 Oct 28.
Article in English | MEDLINE | ID: mdl-36359259

ABSTRACT

Brain development occurs until adulthood, with time-sensitive processes happening during embryo development, childhood, and puberty. During early life and childhood, dynamic changes in the brain are critical for physiological brain maturation, and these changes are tightly regulated by the expression of specific regulatory genetic elements. Early life insults, such as hypoxia, can alter the course of brain maturation, resulting in lifelong neurodevelopmental conditions. MicroRNAs are small non-coding RNAs, which regulate and coordinate gene expression. It is estimated that one single microRNA can regulate the expression of hundreds of protein-coding genes.. Uncovering the miRNome and microRNA-regulated transcriptomes may help to understand the patterns of genes regulating brain maturation, and their contribution to neurodevelopmental pathologies following hypoxia at Postnatal day 7. Here, using a PCR-based platform, we analyzed the microRNA profile postnatally in the hippocampus of control mice at postnatal day 8, 14, and 42 and after hypoxia at postnatal day 7, to elucidate the set of microRNAs which may be key for postnatal hippocampus maturation. We observed that microRNAs can be divided in four groups based on their temporal expression. Further after an early life insult, hypoxia at P7, 15 microRNAs showed a misregulation over time, including Let7a. We speculated that the transcriptional regulator c-myc is a contributor to this process. In conclusion, here, we observed that microRNAs are regulated postnatally in the hippocampus and alteration of their expression after hypoxia at birth may be regulated by the transcriptional regulator c-myc.

17.
FASEB J ; 24(3): 853-61, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19890018

ABSTRACT

The functional significance of neuronal death for pathogenesis of epilepsy and the underlying molecular mechanisms thereof remain incompletely understood. The p53 transcription factor has been implicated in seizure damage, but its target genes and the influence of cell death under its control on epilepsy development are unknown. In the present study, we report that status epilepticus (SE) triggered by intra-amygdala kainic acid in mice causes rapid p53 accumulation and subsequent hippocampal damage. Expression of p53-up-regulated mediator of apoptosis (Puma), a proapoptotic Bcl-2 homology domain 3-only protein under p53 control, was increased within a few hours of SE. Induction of Puma was blocked by pharmacologic inhibition of p53, and hippocampal damage was also reduced. Puma induction was also blocked in p53-deficient mice subject to SE. Compared to Puma-expressing mice, Puma-deficient mice had significantly smaller hippocampal lesions after SE. Long-term, continuous telemetric EEG monitoring revealed a approximately 60% reduction in the frequency of epileptic seizures in the Puma-deficient mice compared to Puma-expressing mice. These are the first data showing genetic deletion of a proapoptotic protein acting acutely to influence neuronal death subsequently alters the phenotype of epilepsy in the long-term, supporting the concept that apoptotic pathway activation is a trigger of epileptogenesis.-Engel, T., Murphy, B. M., Hatazaki, S., Jimenez-Mateos, E. M., Concannon, C. G., Woods, I., Prehn, J. H. M., Henshall, D. C. Reduced hippocampal damage and epileptic seizures after status epilepticus in mice lacking proapoptotic Puma.


Subject(s)
Apoptosis Regulatory Proteins/physiology , Epilepsy/pathology , Hippocampus/pathology , Status Epilepticus/physiopathology , Tumor Suppressor Proteins/physiology , Animals , Apoptosis Regulatory Proteins/genetics , Benzothiazoles/pharmacology , Blotting, Western , Epilepsy/metabolism , Genotype , Hippocampus/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Polymerase Chain Reaction , Signal Transduction/genetics , Signal Transduction/physiology , Status Epilepticus/metabolism , Toluene/analogs & derivatives , Toluene/pharmacology , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/physiology , Tumor Suppressor Proteins/genetics
18.
Front Mol Neurosci ; 14: 732199, 2021.
Article in English | MEDLINE | ID: mdl-34566578

ABSTRACT

Background: Evidence suggests that earlier diagnosis and initiation of treatment immediately after birth is critical for improved neurodevelopmental outcomes following neonatal encephalopathy (NE). Current diagnostic tests are, however, mainly restricted to clinical diagnosis with no molecular tests available. Purines including adenosine are released during brain injury such as hypoxia and are also present in biofluids. Whether blood purine changes can be used to diagnose NE has not been investigated to date. Methods: Blood purines were measured in a mouse model of neonatal hypoxia and infants with NE using a novel point-of-care diagnostic technology (SMARTChip) based on the summated electrochemical detection of adenosine and adenosine metabolites in the blood. Results: Blood purine concentrations were ∼2-3-fold elevated following hypoxia in mice [2.77 ± 0.48 µM (Control) vs. 7.57 ± 1.41 µM (post-hypoxia), p = 0.029]. Data in infants with NE had a 2-3-fold elevation when compared to healthy controls [1.63 ± 0.47 µM (Control, N = 5) vs. 4.87 ± 0.92 µM (NE, N = 21), p = 0.0155]. ROC curve analysis demonstrates a high sensitivity (81%) and specificity (80%) for our approach to identify infants with NE. Moreover, blood purine concentrations were higher in infants with NE and seizures [8.13 ± 3.23 µM (with seizures, N = 5) vs. 3.86 ± 0.56 µM (without seizures, N = 16), p = 0.044]. Conclusion: Our data provides the proof-of-concept that measurement of blood purine concentrations via SMARTChip technology may offer a low-volume bedside test to support a rapid diagnosis of NE.

19.
Cells ; 9(12)2020 12 08.
Article in English | MEDLINE | ID: mdl-33302543

ABSTRACT

Perinatal brain injury or neonatal encephalopathy (NE) is a state of disturbed neurological function in neonates, caused by a number of different aetiologies. The most prominent cause of NE is hypoxic ischaemic encephalopathy, which can often induce seizures. NE and neonatal seizures are both associated with poor neurological outcomes, resulting in conditions such as cerebral palsy, epilepsy, autism, schizophrenia and intellectual disability. The current treatment strategies for NE and neonatal seizures have suboptimal success in effectively treating neonates. Therapeutic hypothermia is currently used to treat NE and has been shown to reduce morbidity and has neuroprotective effects. However, its success varies between developed and developing countries, most likely as a result of lack of sufficient resources. The first-line pharmacological treatment for NE is phenobarbital, followed by phenytoin, fosphenytoin and lidocaine as second-line treatments. While these drugs are mostly effective at halting seizure activity, they are associated with long-lasting adverse neurological effects on development. Over the last years, inflammation has been recognized as a trigger of NE and seizures, and evidence has indicated that this inflammation plays a role in the long-term neuronal damage experienced by survivors. Researchers are therefore investigating the possible neuroprotective effects that could be achieved by using anti-inflammatory drugs in the treatment of NE. In this review we will highlight the current knowledge of the inflammatory response after perinatal brain injury and what we can learn from animal models.


Subject(s)
Brain Injuries/pathology , Inflammation/pathology , Animals , Brain Injuries/complications , Brain Injuries/metabolism , Brain Injuries/therapy , Cytokines/metabolism , Disease Models, Animal , Female , Inflammation/complications , Inflammation/metabolism , Microglia/cytology , Microglia/metabolism , Pregnancy , Receptors, Purinergic/metabolism , Toll-Like Receptors/metabolism
20.
Front Mol Neurosci ; 13: 127, 2020.
Article in English | MEDLINE | ID: mdl-32982684

ABSTRACT

The ionotropic ATP-gated P2X7 receptor is an important contributor to inflammatory signaling cascades via the release of Interleukin-1ß, as well as having roles in cell death, neuronal plasticity and the release of neurotransmitters. Accordingly, there is interest in targeting the P2X7 receptor for the treatment of epilepsy. However, the signaling pathways downstream of P2X7 receptor activation remain incompletely understood. Notably, recent studies showed that P2X7 receptor expression is controlled, in part, by microRNAs (miRNAs). Here, we explored P2X7 receptor-dependent microRNA expression by comparing microRNA expression profiles of wild-type (wt) and P2X7 receptor knockout mice before and after status epilepticus. Genome-wide microRNA profiling was performed using hippocampi from wt and P2X7 receptor knockout mice following status epilepticus induced by intra-amygdala kainic acid. This revealed that the genetic deletion of the P2X7 receptor results in distinct patterns of microRNA expression. Specifically, we found that in vehicle-injected control mice, the lack of the P2X7 receptor resulted in the up-regulation of 50 microRNAs and down-regulation of 35 microRNAs. Post-status epilepticus, P2X7 receptor deficiency led to the up-regulation of 44 microRNAs while 13 microRNAs were down-regulated. Moreover, there was only limited overlap among identified P2X7 receptor-dependent microRNAs between control conditions and post-status epilepticus, suggesting that the P2X7 receptor regulates the expression of different microRNAs during normal physiology and pathology. Bioinformatic analysis revealed that genes targeted by P2X7 receptor-dependent microRNAs were particularly overrepresented in pathways involved in intracellular signaling, inflammation, and cell death; processes that have been repeatedly associated with P2X7 receptor activation. Moreover, whereas genes involved in signaling pathways and inflammation were common among up- and down-regulated P2X7 receptor-dependent microRNAs during physiological and pathological conditions, genes associated with cell death seemed to be restricted to up-regulated microRNAs during both physiological conditions and post-status epilepticus. Taken together, our results demonstrate that the P2X7 receptor impacts on the expression profile of microRNAs in the brain, thereby possibly contributing to both the maintenance of normal cellular homeostasis and pathological processes.

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