Identification of gene regulatory networks affected across drug-resistant epilepsies.

Epilepsy is a chronic and heterogenous disease characterized by recurrent unprovoked seizures, that are commonly resistant to antiseizure medications. This study applies a transcriptome network-based approach across epilepsies aiming to improve understanding of molecular disease pathobiology, recognize affected biological mechanisms and apply causal reasoning to identify therapeutic hypotheses. This study included the most common drug-resistant epilepsies (DREs), such as temporal lobe epilepsy with hippocampal sclerosis (TLE-HS), and mTOR pathway-related malformations of cortical development (mTORopathies). This systematic comparison characterized the global molecular signature of epilepsies, elucidating the key underlying mechanisms of disease pathology including neurotransmission and synaptic plasticity, brain extracellular matrix and energy metabolism. In addition, specific dysregulations in neuroinflammation and oligodendrocyte function were observed in TLE-HS and mTORopathies, respectively. The aforementioned mechanisms are proposed as molecular hallmarks of DRE with the identified upstream regulators offering opportunities for drug-target discovery and development.

Downregulation of Ubiquitin-Specific Protease 15 (USP15) Does Not Provide Therapeutic Benefit in Experimental Mesial Temporal Lobe Epilepsy.

Structural epilepsies display complex immune activation signatures. However, it is unclear which neuroinflammatory pathways drive pathobiology. Transcriptome studies of brain resections from mesial temporal lobe epilepsy (mTLE) patients revealed a dysregulation of transforming growth factor ß, interferon α/ß, and nuclear factor erythroid 2-related factor 2 pathways. Since these pathways are regulated by ubiquitin-specific proteases (USP), in particular USP15, we hypothesized that USP15 blockade may provide therapeutic relief in treatment-resistant epilepsies. For validation, transgenic mice which either constitutively or inducibly lack Usp15 gene expression underwent intrahippocampal kainate injections to induce mTLE. We show that the severity of status epilepticus is unaltered in mice constitutively lacking Usp15 compared to wild types. Cell death, reactive gliosis, and changes in the inflammatory transcriptome were pronounced at 4 days after kainate injection. However, these brain inflammation signatures did not differ between genotypes. Likewise, induced deletion of Usp15 in chronic epilepsy did not affect seizure generation, cell death, gliosis, or the transcriptome. Concordantly, siRNA-mediated knockdown of Usp15 in a microglial cell line did not impact inflammatory responses in the form of cytokine release. Our data show that a lack of USP15 is insufficient to modulate the expression of relevant neuroinflammatory pathways in an mTLE mouse model and do not support targeting USP15 as a therapeutic approach for pharmacoresistant epilepsy.

Seizure-mediated iron accumulation and dysregulated iron metabolism after status epilepticus and in temporal lobe epilepsy.

Neuronal dysfunction due to iron accumulation in conjunction with reactive oxygen species (ROS) could represent an important, yet underappreciated, component of the epileptogenic process. However, to date, alterations in iron metabolism in the epileptogenic brain have not been addressed in detail. Iron-related neuropathology and antioxidant metabolic processes were investigated in resected brain tissue from patients with temporal lobe epilepsy and hippocampal sclerosis (TLE-HS), post-mortem brain tissue from patients who died after status epilepticus (SE) as well as brain tissue from the electrically induced SE rat model of TLE. Magnetic susceptibility of the presumed seizure-onset zone from three patients with focal epilepsy was compared during and after seizure activity. Finally, the cellular effects of iron overload were studied in vitro using an acute mouse hippocampal slice preparation and cultured human fetal astrocytes. While iron-accumulating neurons had a pyknotic morphology, astrocytes appeared to acquire iron-sequestrating capacity as indicated by prominent ferritin expression and iron retention in the hippocampus of patients with SE or TLE. Interictal to postictal comparison revealed increased magnetic susceptibility in the seizure-onset zone of epilepsy patients. Post-SE rats had consistently higher hippocampal iron levels during the acute and chronic phase (when spontaneous recurrent seizures are evident). In vitro, in acute slices that were exposed to iron, neurons readily took up iron, which was exacerbated by induced epileptiform activity. Human astrocyte cultures challenged with iron and ROS increased their antioxidant and iron-binding capacity, but simultaneously developed a pro-inflammatory phenotype upon chronic exposure. These data suggest that seizure-mediated, chronic neuronal iron uptake might play a role in neuronal dysfunction/loss in TLE-HS. On the other hand, astrocytes sequester iron, specifically in chronic epilepsy. This function might transform astrocytes into a highly resistant, pro-inflammatory phenotype potentially contributing to pro-epileptogenic inflammatory processes.

Balloon cells promote immune system activation in focal cortical dysplasia type 2b.

AIMS: Focal cortical dysplasia (FCD) type 2 is an epileptogenic malformation of the neocortex associated with somatic mutations in the mammalian target of rapamycin (mTOR) pathway. Histopathologically, FCD 2 is subdivided into FCD 2a and FCD 2b, the only discriminator being the presence of balloon cells (BCs) in FCD 2b. While pro-epileptogenic immune system activation and inflammatory responses are commonly detected in both subtypes, it is unknown what contextual role BCs play. METHODS: The present study employed RNA sequencing of surgically resected brain tissue from FCD 2a (n = 11) and FCD 2b (n = 20) patients compared to autopsy control (n = 9) focusing on three immune system processes: adaptive immunity, innate immunity and cytokine production. This analysis was followed by immunohistochemistry on a clinically well-characterised FCD 2 cohort. RESULTS: Differential expression analysis revealed stronger expression of components of innate immunity, adaptive immunity and cytokine production in FCD 2b than in FCD 2a, particularly complement activation and antigen presentation. Immunohistochemical analysis confirmed these findings, with strong expression of leukocyte antigen I and II in FCD 2b as compared to FCD 2a. Moreover, T-lymphocyte tissue infiltration was elevated in FCD 2b. Expression of markers of immune system activation in FCD 2b was concentrated in subcortical white matter. Lastly, antigen presentation was strongly correlated with BC load in FCD 2b lesions. CONCLUSION: We conclude that, next to mutation-driven mTOR activation and seizure activity, BCs are crucial drivers of inflammation in FCD 2b. Our findings indicate that therapies targeting inflammation may be beneficial in FCD 2b.

Temporal dynamics of a CSF1R signaling gene regulatory network involved in epilepsy.

Colony Stimulating Factor 1 Receptor (CSF1R) is a potential target for anti-epileptic drugs. However, inhibition of CSF1R is not well tolerated by patients, thereby prompting the need for alternative targets. To develop a framework for identification of such alternatives, we here develop a mathematical model of a pro-inflammatory gene regulatory network (GRN) involved in epilepsy and centered around CSF1R. This GRN comprises validated transcriptional and post-transcriptional regulations involving STAT1, STAT3, NFκB, IL6R, CSF3R, IRF8, PU1, C/EBPα, TNFR1, CSF1 and CSF1R. The model was calibrated on mRNA levels of all GRN components in lipopolysaccharide (LPS)-treated mouse microglial BV-2 cells, and allowed to predict that STAT1 and STAT3 have the strongest impact on the expression of the other GRN components. Microglial BV-2 cells were selected because, the modules from which the GRN was deduced are enriched for microglial marker genes. The function of STAT1 and STAT3 in the GRN was experimentally validated in BV-2 cells. Further, in silico analysis of the GRN dynamics predicted that a pro-inflammatory stimulus can induce irreversible bistability whereby the expression level of GRN components occurs as two distinct states. The irreversibility of the switch may enforce the need for chronic inhibition of the CSF1R GRN in order to achieve therapeutic benefit. The cell-to-cell heterogeneity driven by the bistability may cause variable therapeutic response. In conclusion, our modeling approach uncovered a GRN controlling CSF1R that is predominantly regulated by STAT1 and STAT3. Irreversible inflammation-induced bistability and cell-to-cell heterogeneity of the GRN provide a theoretical foundation to the need for chronic GRN control and the limited potential for disease modification via inhibition of CSF1R.

A systems-level framework for drug discovery identifies Csf1R as an anti-epileptic drug target.

The identification of drug targets is highly challenging, particularly for diseases of the brain. To address this problem, we developed and experimentally validated a general computational framework for drug target discovery that combines gene regulatory information with causal reasoning ("Causal Reasoning Analytical Framework for Target discovery"-CRAFT). Using a systems genetics approach and starting from gene expression data from the target tissue, CRAFT provides a predictive framework for identifying cell membrane receptors with a direction-specified influence over disease-related gene expression profiles. As proof of concept, we applied CRAFT to epilepsy and predicted the tyrosine kinase receptor Csf1R as a potential therapeutic target. The predicted effect of Csf1R blockade in attenuating epilepsy seizures was validated in three pre-clinical models of epilepsy. These results highlight CRAFT as a systems-level framework for target discovery and suggest Csf1R blockade as a novel therapeutic strategy in epilepsy. CRAFT is applicable to disease settings other than epilepsy.

BED: a Biological Entity Dictionary based on a graph data model.

The understanding of molecular processes involved in a specific biological system can be significantly improved by combining and comparing different data sets and knowledge resources. However, these information sources often use different identification systems and an identifier conversion step is required before any integration effort. Mapping between identifiers is often provided by the reference information resources and several tools have been implemented to simplify their use. However, most of these tools do not combine the information provided by individual resources to increase the completeness of the mapping process. Also, deprecated identifiers from former versions of databases are not taken into account. Finally, finding automatically the most relevant path to map identifiers from one scope to the other is often not trivial. The Biological Entity Dictionary (BED) addresses these three challenges by relying on a graph data model describing possible relationships between entities and their identifiers. This model has been implemented using Neo4j and an R package provides functions to query the graph but also to create and feed a custom instance of the database. This design combined with a local installation of the graph database and a cache system make BED very efficient to convert large lists of identifiers.

A computational framework for complex disease stratification from multiple large-scale datasets.

BACKGROUND: Multilevel data integration is becoming a major area of research in systems biology. Within this area, multi-'omics datasets on complex diseases are becoming more readily available and there is a need to set standards and good practices for integrated analysis of biological, clinical and environmental data. We present a framework to plan and generate single and multi-'omics signatures of disease states. METHODS: The framework is divided into four major steps: dataset subsetting, feature filtering, 'omics-based clustering and biomarker identification. RESULTS: We illustrate the usefulness of this framework by identifying potential patient clusters based on integrated multi-'omics signatures in a publicly available ovarian cystadenocarcinoma dataset. The analysis generated a higher number of stable and clinically relevant clusters than previously reported, and enabled the generation of predictive models of patient outcomes. CONCLUSIONS: This framework will help health researchers plan and perform multi-'omics big data analyses to generate hypotheses and make sense of their rich, diverse and ever growing datasets, to enable implementation of translational P4 medicine.

Intrinsic Inflammation Is a Potential Anti-Epileptogenic Target in the Organotypic Hippocampal Slice Model.

Understanding the mechanisms of epileptogenesis is essential to develop novel drugs that could prevent or modify the disease. Neuroinflammation has been proposed as a promising target for therapeutic interventions to inhibit the epileptogenic process that evolves from traumatic brain injury. However, it remains unclear whether cytokine-related pathways, particularly TNFα signaling, have a critical role in the development of epilepsy. In this study, we investigated the role of innate inflammation in an in vitro model of post-traumatic epileptogenesis. We combined organotypic hippocampal slice cultures, representing an in vitro model of post-traumatic epilepsy, with multi-electrode array recordings to directly monitor the development of epileptiform activity and to examine the concomitant changes in cytokine release, cell death, and glial cell activation. We report that synchronized ictal- and interictal-like activities spontaneously evolve in this culture. Dynamic changes in the release of the pro-inflammatory cytokines IL-1ß, TNFα, and IL-6 were observed throughout the culture period (3 to 21 days in vitro) with persistent activation of microglia and astrocytes. We found that neutralizing TNFα with a polyclonal antibody significantly reduced ictal discharges, and this effect lasted for 1 week after antibody washout. Neither phenytoin nor an anti-IL-6 polyclonal antibody was efficacious in inhibiting the development of epileptiform activity. Our data show a sustained effect of the anti-TNFα antibody on the ictal progression in organotypic hippocampal slice cultures supporting the critical role of inflammatory mediators in epilepsy and establishing a proof-of-principle evidence for the utility of this preparation to test the therapeutic effects of anti-inflammatory treatments.

Genome-wide analysis of differential RNA editing in epilepsy.

The recoding of genetic information through RNA editing contributes to proteomic diversity, but the extent and significance of RNA editing in disease is poorly understood. In particular, few studies have investigated the relationship between RNA editing and disease at a genome-wide level. Here, we developed a framework for the genome-wide detection of RNA sites that are differentially edited in disease. Using RNA-sequencing data from 100 hippocampi from mice with epilepsy (pilocarpine-temporal lobe epilepsy model) and 100 healthy control hippocampi, we identified 256 RNA sites (overlapping with 87 genes) that were significantly differentially edited between epileptic cases and controls. The degree of differential RNA editing in epileptic mice correlated with frequency of seizures, and the set of genes differentially RNA-edited between case and control mice were enriched for functional terms highly relevant to epilepsy, including "neuron projection" and "seizures." Genes with differential RNA editing were preferentially enriched for genes with a genetic association to epilepsy. Indeed, we found that they are significantly enriched for genes that harbor nonsynonymous de novo mutations in patients with epileptic encephalopathy and for common susceptibility variants associated with generalized epilepsy. These analyses reveal a functional convergence between genes that are differentially RNA-edited in acquired symptomatic epilepsy and those that contribute risk for genetic epilepsy. Taken together, our results suggest a potential role for RNA editing in the epileptic hippocampus in the occurrence and severity of epileptic seizures.

Pathway analysis from lists of microRNAs: common pitfalls and alternative strategy.

MicroRNAs (miRNAs) are involved in the regulation of gene expression at a post-transcriptional level. As such, monitoring miRNA expression has been increasingly used to assess their role in regulatory mechanisms of biological processes. In large scale studies, once miRNAs of interest have been identified, the target genes they regulate are often inferred using algorithms or databases. A pathway analysis is then often performed in order to generate hypotheses about the relevant biological functions controlled by the miRNA signature. Here we show that the method widely used in scientific literature to identify these pathways is biased and leads to inaccurate results. In addition to describing the bias and its origin we present an alternative strategy to identify potential biological functions specifically impacted by a miRNA signature. More generally, our study exemplifies the crucial need of relevant negative controls when developing, and using, bioinformatics methods.

Nrf2 defense pathway: Experimental evidence for its protective role in epilepsy.

OBJECTIVE: Epigenetic mechanisms involved in transcriptional regulation of multiple molecular pathways are potentially attractive therapeutic interventions for epilepsy, because single target therapies are unlikely to provide both anticonvulsant and disease-modifying effects. METHODS: A selection of epilepsy-related gene expression data sets were retrieved using NextBio software and imported to Ingenuity Pathway Analysis for transcription factor enrichment analysis. Nuclear factor erythroid 2-related factor 2 (Nrf2)-a transcription factor that promotes the expression of numerous antioxidant, anti-inflammatory, and neuroprotective proteins-was identified as a candidate for confirmation of mRNA expression in hippocampal tissue from patients with temporal lobe epilepsy and in mice following pilocarpine-induced status epilepticus (SE). Human Nrf2 was overexpressed via an adeno-associated virus (AAV) vector after the onset of spontaneous recurrent seizures (SRS) in the animals. At the end of a 5-week continuous monitoring period for SRS, quantitative immunohistochemistry using neuronal (neuronal-specific nuclear protein), astrocytic (glial fibrillary acidic protein), and microglial (ionized calcium binding adaptor molecule 1) markers was performed. RESULTS: A significant increase in Nrf2 mRNA expression was observed in human epileptic hippocampal tissue. Nrf2 expression levels increased progressively in mice, reaching a peak at 72 hours after SE, and then declined. Similar expression patterns were observed for 3 Nrf2-regulated genes: HO-1, NQO1, and mGST. Remarkably, mice injected with AAV Nrf2 displayed significantly fewer generalized seizures, with profound reduction in microglia activation. Hippocampal neurons were preserved, whereas the number of astrocytes was unchanged. INTERPRETATION: These findings extend the potential of Nrf2-based therapies to epilepsy and add to the rapidly accumulating evidence from other neurodegenerative and inflammatory disease models.

Mechanism of primitive duct formation in the pancreas and submandibular glands: a role for SDF-1.

BACKGROUND: The exocrine pancreas is composed of a branched network of ducts connected to acini. They are lined by a monolayered epithelium that derives from the endoderm and is surrounded by mesoderm-derived mesenchyme. The morphogenic mechanisms by which the ductal network is established as well as the signaling pathways involved in this process are poorly understood. RESULTS: By morphological analyzis of wild-type and mutant mouse embryos and using cultured embryonic explants we investigated how epithelial morphogenesis takes place and is regulated by chemokine signaling. Pancreas ontogenesis displayed a sequence of two opposite epithelial transitions. During the first transition, the monolayered and polarized endodermal cells give rise to tissue buds composed of a mass of non polarized epithelial cells. During the second transition the buds reorganize into branched and polarized epithelial monolayers that further differentiate into tubulo-acinar glands. We found that the second epithelial transition is controlled by the chemokine Stromal cell-Derived Factor (SDF)-1. The latter is expressed by the mesenchyme, whereas its receptor CXCR4 is expressed by the epithelium. Reorganization of cultured pancreatic buds into monolayered epithelia was blocked in the presence of AMD3100, a SDF-1 antagonist. Analyzis of sdf1 and cxcr4 knockout embryos at the stage of the second epithelial transition revealed transient defective morphogenesis of the ventral and dorsal pancreas. Reorganization of a globular mass of epithelial cells in polarized monolayers is also observed during submandibular glands development. We found that SDF-1 and CXCR4 are expressed in this organ and that AMD3100 treatment of submandibular gland explants blocks its branching morphogenesis. CONCLUSION: In conclusion, our data show that the primitive pancreatic ductal network, which is lined by a monolayered and polarized epithelium, forms by remodeling of a globular mass of non polarized epithelial cells. Our data also suggest that SDF-1 controls the branching morphogenesis of several exocrine tissues.

Eph receptors and their ephrin ligands are expressed in developing mouse pancreas.

Pancreas development involves branching morphogenesis concomitantly to differentiation of endocrine, exocrine and ductal cell types from a single population of pancreatic precursors. These processes depend on many signals and factors that also control development of the central nervous system. In the latter, Eph receptors and their class-A (GPI-anchored) and class-B (transmembrane) ephrin ligands control cell migration and axon-pathfinding, help establish regional patterns and act as labels for cell positioning. This raised the question as to whether and where Ephs and ephrins are expressed during pancreas development. Here we have identified the Eph and ephrin genes that are expressed in mouse embryonic pancreas, as detected by RT-PCR analysis. In situ hybridization experiments showed that Ephs and ephrins are mainly expressed in the burgeoning structures of the epithelium which differentiate into exocrine acini. Binding experiments on whole pancreas demonstrated the presence of functional Eph receptors. They showed that EphBs are expressed by the pancreatic epithelium at embryonic day (e) 12.5 and that, from e14.5 on, Ephs of both classes are expressed by the pancreatic epithelium and then become restricted to developing acini. We conclude that specific members of the Eph/ephrin family are expressed in embryonic pancreas according to a dynamic temporal and regional pattern.

Shh-dependent differentiation of intestinal tissue from embryonic pancreas by activin A.

The pancreas develops from the endoderm to give rise to ducts, acini and islets of Langerhans. This process involves extracellular signals of the Transforming Growth Factor beta (TGFbeta) family. The aim of this work was to study the effects of activin A, a member of this family, whose potential role in pancreas differentiation is controversial. To this end, we used pancreatic explants from E12.5 mouse embryos. In culture these explants exhibited spontaneous growth, epithelial morphogenesis and endocrine and exocrine differentiation. Exposure to activin A did not affect exocrine or endocrine differentiation. Surprisingly, activin A induced in the explants the appearance of a large contractile structure surrounded by a cylindrical epithelium, a thick basal lamina and a smooth muscle layer. This structure, the formation of which was prevented by follistatin, was typical of an intestinal wall. Consistent with this interpretation, activin A rapidly induced in the explants the mRNAs for fatty acid binding proteins (FABPs), which are markers of the intestine, but not of the pancreas. We also found that induction of the FABPs was preceded by induction of Sonic hedgehog (Shh), a known inducer of intestinal differentiation in the endoderm. Activin B induced neither Shh nor intestinal differentiation. The activin A-mediated intestinal differentiation was blocked by cyclopamine, an inhibitor of Hedgehog signaling, and it was mimicked by Shh. We conclude that activin A does not appear to affect the exocrine or endocrine components of the pancreas, but that it can promote differentiation of pancreatic tissue into intestine via a Shh-dependent mechanism. These findings illustrate the plasticity of differentiation programs in response to extracellular signals in the pancreas and they shed new light on the regulation of pancreas and intestinal development.

Cloning and embryonic expression pattern of the mouse Onecut transcription factor OC-2.

Onecut (OC) transcription factors are evolutionarily conserved proteins with important developmental functions. They contain a bipartite DNA-binding domain composed of a single cut domain associated with a divergent homeodomain. The human genome contains three Onecut paralogues, Hnf6 (also called Oc1), Oc2 and Oc3. We describe here the cloning of mouse (m) OC-2 and its expression pattern in the mouse embryo. The mOc2 gene was localized on chromosome 18. Analysis of the mOC-2 amino acid sequence revealed overall identities of 67% with mHNF-6 and of 56% with mOC-3, and the presence of functional domains delineated earlier in HNF-6. The sequence of the 153 residue-long cut-homeodomain was very conserved, as it was 92% identical to that of mHNF-6 and 89% identical to that of mOC-3. In situ hybridization showed expression of mOc2 in the developing nervous system and gut endoderm. Like Hnf6, Oc2 was expressed in developing liver and pancreas. As many genes that are targeted by Onecut factors are recognized by both OC-2 and HNF-6, this overlap of expression patterns may have functional implications.