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.

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.

Rare and common epilepsies converge on a shared gene regulatory network providing opportunities for novel antiepileptic drug discovery.

BACKGROUND: The relationship between monogenic and polygenic forms of epilepsy is poorly understood and the extent to which the genetic and acquired epilepsies share common pathways is unclear. Here, we use an integrated systems-level analysis of brain gene expression data to identify molecular networks disrupted in epilepsy. RESULTS: We identified a co-expression network of 320 genes (M30), which is significantly enriched for non-synonymous de novo mutations ascertained from patients with monogenic epilepsy and for common variants associated with polygenic epilepsy. The genes in the M30 network are expressed widely in the human brain under tight developmental control and encode physically interacting proteins involved in synaptic processes. The most highly connected proteins within the M30 network were preferentially disrupted by deleterious de novo mutations for monogenic epilepsy, in line with the centrality-lethality hypothesis. Analysis of M30 expression revealed consistent downregulation in the epileptic brain in heterogeneous forms of epilepsy including human temporal lobe epilepsy, a mouse model of acquired temporal lobe epilepsy, and a mouse model of monogenic Dravet (SCN1A) disease. These results suggest functional disruption of M30 via gene mutation or altered expression as a convergent mechanism regulating susceptibility to epilepsy broadly. Using the large collection of drug-induced gene expression data from Connectivity Map, several drugs were predicted to preferentially restore the downregulation of M30 in epilepsy toward health, most notably valproic acid, whose effect on M30 expression was replicated in neurons. CONCLUSIONS: Taken together, our results suggest targeting the expression of M30 as a potential new therapeutic strategy in epilepsy.

Systems genetics identifies a convergent gene network for cognition and neurodevelopmental disease.

Genetic determinants of cognition are poorly characterized, and their relationship to genes that confer risk for neurodevelopmental disease is unclear. Here we performed a systems-level analysis of genome-wide gene expression data to infer gene-regulatory networks conserved across species and brain regions. Two of these networks, M1 and M3, showed replicable enrichment for common genetic variants underlying healthy human cognitive abilities, including memory. Using exome sequence data from 6,871 trios, we found that M3 genes were also enriched for mutations ascertained from patients with neurodevelopmental disease generally, and intellectual disability and epileptic encephalopathy in particular. M3 consists of 150 genes whose expression is tightly developmentally regulated, but which are collectively poorly annotated for known functional pathways. These results illustrate how systems-level analyses can reveal previously unappreciated relationships between neurodevelopmental disease-associated genes in the developed human brain, and provide empirical support for a convergent gene-regulatory network influencing cognition and neurodevelopmental disease.

Systems genetics identifies Sestrin 3 as a regulator of a proconvulsant gene network in human epileptic hippocampus.

Gene-regulatory network analysis is a powerful approach to elucidate the molecular processes and pathways underlying complex disease. Here we employ systems genetics approaches to characterize the genetic regulation of pathophysiological pathways in human temporal lobe epilepsy (TLE). Using surgically acquired hippocampi from 129 TLE patients, we identify a gene-regulatory network genetically associated with epilepsy that contains a specialized, highly expressed transcriptional module encoding proconvulsive cytokines and Toll-like receptor signalling genes. RNA sequencing analysis in a mouse model of TLE using 100 epileptic and 100 control hippocampi shows the proconvulsive module is preserved across-species, specific to the epileptic hippocampus and upregulated in chronic epilepsy. In the TLE patients, we map the trans-acting genetic control of this proconvulsive module to Sestrin 3 (SESN3), and demonstrate that SESN3 positively regulates the module in macrophages, microglia and neurons. Morpholino-mediated Sesn3 knockdown in zebrafish confirms the regulation of the transcriptional module, and attenuates chemically induced behavioural seizures in vivo.

Different microRNA profiles in chronic epilepsy versus acute seizure mouse models.

Epilepsy affects around 50 million people worldwide, and in about 65% of patients, the etiology of disease is unknown. MicroRNAs are small non-coding RNAs that have been suggested to play a role in the pathophysiology of epilepsy. Here, we compared microRNA expression patterns in the hippocampus using two chronic models of epilepsy characterised by recurrent spontaneous seizures (pilocarpine and self-sustained status epilepticus (SSSE)) and an acute 6-Hz seizure model. The vast majority of microRNAs deregulated in the acute model exhibited increased expression with 146 microRNAs up-regulated within 6 h after a single seizure. In contrast, in the chronic models, the number of up-regulated microRNAs was similar to the number of down-regulated microRNAs. Three microRNAs-miR-142-5p, miR-331-3p and miR-30a-5p-were commonly deregulated in all three models. However, there is a clear overlap of differentially expressed microRNAs within the chronic models with 36 and 15 microRNAs co-regulated at 24 h and at 28 days following status epilepticus, respectively. Pathway analysis revealed that the altered microRNAs are associated with inflammation, innate immunity and cell cycle regulation. Taken together, the identified microRNAs and the pathways they modulate might represent candidates for novel molecular approaches for the treatment of patients with epilepsy.

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.

Rapid epileptogenesis in the mouse pilocarpine model: video-EEG, pharmacokinetic and histopathological characterization.

Temporal lobe epilepsy (TLE) is the most common form of drug-resistant epilepsy and several rodent models allow studying the pathophysiology of this disorder. One of the best characterized models of TLE is the pilocarpine model. The model has been widely used in rats, but relatively few studies report data obtained with mice. This triggered the present study to perform a comprehensive characterization of the mouse pilocarpine model. We used male NMRI mice (28-32 g) and first established the dose-response relationship for pilocarpine (250-400 mg/kg; ip) to induce status epilepticus (SE). This enabled to define the optimal dose (300 mg/kg) producing the highest SE response (50%) associated with the best survival rate of the animals (90%). The impact of different durations of SE (0.5-3.0 h) on the time to the onset of the first spontaneous recurrent seizures (SRS) was recorded during 5-day continuous video monitoring following the SE. Virtually no "latent" period was observed as the seizures appeared already within 24-48 h after the pilocarpine-induced SE and 0.5 h duration of the SE was sufficient to trigger SRS. Pharmacokinetics assessment showed that these seizures were not associated with residual pilocarpine exposure as it was cleared from the blood and brain already within 24 h post-injection. Consistent with previous reports from the rat pilocarpine model we observed that the extent of hippocampal reorganization and neuronal loss correlates with the duration of SE. However, the shorter durations of SE (0.5-2.0 h) appeared to produce cell loss restricted mainly to the hilus of the dentate gyrus. Interestingly, we also observed that the number of seizures occurring within 5 days after SE appeared to correlate with the degree of hippocampal damage. Continuous 7-week video-EEG monitoring after the SE revealed that SRS were expressed in a particular pattern of clusters. Taken together, the current study provides an in-depth characterization of the mouse pilocarpine model and confirms several features of the epileptogenesis process previously reported from the rat pilocarpine model. However, the mouse pilocarpine model differs by the rapid onset of seizures and an apparent correlation between their numbers and the degree of histopathological changes. Our findings highlight that the pilocarpine model of TLE in mice is associated with brain pathology akin to different stages of human disease and may provide a valuable tool for the discovery of future antiepileptic drugs with disease-modifying properties.

11-Deoxycortisol impedes GABAergic neurotransmission and induces drug-resistant status epilepticus in mice.

Systemic injection of high doses of 11-deoxycortisol succinate had been reported to induce status epilepticus in rats and cats that was associated with paroxysmal epileptiform activity refractory to first generation antiepileptic drugs (AEDs). Using patch clamp recordings we have investigated the mechanisms of 11-deoxycortisol-induced excitability and we have discovered that this molecule accelerates the decay time of the inhibitory postsynaptic currents (IPSCs) mediated by GABA(A) receptors, both in neuronal cultures and in hippocampal slices. In addition, it reduces the amplitude and frequency of IPSCs. Thus, 11-deoxycortisol action on GABAergic neurotransmission may be one of the underlying causes of convulsive seizures that had been observed in rats. In the present study, we have reproduced the ability of 11-deoxycortisol to induce convulsive seizures after intravenous infusion in mice. The threshold dose of 11-deoxycortisol necessary for seizure induction was also determined (0.95 mmol/kg). Furthermore, we have established that these seizures are completely refractory to several AEDs such as phenytoin (up to 100 mg/kg), carbamazepine (up to 56 mg/kg), and valproate (up to 300 mg/kg). Levetiracetam and diazepam afforded only limited protection at high doses, 540 and 3-10 mg/kg, respectively. Interestingly, long-lasting seizures induced by 11-deoxycortisol in mice were not associated with typical neuropathological changes observed in other models of status epilepticus. We propose that 11-deoxycortisol-induced seizures may be an advantageous experimental model of drug-resistant epilepsy. Finally, better understanding of the pro-epileptic properties of 11-deoxycortisol is very important, because this endogenous steroid precursor may play a role in the pathophysiology of epilepsy. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Enhancement of GABA(A)-current run-down in the hippocampus occurs at the first spontaneous seizure in a model of temporal lobe epilepsy.

Refractory temporal lobe epilepsy (TLE) is associated with a dysfunction of inhibitory signaling mediated by GABA(A) receptors. In particular, the use-dependent decrease (run-down) of the currents (I(GABA)) evoked by the repetitive activation of GABA(A) receptors is markedly enhanced in hippocampal and cortical neurons of TLE patients. Understanding the role of I(GABA) run-down in the disease, and its mechanisms, may allow development of medical alternatives to surgical resection, but such mechanistic insights are difficult to pursue in surgical human tissue. Therefore, we have used an animal model (pilocarpine-treated rats) to identify when and where the increase in I(GABA) run-down occurs in the natural history of epilepsy. We found: (i) that the increased run-down occurs in the hippocampus at the time of the first spontaneous seizure (i.e., when the diagnosis of epilepsy is made), and then extends to the neocortex and remains constant in the course of the disease; (ii) that the phenomenon is strictly correlated with the occurrence of spontaneous seizures, because it is not observed in animals that do not become epileptic. Furthermore, initial exploration of the molecular mechanism disclosed a relative increase in alpha4-, relative to alpha1-containing GABA(A) receptors, occurring at the same time when the increased run-down appears, suggesting that alterations in the molecular composition of the GABA receptors may be responsible for the occurrence of the increased run-down. These observations disclose research opportunities in the field of epileptogenesis that may lead to a better understanding of the mechanism whereby a previously normal tissue becomes epileptic.

The biocompatibility of materials used in printed circuit board technologies with respect to primary neuronal and K562 cells.

Printed circuit board (PCB) technology can be used for producing lab-on-a-chip (LOAC) devices. PCBs are characterized by low production costs and large-scale development, both essential elements in the frame of disposable applications. LOAC platforms have been employed not only for diagnostic and/or analytical purposes, but also for identification and isolation of eukaryotic cells, including cancer and stem cells. Accordingly, the compatibility of the employed materials with the biological system under analysis is critical for the development of LOAC devices to be proposed for efficient and safe cell isolation. In this study, we analyzed the in-vitro compatibility of a large set of materials and surface treatments used for LOAC development and evaluation with quasi-standard PCB processes. Biocompatibility was analyzed on hippocampal primary cells (a model of attached cell cultures), in comparison with the reference K562 cell line (a model of cells growing in suspension). We demonstrate here that some of the materials under study alter survival, organization, morphology and adhesion capacity of hippocampal cells, and inhibit growth and differentiation of K562 cells. Nonetheless, a subset of the materials tested did not negatively affect these functions, thus demonstrating that PCB technology, with some limitations, is suitable for the realization of LOAC devices well compatible at least with these preparations.

Localized delivery of fibroblast growth factor-2 and brain-derived neurotrophic factor reduces spontaneous seizures in an epilepsy model.

A loss of neurons is observed in the hippocampus of many patients with epilepsies of temporal lobe origin. It has been hypothesized that damage limitation or repair, for example using neurotrophic factors (NTFs), may prevent the transformation of a normal tissue into epileptic (epileptogenesis). Here, we used viral vectors to locally supplement two NTFs, fibroblast growth factor-2 (FGF-2) and brain-derived neurotrophic factor (BDNF), when epileptogenic damage was already in place. These vectors were first characterized in vitro, where they increased proliferation of neural progenitors and favored their differentiation into neurons, and they were then tested in a model of status epilepticus-induced neurodegeneration and epileptogenesis. When injected in a lesioned hippocampus, FGF-2/BDNF expressing vectors increased neuronogenesis, embanked neuronal damage, and reduced epileptogenesis. It is concluded that reduction of damage reduces epileptogenesis and that supplementing specific NTFs in lesion areas represents a new approach to the therapy of neuronal damage and of its consequences.

GABA(A)-current rundown of temporal lobe epilepsy is associated with repetitive activation of GABA(A) "phasic" receptors.

A study was made of the "rundown" of GABA(A) receptors, microtransplanted to Xenopus oocytes from surgically resected brain tissues of patients afflicted with drug-resistant human mesial temporal lobe epilepsy (mTLE). Cell membranes, isolated from mTLE neocortex specimens, were injected into frog oocytes that rapidly incorporated functional GABA(A) receptors. Upon repetitive activation with GABA (1 mM), "epileptic" GABA(A) receptors exhibited a GABA(A)-current (I(GABA)) rundown that was significantly enhanced by Zn(2+) (

Synthesis and anticonvulsant activity of a class of 2-amino 3-hydroxypropanamide and 2-aminoacetamide derivatives.

Several studies have demonstrated that N-substituted amino acid derivatives exhibit weak anticonvulsant activities in vivo. In the present study, a series of amides of aminoacids structurally related to aminoacetamide have been synthesised and investigated for anticonvulsant activity. Among the molecules investigated, those containing a bicyclic (tetralinyl, indanyl) group linked to the aminoacetamide chain (40, 47 and 59) were among the most active as anticonvulsants (ED50 > 10, <100 mg/kg after oral administration) against tonic seizures in the mouse maximal electroshock, bicuculline and picrotoxin tests at doses devoid of neurotoxic activity. Altogether, these results suggest the described compounds as a class of orally available anticonvulsants. The ability of these compounds to partially block veratridine-induced aspartate efflux from rat cortical synaptosomes suggests that their anticonvulsant activity may be only partly the consequence of an interaction with neuronal voltage-dependent sodium channels. Some of the most potent compounds appear worthy of a further investigation aimed at assessing their anticonvulsant activity in other models and at elucidating the underlying mechanism of action.

BODIPY-conjugated neuropeptide Y ligands: new fluorescent tools to tag Y1, Y2, Y4 and Y5 receptor subtypes.

N-terminal labelled fluorescent BODIPY-NPY peptide analogues were tested in Y1, Y2, Y4 and Y5 receptor-binding assays performed in rat brain membrane preparations and HEK293 cells expressing the rat Y1, Y2, Y4 and Y5 receptors. BODIPY TMR/FL-[Leu31, Pro34]NPY/PYY were able to compete for specific [125][Leu31, Pro34]PYY-binding sites with an affinity similar to that observed for the native peptide at the Y1 (Ki=1-6 nM), Y2 (Ki>1000 nM), Y4 (Ki=10 nM) and Y5 (Ki=1-4 nM) receptor subtypes. BODIPY FL-PYY(3-36) was able to compete for specific Y2 (Ki=10 nM) and Y5 (Ki=30 nM) binding sites, but had almost no affinity in Y1 and Y4 assays. BODIPY FL-hPP was able to compete with high affinity (Ki; 1 and 15 nM) only in Y4 and Y5 receptor-binding assays. BODIPY TMR-[cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP and BODIPY TMR-[hPP(1-17), Ala31, Aib32]NPY were potent competitors only on specific Y5-binding sites (Ki=0.1-0.6 nM). As expected, these fluorescent peptides inhibited forskolin-induced cAMP accumulation, demonstrating that they retained their agonist properties. When tested in confocal microscopy imaging, fluorescent Y1 and Y5 agonists internalized in a time-dependent manner in Y1 and Y5 transfected cells, respectively. These results demonstrate that BODIPY-conjugated NPY analogues retain their selectivity, affinity and agonist properties for the Y1, Y2, Y4 and Y5 receptor subtypes, respectively. Thus, they represent novel tools to study and visualize NPY receptors in living cells.

Kainate seizures increase nociceptin/orphanin FQ release in the rat hippocampus and thalamus: a microdialysis study.

The neuropeptide nociceptin/orphanin FQ (N/OFQ) has been suggested to play a facilitatory role in kainate seizure expression. Furthermore, mRNA levels for the N/OFQ precursor are increased following kainate seizures, while its receptor (NOP) density is decreased. These data suggest increased N/OFQ release. To obtain direct evidence that this is the case, we have developed a microdialysis technique, coupled with a sensitive radioimmunoassay, that allows measurement of N/OFQ release from the hippocampus and thalamus of awake, freely moving animals. In both these brain areas, the spontaneous N/OFQ efflux decreased by approximately 50% and 65% when Ca2+ was omitted and when tetrodotoxin was added to the perfusion medium, respectively. Perfusion of the dialysis probe with high K+ increased N/OFQ release (approximately threefold) in a Ca2+-dependent and tetrodotoxin-sensitive manner. Kainate seizures caused a twofold increase in N/OFQ release followed, within 3 h, by a return to baseline levels. Approximately 5 h after kainate, a late increase in N/OFQ release was observed. On the following day, when animals were having only low grade seizures, N/OFQ release was not significantly different from normal. These phenomena were observed with similar patterns in the hippocampus and in the thalamus. The present data indicate that acute limbic seizures are associated with increased N/OFQ release, which may prime the molecular changes described above, i.e. cause down-regulation of NOP receptors and activation of N/OFQ biosynthesis.

Delayed epileptogenesis in nociceptin/orphanin FQ-deficient mice.

The neuropeptide nociceptin/orphanin FQ (N/OFQ) is implicated in many biological functions, including nociception, locomotor activity, stress and anxiety, drinking and food-intake. N/OFQ has also been reported to play a facilitatory role in acute kainate-induced seizures. The aim of the present study was to investigate its involvement in a chronic model of temporal lobe epilepsy, kindling epileptogenesis, using N/OFQ knock-out mice and their wild-type littermates as controls. Kindling development was retarded in N/OFQ-deficient mice, in that (compared with controls) they required a significantly greater number of stimulations and a significantly longer time in electrical seizures to reach kindling criteria. These data indicate that N/OFQ is involved in the development of kindling and that it may play a pro-epileptogenic role.

Changes in NPY-mediated modulation of hippocampal [3H]D-aspartate outflow in the kindling model of epilepsy.

The anticonvulsant effect of NPY may depend on Y(2) and/or Y(5) receptor-mediated inhibition of glutamate release in critical areas, such as the hippocampus. However, Y(2) and Y(5) receptor levels have been reported to increase and decrease, respectively, in the epileptic hippocampus, implicating that the profile of NPY effects may change accordingly. The aim of this study was to evaluate the differential effects of NPY on glutamate release in the normal and in the epileptic hippocampus. Thus, we pharmacologically characterized the effects of NPY on the release of [(3)H]D-aspartate, a valid marker of endogenous glutamate, from synaptosomes prepared from the whole hippocampus and from the three hippocampal subregions (dentate gyrus and CA1 and CA3 subfields) of control and kindled rats, killed 1 week after the last stimulus-evoked seizure. In the whole hippocampus, NPY does not significantly affect stimulus-evoked [(3)H]D-aspartate overflow. In synaptosomes prepared from control rats, NPY significantly inhibited 15 mM K(+)-evoked [(3)H]D-aspartate overflow only in the CA1 subfield (approx. -30%). Both Y(2) and Y(5) receptor antagonists (respectively, 1 microM BIIE0246 and 1 microM CGP71683A) prevented this effect, suggesting the involvement of both receptor types. In contrast, in synaptosomes prepared from kindled rats NPY significantly inhibited 15 mM K(+)-evoked [(3)H]D-aspartate overflow in the CA1 subfield and in the dentate gyrus (approx. -30%). Only the Y(2) (not the Y(5)) antagonist prevented these effects. These data indicate a critical role for the Y(2) receptor in the inhibitory control of glutamate release in the kindled hippocampus and, thus, suggest that the anticonvulsant effect of NPY in the epileptic brain is most likely Y(2), but not Y(5), receptor-mediated.