Amaya Sanz-Rodriguez

miRNA Expression Profile after Status Epilepticus and Hippocampal Neuroprotection by Targeting miR-132

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.

Seizure suppression and neuroprotection by targeting the purinergic P2X7 receptor during status epilepticus in mice

Prolonged seizures [status epilepticus (SE)] constitute a neurological emergency that can permanently damage the brain. SE results from a failure of the normal mechanisms to terminate seizures; in particular, γ‐amino butyric acid‐mediated inhibition, and benzodiazepine anticonvulsants are often incompletely effective. ATP acts as a fast neurotransmitter via ionotropic ligandgated P2X receptors. Here we report that SE induced by intra‐amygdala kainic acid in mice selectively increased hippocampal levels of P2X7 receptors relative to other P2X receptors. Using transgenic P2X7 reporter mice expressing enhanced green fluorescent protein, we identify dentate granule neurons as the major cell population transcribing the P2X7 receptor after SE. Pretreatment of mice with an intracerebroventricular microinjection of 1.75 nmol A438079, a P2X7 receptor antagonist, reduced seizure duration by 58% and reduced seizure‐induced neuronal death by 61%. Injection of brilliant blue G (1 pmol), another selective antagonist, reduced seizure duration by 48% and was also neuroprotective. A438079 was seizure‐suppressive when injected shortly after induction of SE, and coinjection of A438079 with lorazepam 60 min after triggering SE, when electrographic seizure‐responsiveness to lorazepam had decreased, also terminated SE. Our results suggest that P2X7 receptor antagonists may be a promising class of drug for seizure abrogation and neuroprotection in SE.—Engel, T., Gomez‐Villafuertes, R., Tanaka, K., Mesuret, G., Sanz‐Rodriguez, A., Garcia‐Huerta, P., Miras‐Portugal, M. T., Henshall, D. C., Diaz‐Hernandez, M. Seizure suppression and neuroprotection by targeting the purinergic P2X7 receptor during status epilepticus in mice. FASEB J. 26, 1616‐1628 (2012). www.fasebj.org

Increased neocortical expression of the P2X7 receptor after status epilepticus and anticonvulsant effect of P2X7 receptor antagonist A-438079

ATP is an essential transmitter/cotransmitter in neuron function and pathophysiology and has recently emerged as a potential contributor to prolonged seizures (status epilepticus) through the activation of the purinergic ionotropic P2X7 receptor (P2X7R). Increased P2X7R expression has been reported in the hippocampus, and P2X7R antagonists reduced seizure‐induced damage to this brain region. However, status epilepticus also produces damage to the neocortex. The present study was designed to characterize P2X7R in the neocortex and assess effects of P2X7R antagonists on cortical injury after status epilepticus.

microRNA targeting of the P2X7 purinoceptor opposes a contralateral epileptogenic focus in the hippocampus

The ATP-gated ionotropic P2X7 receptor (P2X7R) modulates glial activation, cytokine production and neurotransmitter release following brain injury. Levels of the P2X7R are increased in experimental and human epilepsy but the mechanisms controlling P2X7R expression remain poorly understood. Here we investigated P2X7R responses after focal-onset status epilepticus in mice, comparing changes in the damaged, ipsilateral hippocampus to the spared, contralateral hippocampus. P2X7R-gated inward currents were suppressed in the contralateral hippocampus and P2rx7 mRNA was selectively uploaded into the RNA-induced silencing complex (RISC), suggesting microRNA targeting. Analysis of RISC-loaded microRNAs using a high-throughput platform, as well as functional assays, suggested the P2X7R is a target of microRNA-22. Inhibition of microRNA-22 increased P2X7R expression and cytokine levels in the contralateral hippocampus after status epilepticus and resulted in more frequent spontaneous seizures in mice. The major pro-inflammatory and hyperexcitability effects of microRNA-22 silencing were prevented in P2rx7−/− mice or by treatment with a specific P2X7R antagonist. Finally, in vivo injection of microRNA-22 mimics transiently suppressed spontaneous seizures in mice. The present study supports a role for post-transcriptional regulation of the P2X7R and suggests therapeutic targeting of microRNA-22 may prevent inflammation and development of a secondary epileptogenic focus in the brain.

Transient P2X7 Receptor Antagonism Produces Lasting Reductions in Spontaneous Seizures and Gliosis in Experimental Temporal Lobe Epilepsy.

Neuroinflammation is thought to contribute to the pathogenesis and maintenance of temporal lobe epilepsy, but the underlying cell and molecular mechanisms are not fully understood. The P2X7 receptor is an ionotropic receptor predominantly expressed on the surface of microglia, although neuronal expression has also been reported. The receptor is activated by the release of ATP from intracellular sources that occurs during neurodegeneration, leading to microglial activation and inflammasome-mediated interleukin 1β release that contributes to neuroinflammation. Using a reporter mouse in which green fluorescent protein is induced in response to the transcription of P2rx7, we show that expression of the receptor is selectively increased in CA1 pyramidal and dentate granule neurons, as well as in microglia in mice that developed epilepsy after intra-amygdala kainic acid-induced status epilepticus. P2X7 receptor levels were increased in hippocampal subfields in the mice and in resected hippocampus from patients with pharmacoresistant temporal lobe epilepsy. Cells transcribing P2rx7 in hippocampal slices from epileptic mice displayed enhanced agonist-evoked P2X7 receptor currents, and synaptosomes from these animals showed increased P2X7 receptor levels and altered calcium responses. A 5 d treatment of epileptic mice with systemic injections of the centrally available, potent, and specific P2X7 receptor antagonist JNJ-47965567 (30 mg/kg) significantly reduced spontaneous seizures during continuous video-EEG monitoring that persisted beyond the time of drug presence in the brain. Hippocampal sections from JNJ-47965567-treated animals obtained >5 d after treatment ceased displayed strongly reduced microgliosis and astrogliosis. The present study suggests that targeting the P2X7 receptor has anticonvulsant and possibly disease-modifying effects in experimental epilepsy. SIGNIFICANCE STATEMENT Temporal lobe epilepsy is the most common and drug-resistant form of epilepsy in adults. Neuroinflammation is implicated as a pathomechanism, but the upstream mechanisms driving gliosis and how important this is for seizures remain unclear. In our study, we show that the ATP-gated P2X7 receptor is upregulated in experimental epilepsy and resected hippocampus from epilepsy patients. Targeting the receptor with a new centrally available antagonist, JNJ-47965567, suppressed epileptic seizures well beyond the time of treatment and reduced underlying gliosis in the hippocampus. The findings suggest a potential disease-modifying treatment for epilepsy based on targeting the P2X7 receptor.

P2X7 receptor inhibition interrupts the progression of seizures in immature rats and reduces hippocampal damage.

Early‐life seizures, particularly when prolonged, may be harmful to the brain. Current pharmacotherapy is often ineffective; therefore, novel neuro‐ and/or glio‐transmitter systems should be explored for targeting. The P2X7 receptor is a cation‐permeable channel with trophic and excitability effects on neurons and glia which is activated by high amounts of ATP that may be released in the setting of injury after severe seizures. Here, we tested the effects of A‐438079, a potent and selective P2X7 receptor antagonist in a lesional model of early‐life status epilepticus.

Protective neuronal induction of ATF5 in endoplasmic reticulum stress induced by status epilepticus

Activating transcription factor 5 (ATF5) is a basic-leucine-zipper transcription factor of the ATF/CREB family. The Atf5 gene generates two transcripts, Atf5α and Atf5β, of which Atf5α is known to be selectively translated upon endoplasmic reticulum stress response in non-neuronal cells. ATF5 is highly expressed in the developing brain where it modulates proliferation of neural progenitor cells. These cells show a high level of ATF5 that has to decrease to allow them to differentiate into mature neurons or glial cells. This has led to the extended notion that differentiated neural cells do not express ATF5 unless they undergo tumourigenic transformation. However, no systematic analysis of the distribution of ATF5 in adult brain or of its potential role in neuronal endoplasmic reticulum stress response has been reported. By immunostaining here we confirm highest ATF5 levels in neuroprogenitor cells of the embryonic and adult subventricular zone but also found ATF5 in a large variety of neurons in adult mouse brain. By combining Atf5 in situ hybridization and immunohistochemistry for the neuronal marker NeuN we further confirmed Atf5 messenger RNA in adult mouse neurons. Quantitative reverse transcriptase polymerase chain reaction demonstrated that Atf5α is the most abundant transcript in adult mouse encephalon and injection of the endoplasmic reticulum stress inducer tunicamycin into adult mouse brain increased neuronal ATF5 levels. Accordingly, ATF5 levels increased in hippocampal neurons of a mouse model of status epilepticus triggered by intra-amygdala injection of kainic acid, which leads to abnormal hippocampal neuronal activity and endoplasmic reticulum stress. Interestingly, ATF5 upregulation occurred mainly in hippocampal neuronal fields that do not undergo apoptosis in this status epilepticus model such as CA1 and dentate gyrus, thus suggesting a neuroprotective role. This was confirmed in a primary neuronal culture model in which ATF5 overexpression resulted in decreased endoplasmic reticulum stress-induced apoptosis and the opposite result was achieved by Atf5 RNA interference. Furthermore, in vivo administration of the eIF2α phosphatase inhibitor salubrinal resulted in increased ATF5 hippocampal levels and attenuated status epilepticus-induced neuronal death in the vulnerable CA3 subfield. In good agreement with the neuroprotective effect of increased ATF5, we found that apoptosis-resistant epileptogenic foci from patients with temporal lobe epilepsy also showed increased levels of ATF5. Thus, our results demonstrate that adult neurons express ATF5 and that they increase its levels upon endoplasmic reticulum stress as a pro-survival mechanism, thus opening a new field for neuroprotective strategies focused on ATF5 modulation.

High Throughput qPCR Expression Profiling of Circulating MicroRNAs Reveals Minimal Sex- and Sample Timing-Related Variation in Plasma of Healthy Volunteers.

MicroRNAs are a class of small non-coding RNA that regulate gene expression at a post-transcriptional level. MicroRNAs have been identified in various body fluids under normal conditions and their stability as well as their dysregulation in disease opens up a new field for biomarker study. However, diurnal and day-to-day variation in plasma microRNA levels, and differential regulation between males and females, may affect biomarker stability. A QuantStudio 12K Flex Real-Time PCR System was used to profile plasma microRNA levels using OpenArray in male and female healthy volunteers, in the morning and afternoon, and at four time points over a one month period. Using this system we were able to run four OpenArray plates in a single run, the equivalent of 32 traditional 384-well qPCR plates or 12,000 data points. Up to 754 microRNAs can be identified in a single plasma sample in under two hours. 108 individual microRNAs were identified in at least 80% of all our samples which compares favourably with other reports of microRNA profiles in serum or plasma in healthy adults. Many of these microRNAs, including miR-16-5p, miR-17-5p, miR-19a-3p, miR-24-3p, miR-30c-5p, miR-191-5p, miR-223-3p and miR-451a are highly expressed and consistent with previous studies using other platforms. Overall, microRNA levels were very consistent between individuals, males and females, and time points and we did not detect significant differences in levels of microRNAs. These results suggest the suitability of this platform for microRNA profiling and biomarker discovery and suggest minimal confounding influence of sex or sample timing. However, the platform has not been subjected to rigorous validation which must be demonstrated in future biomarker studies where large differences may exist between disease and control samples.

Potent Anti-seizure Effects of Locked Nucleic Acid Antagomirs Targeting miR-134 in Multiple Mouse and Rat Models of Epilepsy

Current anti-epileptic drugs (AEDs) act on a limited set of neuronal targets, are ineffective in a third of patients with epilepsy, and do not show disease-modifying properties. MicroRNAs are small noncoding RNAs that regulate levels of proteins by post-transcriptional control of mRNA stability and translation. MicroRNA-134 is involved in controlling neuronal microstructure and brain excitability and previous studies showed that intracerebroventricular injections of locked nucleic acid (LNA), cholesterol-tagged antagomirs targeting microRNA-134 (Ant-134) reduced evoked and spontaneous seizures in mouse models of status epilepticus. Translation of these findings would benefit from evidence of efficacy in non-status epilepticus models and validation in another species. Here, we report that electrographic seizures and convulsive behavior are strongly reduced in adult mice pre-treated with Ant-134 in the pentylenetetrazol model. Pre-treatment with Ant-134 did not affect the severity of status epilepticus induced by perforant pathway stimulation in adult rats, a toxin-free model of acquired epilepsy. Nevertheless, Ant-134 post-treatment reduced the number of rats developing spontaneous seizures by 86% in the perforant pathway stimulation model and Ant-134 delayed epileptiform activity in a rat ex vivo hippocampal slice model. The potent anticonvulsant effects of Ant-134 in multiple models may encourage pre-clinical development of this approach to epilepsy therapy.

Mitochondrial localization of the forkhead box class O transcription factor FOXO3a in brain.

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.