MicroRNA-335-5p suppresses voltage-gated sodium channel expression and may be a target for seizure control.

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.

Impact of JQ1 treatment on seizures, hippocampal gene expression, and gliosis in a mouse model of temporal lobe epilepsy.

Epilepsy is a neurological disease characterised by recurrent seizures with complex aetiology. Temporal lobe epilepsy, the most common form in adults, can be acquired following brain insults including trauma, stroke, infection or sustained status epilepticus. The mechanisms that give rise to the formation and maintenance of hyperexcitable networks following acquired insults remain unknown, yet an extensive body of literature points towards persistent gene and epigenomic dysregulation as a potential mediator of this dysfunction. While much is known about the function of specific classes of epigenetic regulators (writers and erasers) in epilepsy, much less is known about the enzymes, which read the epigenome and modulate gene expression accordingly. Here, we explore the potential role for the epigenetic reader bromodomain and extra-terminal domain (BET) proteins in epilepsy. Using the intra-amygdala kainic acid model of temporal lobe epilepsy, we initially identified widespread dysregulation of important epigenetic regulators including EZH2 and REST as well as altered BRD4 expression in chronically epileptic mice. BRD4 activity was also notably affected by epilepsy-provoking insults as seen by elevated binding to and transcriptional regulation of the immediate early gene Fos. Despite influencing early aspects of epileptogenesis, blocking BET protein activity with JQ1 had no overt effects on epilepsy development in mice but did alter glial reactivity and influence gene expression patterns, promoting various neurotransmitter signalling mechanisms and inflammatory pathways in the hippocampus. Together, these results confirm that epigenetic reader activity is affected by epilepsy-provoking brain insults and that BET activity may exert cell-specific actions on inflammation in epilepsy.

Opposing effects of the purinergic P2X7 receptor on seizures in neurons and microglia in male mice.

BACKGROUND: The purinergic ATP-gated P2X7 receptor (P2X7R) is increasingly recognized to contribute to pathological neuroinflammation and brain hyperexcitability. P2X7R expression has been shown to be increased in the brain, including both microglia and neurons, in experimental models of epilepsy and patients. To date, the cell type-specific downstream effects of P2X7Rs during seizures remain, however, incompletely understood. METHODS: Effects of P2X7R signaling on seizures and epilepsy were analyzed in induced seizure models using male mice including the kainic acid model of status epilepticus and pentylenetetrazole model and in male and female mice in a genetic model of Dravet syndrome. RNA sequencing was used to analyze P2X7R downstream signaling during seizures. To investigate the cell type-specific role of the P2X7R during seizures and epilepsy, we generated mice lacking exon 2 of the P2rx7 gene in either microglia (P2rx7:Cx3cr1-Cre) or neurons (P2rx7:Thy-1-Cre). To investigate the protective potential of overexpressing P2X7R in GABAergic interneurons, P2X7Rs were overexpressed using adeno-associated virus transduction under the mDlx promoter. RESULTS: RNA sequencing of hippocampal tissue from wild-type and P2X7R knock-out mice identified both glial and neuronal genes, in particular genes involved in GABAergic signaling, under the control of the P2X7R following seizures. Mice with deleted P2rx7 in microglia displayed less severe acute seizures and developed a milder form of epilepsy, and microglia displayed an anti-inflammatory molecular profile. In contrast, mice lacking P2rx7 in neurons showed a more severe seizure phenotype when compared to epileptic wild-type mice. Analysis of single-cell expression data revealed that human P2RX7 expression is elevated in the hippocampus of patients with temporal lobe epilepsy in excitatory and inhibitory neurons. Functional studies determined that GABAergic interneurons display increased responses to P2X7R activation in experimental epilepsy. Finally, we show that viral transduction of P2X7R in GABAergic interneurons protects against evoked and spontaneous seizures in experimental temporal lobe epilepsy and in mice lacking Scn1a, a model of Dravet syndrome. CONCLUSIONS: Our results suggest a dual and opposing action of P2X7R in epilepsy and suggest P2X7R overexpression in GABAergic interneurons as a novel therapeutic strategy for acquired and, possibly, genetic forms of epilepsy.

WONOEP appraisal: The role of glial cells in focal malformations associated with early onset epilepsies.

Epilepsy represents a common neurological disorder in patients with developmental brain lesions, particularly in association with malformations of cortical development and low-grade glioneuronal tumors. In these diseases, genetic and molecular alterations in neurons are increasingly discovered that can trigger abnormalities in the neuronal network, leading to higher neuronal excitability levels. However, the mechanisms underlying epilepsy cannot rely solely on assessing the neuronal component. Growing evidence has revealed the high degree of complexity underlying epileptogenic processes, in which glial cells emerge as potential modulators of neuronal activity. Understanding the role of glial cells in developmental brain lesions such as malformations of cortical development and low-grade glioneuronal tumors is crucial due to the high degree of pharmacoresistance characteristic of these lesions. This has prompted research to investigate the role of glial and immune cells in epileptiform activity to find new therapeutic targets that could be used as combinatorial drug therapy. In a special session of the XVI Workshop of the Neurobiology of Epilepsy (WONOEP, Talloires, France, July 2022) organized by the Neurobiology Commission of the International League Against Epilepsy, we discussed the evidence exploring the genetic and molecular mechanisms of glial cells and immune response and their implications in the pathogenesis of neurodevelopmental pathologies associated with early life epilepsies.

WONOEP 2022: Neurotechnology for the diagnosis of epilepsy.

Epilepsy's myriad causes and clinical presentations ensure that accurate diagnoses and targeted treatments remain a challenge. Advanced neurotechnologies are needed to better characterize individual patients across multiple modalities and analytical techniques. At the XVIth Workshop on Neurobiology of Epilepsy: Early Onset Epilepsies: Neurobiology and Novel Therapeutic Strategies (WONOEP 2022), the session on "advanced tools" highlighted a range of approaches, from molecular phenotyping of genetic epilepsy models and resected tissue samples to imaging-guided localization of epileptogenic tissue for surgical resection of focal malformations. These tools integrate cutting edge research, clinical data acquisition, and advanced computational methods to leverage the rich information contained within increasingly large datasets. A number of common challenges and opportunities emerged, including the need for multidisciplinary collaboration, multimodal integration, potential ethical challenges, and the multistage path to clinical translation. Despite these challenges, advanced epilepsy neurotechnologies offer the potential to improve our understanding of the underlying causes of epilepsy and our capacity to provide patient-specific treatment.

WONOEP appraisal: Genetic insights into early onset epilepsies.

Early onset epilepsies occur in newborns and infants, and to date, genetic aberrations and variants have been identified in approximately one quarter of all patients. With technological sequencing advances and ongoing research, the genetic diagnostic yield for specific seizure disorders and epilepsies is expected to increase. Genetic variants associated with epilepsy include chromosomal abnormalities and rearrangements of various sizes as well as single gene variants. Among these variants, a distinction can be made between germline and somatic, with the latter being increasingly identified in epilepsies with focal cortical malformations in recent years. The identification of the underlying genetic mechanisms of epilepsy syndromes not only revolutionizes the diagnostic schemes but also leads to a better understanding of the diseases and their interrelationships, ultimately providing new opportunities for therapeutic targeting. At the XVI Workshop on Neurobiology of Epilepsy (WONOEP 2022, Talloires, France, July 2022), various etiologies, research models, and mechanisms of genetic early onset epilepsies were presented and discussed.

WONOEP appraisal: Modeling early onset epilepsies.

Epilepsy has a peak incidence during the neonatal to early childhood period. These early onset epilepsies may be severe conditions frequently associated with comorbidities such as developmental deficits and intellectual disability and, in a significant percentage of patients, may be medication-resistant. The use of adult rodent models in the exploration of mechanisms and treatments for early life epilepsies is challenging, as it ignores significant age-specific developmental differences. More recently, models developed in immature animals, such as rodent pups, or in three-dimensional organoids may more closely model aspects of the immature brain and could result in more translatable findings. Although models are not perfect, they may offer a more controlled screening platform in studies of mechanisms and treatments, which cannot be done in pediatric patient cohorts. On the other hand, more simplified models with higher throughput capacities are required to deal with the large number of epilepsy candidate genes and the need for new treatment options. Therefore, a combination of different modeling approaches will be beneficial in addressing the unmet needs of pediatric epilepsy patients. In this review, we summarize the discussions on this topic that occurred during the XVI Workshop on Neurobiology of Epilepsy, organized in 2022 by the Neurobiology Commission of the International League Against Epilepsy. We provide an overview of selected models of early onset epilepsies, discussing their advantages and disadvantages. Heterologous expression models provide initial functional insights, and zebrafish, rodent models, and brain organoids present increasingly complex platforms for modeling and validating epilepsy-related phenomena. Together, these models offer valuable insights into early onset epilepsies and accelerate hypothesis generation and therapy discovery.

Somatic variants as a cause of drug-resistant epilepsy including mesial temporal lobe epilepsy with hippocampal sclerosis.

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.

Increased uptake of the P2X7 receptor radiotracer 18 F-JNJ-64413739 in the brain and peripheral organs according to the severity of status epilepticus in male mice.

OBJECTIVE: The P2X7 receptor (P2X7R) is an important contributor to neuroinflammation, responding to extracellularly released adenosine triphosphate. Expression of the P2X7R is increased in the brain in experimental and human epilepsy, and genetic or pharmacologic targeting of the receptor can reduce seizure frequency and severity in preclinical models. Experimentally induced seizures also increase levels of the P2X7R in blood. Here, we tested 18 F-JNJ-64413739, a positron emission tomography (PET) P2X7R antagonist, as a potential noninvasive biomarker of seizure-damage and epileptogenesis. METHODS: Status epilepticus was induced via an intra-amygdala microinjection of kainic acid. Static PET studies (30 min duration, initiated 30 min after tracer administration) were conducted 48 h after status epilepticus via an intravenous injection of 18 F-JNJ-64413739. PET images were coregistered with a brain magnetic resonance imaging atlas, tracer uptake was determined in the different brain regions and peripheral organs, and values were correlated to seizure severity during status epilepticus. 18 F-JNJ-64413739 was also applied to ex vivo human brain slices obtained following surgical resection for intractable temporal lobe epilepsy. RESULTS: P2X7R radiotracer uptake correlated strongly with seizure severity during status epilepticus in brain structures including the cerebellum and ipsi- and contralateral cortex, hippocampus, striatum, and thalamus. In addition, a correlation between radiotracer uptake and seizure severity was also evident in peripheral organs such as the heart and the liver. Finally, P2X7R radiotracer uptake was found elevated in brain sections from patients with temporal lobe epilepsy when compared to control. SIGNIFICANCE: Taken together, our data suggest that P2X7R-based PET imaging may help to identify seizure-induced neuropathology and temporal lobe epilepsy patients with increased P2X7R levels possibly benefitting from P2X7R-based treatments.

CPEB4-CLOCK crosstalk during temporal lobe epilepsy.

OBJECTIVE: Posttranscriptional mechanisms are increasingly recognized as important contributors to the formation of hyperexcitable networks in epilepsy. Messenger RNA (mRNA) polyadenylation is a key regulatory mechanism governing protein expression by enhancing mRNA stability and translation. Previous studies have shown large-scale changes in mRNA polyadenylation in the hippocampus of mice during epilepsy development. The cytoplasmic polyadenylation element-binding protein CPEB4 was found to drive epilepsy-induced poly(A) tail changes, and mice lacking CPEB4 develop a more severe seizure and epilepsy phenotype. The mechanisms controlling CPEB4 function and the downstream pathways that influence the recurrence of spontaneous seizures in epilepsy remain poorly understood. METHODS: Status epilepticus was induced in wild-type and CPEB4-deficient male mice via an intra-amygdala microinjection of kainic acid. CLOCK binding to the CPEB4 promoter was analyzed via chromatin immunoprecipitation assay and melatonin levels via high-performance liquid chromatography in plasma. RESULTS: Here, we show increased binding of CLOCK to recognition sites in the CPEB4 promoter region during status epilepticus in mice and increased Cpeb4 mRNA levels in N2A cells overexpressing CLOCK. Bioinformatic analysis of CPEB4-dependent genes undergoing changes in their poly(A) tail during epilepsy found that genes involved in the regulation of circadian rhythms are particularly enriched. Clock transcripts displayed a longer poly(A) tail length in the hippocampus of mice post-status epilepticus and during epilepsy. Moreover, CLOCK expression was increased in the hippocampus in mice post-status epilepticus and during epilepsy, and in resected hippocampus and cortex of patients with drug-resistant temporal lobe epilepsy. Furthermore, CPEB4 is required for CLOCK expression after status epilepticus, with lower levels in CPEB4-deficient compared to wild-type mice. Last, CPEB4-deficient mice showed altered circadian function, including altered melatonin blood levels and altered clustering of spontaneous seizures during the day. SIGNIFICANCE: Our results reveal a new positive transcriptional-translational feedback loop involving CPEB4 and CLOCK, which may contribute to the regulation of the sleep-wake cycle during epilepsy.

Functional expression of the ATP-gated P2X7 receptor in human iPSC-derived astrocytes.

Activation of the ATP-gated P2X7 receptor (P2X7R), implicated in numerous diseases of the brain, can trigger diverse responses such as the release of pro-inflammatory cytokines, modulation of neurotransmission, cell proliferation or cell death. However, despite the known species-specific differences in its pharmacological properties, to date, most functional studies on P2X7R responses have been analyzed in cells from rodents or immortalised cell lines. To assess the endogenous and functional expression of P2X7Rs in human astrocytes, we differentiated human-induced pluripotent stem cells (hiPSCs) into GFAP and S100 ß-expressing astrocytes. Immunostaining revealed prominent punctate P2X7R staining. P2X7R protein expression was also confirmed by Western blot. Importantly, stimulation with the potent non-selective P2X7R agonist 2',3'-O-(benzoyl-4-benzoyl)-adenosine 5'- triphosphate (BzATP) or endogenous agonist ATP induced robust calcium rises in hiPSC-derived astrocytes which were blocked by the selective P2X7R antagonists AFC-5128 or JNJ-47965567. Our findings provide evidence for the functional expression of P2X7Rs in hiPSC-derived astrocytes and support their in vitro utility in investigating the role of the P2X7R and drug screening in disorders of the central nervous system (CNS).

A systems approach delivers a functional microRNA catalog and expanded targets for seizure suppression in temporal lobe epilepsy.

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.

MicroRNA inhibition using antimiRs in acute human brain tissue sections.

Antisense inhibition of microRNAs is an emerging preclinical approach to pharmacoresistant epilepsy. A leading candidate is an "antimiR" targeting microRNA-134 (ant-134), but testing to date has used rodent models. Here, we develop an antimiR testing platform in human brain tissue sections. Brain specimens were obtained from patients undergoing resective surgery to treat pharmacoresistant epilepsy. Neocortical specimens were submerged in modified artificial cerebrospinal fluid (ACSF) and dissected for clinical neuropathological examination, and unused material was transferred for sectioning. Individual sections were incubated in oxygenated ACSF, containing either ant-134 or a nontargeting control antimiR, for 24 h at room temperature. RNA integrity was assessed using BioAnalyzer processing, and individual miRNA levels were measured using quantitative reverse transcriptase polymerase chain reaction. Specimens transported in ACSF could be used for neuropathological diagnosis and had good RNA integrity. Ant-134 mediated a dose-dependent knockdown of miR-134, with approximately 75% reduction of miR-134 at 1 µmol L-1 and 90% reduction at 3 µmol L-1 . These doses did not have off-target effects on expression of a selection of three other miRNAs. This is the first demonstration of ant-134 effects in live human brain tissues. The findings lend further support to the preclinical development of a therapy that targets miR-134 and offer a flexible platform for the preclinical testing of antimiRs, and other antisense oligonucleotide therapeutics, in human brain.

Systemic delivery of antagomirs during blood-brain barrier disruption is disease-modifying in experimental epilepsy.

Oligonucleotide therapies offer precision treatments for a variety of neurological diseases, including epilepsy, but their deployment is hampered by the blood-brain barrier (BBB). Previous studies showed that intracerebroventricular injection of an antisense oligonucleotide (antagomir) targeting microRNA-134 (Ant-134) reduced evoked and spontaneous seizures in animal models of epilepsy. In this study, we used assays of serum protein and tracer extravasation to determine that BBB disruption occurring after status epilepticus in mice was sufficient to permit passage of systemically injected Ant-134 into the brain parenchyma. Intraperitoneal and intravenous injection of Ant-134 reached the hippocampus and blocked seizure-induced upregulation of miR-134. A single intraperitoneal injection of Ant-134 at 2 h after status epilepticus in mice resulted in potent suppression of spontaneous recurrent seizures, reaching a 99.5% reduction during recordings at 3 months. The duration of spontaneous seizures, when they occurred, was also reduced in Ant-134-treated mice. In vivo knockdown of LIM kinase-1 (Limk-1) increased seizure frequency in Ant-134-treated mice, implicating de-repression of Limk-1 in the antagomir mechanism. These studies indicate that systemic delivery of Ant-134 reaches the brain and produces long-lasting seizure-suppressive effects after systemic injection in mice when timed with BBB disruption and may be a clinically viable approach for this and other disease-modifying microRNA therapies.

Epigenetic principles underlying epileptogenesis and epilepsy syndromes.

Epilepsy is a network disorder driven by fundamental changes in the function of the cells which compose these networks. Driving this aberrant cellular function are large scale changes in gene expression and gene expression regulation. Recent studies have revealed rapid and persistent changes in epigenetic control of gene expression as a critical regulator of the epileptic transcriptome. Epigenetic-mediated gene output regulates many aspects of cellular physiology including neuronal structure, neurotransmitter assembly and abundance, protein abundance of ion channels and other critical neuronal processes. Thus, understanding the contribution of epigenetic-mediated gene regulation could illuminate novel regulatory mechanisms which may form the basis of novel therapeutic approaches to treat epilepsy. In this review we discuss the effects of epileptogenic brain insults on epigenetic regulation of gene expression, recent efforts to target epigenetic processes to block epileptogenesis and the prospects of an epigenetic-based therapy for epilepsy, and finally we discuss technological advancements which have facilitated the interrogation of the epigenome.

A microRNA-129-5p/Rbfox crosstalk coordinates homeostatic downscaling of excitatory synapses.

Synaptic downscaling is a homeostatic mechanism that allows neurons to reduce firing rates during chronically elevated network activity. Although synaptic downscaling is important in neural circuit development and epilepsy, the underlying mechanisms are poorly described. We performed small RNA profiling in picrotoxin (PTX)-treated hippocampal neurons, a model of synaptic downscaling. Thereby, we identified eight microRNAs (miRNAs) that were increased in response to PTX, including miR-129-5p, whose inhibition blocked synaptic downscaling in vitro and reduced epileptic seizure severity in vivo Using transcriptome, proteome, and bioinformatic analysis, we identified the calcium pump Atp2b4 and doublecortin (Dcx) as miR-129-5p targets. Restoring Atp2b4 and Dcx expression was sufficient to prevent synaptic downscaling in PTX-treated neurons. Furthermore, we characterized a functional crosstalk between miR-129-5p and the RNA-binding protein (RBP) Rbfox1. In the absence of PTX, Rbfox1 promoted the expression of Atp2b4 and Dcx. Upon PTX treatment, Rbfox1 expression was downregulated by miR-129-5p, thereby allowing the repression of Atp2b4 and Dcx. We therefore identified a novel activity-dependent miRNA/RBP crosstalk during synaptic scaling, with potential implications for neural network homeostasis and epileptogenesis.

Elevated blood purine levels as a biomarker of seizures and epilepsy.

OBJECTIVE: There is a major unmet need for a molecular biomarker of seizures or epilepsy that lends itself to fast, affordable detection in an easy-to-use point-of-care device. Purines such as adenosine triphosphate and adenosine are potent neuromodulators released during excessive neuronal activity that are also present in biofluids. Their biomarker potential for seizures and epilepsy in peripheral blood has, however, not yet been investigated. The aim of the present study was to determine whether blood purine nucleoside measurements can serve as a biomarker for the recent occurrence of seizures and to support the diagnosis of epilepsy. METHODS: Blood purine concentrations were measured via a point-of-care diagnostic technology based on the summated electrochemical detection of adenosine and adenosine breakdown products (inosine, hypoxanthine, and xanthine; SMARTChip). Measurements of blood purine concentrations were carried out using samples from mice subjected to intra-amygdala kainic acid-induced status epilepticus and in video-electroencephalogram (EEG)-monitored adult patients with epilepsy. RESULTS: In mice, blood purine concentrations were rapidly increased approximately two- to threefold after status epilepticus (2.32 ± .40 µmol·L-1 [control] vs. 8.93 ± 1.03 µmol·L-1 [after status epilepticus]), and levels correlated with seizure burden and postseizure neurodegeneration in the hippocampus. Blood purine concentrations were also elevated in patients with video-EEG-diagnosed epilepsy (2.39 ± .34 µmol·L-1 [control, n = 13] vs. 4.35 ± .38 µmol·L-1 [epilepsy, n = 26]). SIGNIFICANCE: Our data provide proof of concept that the measurement of blood purine concentrations may offer a rapid, low-volume bedside test to support the diagnosis of seizures and epilepsy.

Polyadenylation of mRNA as a novel regulatory mechanism of gene expression in temporal lobe epilepsy.

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.

Precise Targeting of miRNA Sites Restores CFTR Activity in CF Bronchial Epithelial Cells.

MicroRNAs that are overexpressed in cystic fibrosis (CF) bronchial epithelial cells (BEC) negatively regulate CFTR and nullify the beneficial effects of CFTR modulators. We hypothesized that it is possible to reverse microRNA-mediated inhibition of CFTR using CFTR-specific target site blockers (TSBs) and to develop a drug-device combination inhalation therapy for CF. Lead microRNA expression was quantified in a series of human CF and non-CF samples and in vitro models. A panel of CFTR 3' untranslated region (UTR)-specific locked nucleic acid antisense oligonucleotide TSBs was assessed for their ability to increase CFTR expression. Their effects on CFTR activity alone or in combination with CFTR modulators were measured in CF BEC models. TSB encapsulation in poly-lactic-co-glycolic acid (PLGA) nanoparticles was assessed as a proof of principle of delivery into CF BECs. TSBs targeting the CFTR 3' UTR 298-305:miR-145-5p or 166-173:miR-223-3p sites increased CFTR expression and anion channel activity and enhanced the effects of ivacaftor/lumacaftor or ivacaftor/tezacaftor in CF BECs. Biocompatible PLGA-TSB nanoparticles promoted CFTR expression in primary BECs and retained desirable biophysical characteristics following nebulization. Alone or in combination with CFTR modulators, aerosolized CFTR-targeting TSBs encapsulated in PLGA nanoparticles could represent a promising drug-device combination therapy for the treatment for CFTR dysfunction in the lung.

CHD2-Related CNS Pathologies.

Epileptic encephalopathies (EE) are severe epilepsy syndromes characterized by multiple seizure types, developmental delay and even regression. This class of disorders are increasingly being identified as resulting from de novo genetic mutations including many identified mutations in the family of chromodomain helicase DNA binding (CHD) proteins. In particular, several de novo pathogenic mutations have been identified in the gene encoding chromodomain helicase DNA binding protein 2 (CHD2), a member of the sucrose nonfermenting (SNF-2) protein family of epigenetic regulators. These mutations in the CHD2 gene are causative of early onset epileptic encephalopathy, abnormal brain function, and intellectual disability. Our understanding of the mechanisms by which modification or loss of CHD2 cause this condition remains poorly understood. Here, we review what is known and still to be elucidated as regards the structure and function of CHD2 and how its dysregulation leads to a highly variable range of phenotypic presentations.