In the 7th issue of the EPITARGET newsletter, we would like to update you about the latest EPITARGET meeting, EPITARGET awardees, EPITARGET publications as well as EPITARGET presentations.

6th General Assembly Meeting in Paris, 17-19 October 2018

The EPITARGET Consortium met in October 2018 in Paris. The meeting was hosted by the EPITARGET partner ARTTIC and took place at Paris International University Campus. At the meeting, all partners updated each other on the work done since the last meeting and discussed the next steps and potential challenges.

The meeting was held back-to-back with the 4^th EPITARGET Young Researchers’ Symposium during which the young investigators of EPITARGET got the chance to present their work in presentations as well as posters. The best oral presentation as well as the best poster were awarded by a committee of experts within the consortium nominated by the coordinator, Merab Kokaia.

 

 

 

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Rossella Di Sapia and Martina Di Nunzio, both PhD students at the Istituto di Ricerche Farmacologiche Mario Negri (IRFMN), were rewarded for posters presenting their work performed within EPITARGET.

 

Rossella Di Sapia won the best poster award at the Italian Society of Neuroscience (Naples, Italy), in the field of "Neuronal excitability and Synaptic Plasticity".

 

Martina Di Nunzio won the best poster award at the meeting "More than Neurons: toward a less neurocentric view of brain disorders" (Torino, Italy).

 

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When an epileptic seizure occurs in the brain, the nerve cells lose their usual pattern and fire in a very fast rhythm. The cause is a complex interplay of various factors. Scientists at the University of Bonn have now discovered the important role of one of the participants: α2δ4 (alpha2delta4). It is a central player between the nerve cells, a puzzle piece that plays a decisive role in the development of epilepsies and is a possible starting point for therapies. The results now appear in The Journal of Neuroscience.

During an epileptic seizure, the nerve cells fire simultaneously in a very fast rhythm - like a thunderstorm in the brain. This results in seizures. Due to changes in the brain, such a short seizure can develop into chronic epilepsy in the long term. "The search for new therapies is above all about preventing the gradual development of such severe types of seizures," says Prof. Dr. Albert J. Becker from the Institute of Neuropathology at the University Hospital Bonn (UKB).

When an epileptic seizure occurs, genes and their regulation are also involved. Genes are blueprints of DNA. Transcripts are made of genes during transcription; these then reach different locations in the nerve cells and influence their function. As the scientists at the University of Bonn were able to demonstrate several years ago, the transcription factor Early growth response 1 (Egr1) upregulates the calcium channels in the nerve cells. Calcium can then increasingly flow into the pores of the nerve cell channels. This causes them to start firing in unison - an epileptic seizure begins.

Search for epilepsy genes

"However, as we have now discovered, the process is much more complex," says Dr. Karen M. J. van Loo, junior research group leader at the UKB. "There are other factors involved." Like in a grid search, the scientist and her UKB colleagues Prof. Dr. Dirk Dietrich from Neurosurgery and Prof. Dr. Sandra Blaess from the Institute of Reconstructive Neurobiology used bioinformatic methods to search for additional epilepsy genes involved in the seizures. The researchers then observed the interaction of the epileptic factors in human tissue obtained during surgical removal of epileptic foci from patients' brains and in mice.

The main focus is on the triangular relationship of the already known transcription factor Egr1 and the specialized calcium channel pores Cav3.2 and α2δ4 (alpha2delta4). Van Loo: "Especially the role of α2δ4 has been underestimated so far." By upregulating the transcription factor in mice, the scientists ensured that their nerve cells produced considerably more α2δ4 in the brain. "The more α2δ4 was present, the greater the tendency to have seizures," summarizes Prof. Dr. Susanne Schoch-McGovern from the Institute of Neuropathology at the University Hospital Bonn. This connection was also confirmed by investigations using human brain tissue.

Like the anti-lock brake system in a car

The nerve cells in the brain normally protect themselves from an epileptic seizure with a stable rhythm. "This can be compared to the anti-lock braking system in a car, which also protects against overreaction," says Becker. Various sensors measure whether a wheel is blocked and then use this information to perform an optimized adjustment of the vehicle's braking force. If α2δ4 is upregulated, the anti-lock braking system in the nerve cell network figuratively fails and the accelerator pedal is fully depressed: The usual nerve cell rhythm gets out of sync and accelerates - an epileptic seizure is imminent.

"It is not enough to look at individual molecules in the brain to understand the onset of an epileptic seizure," says Karen van Loo. "Rather, the whole network must be considered." The scientists regard the calcium channel pores α2δ4 and Cav3.2 in combination with the transcription factor Egr1 to be a promising biomarker for epileptogenesis as well as future therapeutic approach to potentially inhibiting the onset of epilepsies. "But intensive research is still required," emphasizes Susanne Schoch-McGovern.

Figure: Fluorescent signals of Egr1 signaling molecules (mRNA) in green color in neurons within the seizure origin region of the brain. Note the strong co-localization with the Cacna2d4 mRNA, which is well in line with the described functional interplay of these molecules.

 

The study was funded by the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement n°602102 (EPITARGET; AJB,SS) and others.

Publication: Karen M.J. van Loo, Christine K. Rummel, Julika Pitsch, Johannes Alexander Müller, Arthur F. Bikbaev, Erick Martinez-Chavez, Sandra Blaess, Dirk Dietrich, Martin Heine, Albert J. Becker, Susanne Schoch: Calcium channel subunit α2δ4 is regulated by early growth response 1 and facilitates epileptogenesis, The Journal of Neuroscience, DOI: https://doi.org/10.1523/JNEUROSCI.1731-18.2019

 

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Several EPITARGET partners took part in the Jasper's Basic Mechanisms of the Epilepsies Workshops held in Yosemite National Park, March 4-8, 2019 and presented their work in relation to EPITARGET:

Multimodal profiling of epileptogenic phenotype after TBI: machine-learning approach to discover novel biomarkers

Robert Ciszek, Niina Lapinlampi, Eppu Manninen, Pedro Andrade, Ezrie Samuel, Tomi Paananen, Noora Puhakka, Olli Gröhn, Jussi Tohka, Asla Pitkänen*

A.I.Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland. *presenting author: asla.pitkanen@uef.fi

BACKGROUND: Traumatic brain injury (TBI) causes 10-20% of structural epilepsies. The risk of epilepsy varies between 4-53%, depending on the type and severity of TBI. Lack of biomarkers hinder the enrichment of TBI cohorts with subjects at the highest risk of epileptogenesis.

OBJECTIVE: To identify plasma molecular, brain histology, behavioral, magnetic resonance imaging, or electro-encephalogram -derived single or combinatory diagnostic biomarkers for posttraumatic epileptogenesis.

HYPOTHESIS: Parameters signaling from altered network activity alone or together with molecular/structural indicators of brain pathology will indicate subjects who will develop epilepsy with a high sensitivity and specificity.

DATASETS:TBI was induced with lateral fluid-percussion injury. Altogether 154 rats (117 TBI, 25 sham-operated controls, 14 naïve) underwent a 6-month post-TBI follow-up. Molecular, histologic, behavioral, imaging and EEG datasets were collected using common data elements and stored in RedCap electronic database. Normality of data was tested with the Shapiro–Wilk test and groups were compared with t-test or Wilcoxon-Mann-Whitney rank-sum test.

MACHINE LEARNING (ML) PIPELINE: ML pipeline consisted of feature selection, classification, and interpretation steps. Candidate features were selected by utilizing a combination of filter, wrapper and embedding methods. The selected features were used to train gradient boosted trees, support vector machines, logistic regression, k-nearest neighbor, and Gaussian naïve Bayes classifiers to differentiate epileptic rats from non-epileptic. Classification was performed using 10fold cross-validation, with classifier hyper-parameter optimization performed via 10-fold nested grid search. Classifier performance was evaluated in terms of area under the receiver operating characteristic curve (AUC), accuracy, specificity and sensitivity. The feature usage of the classifiers was interpreted to retrace the differentiability of epileptic rats with ML to potential biomarkers in the data.

CURRENT STATUS OF INTERIM BIOMARKER ANALYSIS: From TBI animals, 28% (n=33) developed epilepsy.  From each animal, we derived > 300 features for subsequent analysis. We will present the interim analysis of data currently being entered to the multimodal database.

Cerebro-spinal fluid and blood microRNAs as biomarkers of epilepsy

Michele Simonato

University of Ferrara, Italy University Vita-Salute San Raffaele, Milan, Italy

There is a major unmet need for biomarkers of epilepsy. For example, biomarkers would facilitate epilepsy diagnosis and monitoring, ease antiepileptogenic and antiseizure therapy discovery and reduce the cost of clinical trials by enriching subject populations. Biofluids such as blood and cerebro-spinal fluid (CSF) offer a potential source of molecular biomarkers. In particular, biofluid microRNAs (miRNAs) possess attractive properties, including stability and cheap assay techniques. I will summarize and discuss findings from my lab and others, from both animal models and epilepsy patients. Altered miRNA profiles in biofluids have been found in association with epileptogenesis and epilepsy in animal models, and blood levels of several miRNAs were found altered in patients with epilepsy and in patients with drug-resistant compared to drug-responsive seizures. Although often associated with encouraging receiver-operating characteristic (ROC) curve analyses, these results remain preliminary. In addition, available studies display significant pre-analytical and analytical limitations. I will describe some relevant knowledge gaps and propose experiments to close these. There is an urgent need to define a strategic roadmap to facilitate epilepsy biomarker identification, characterization and clinical validation.

Microvascular Pathology in Seizures and Epileptogenesis: A Mechanism-Driven Biomarker and New Target for Treatment?

Alon Friedman^1,2 and Daniela Kaufer³

1 Ben-Gurion University of the Negev, Beer-Sheva 2 Dalhousie University, Halifax, NS, 3 Berkeley University, CA

Our research on the role blood-brain barrier (BBB) dysfunction in epileptogenesis, revealed a role for leaky BBB and serum albumin in the activation of astrocytes, excitatory synaptogenesis and pathological plasticity in reducing seizure threshold and ictogenesis. Recent studies further indicate that BBB permeability is increased within minutes after seizures, highlighting a new mechanism for their role in epileptogenesis. To test the potential of BBB imaging as a biomarker for the epileptogenic brain, we developed contrast-enhanced magnetic resonance imaging (CE-MRI) protocols for the quantitative assessment of vascular permeability. In the rodent status-epilepticus model, we found that early BBB pathology in specific brain regions (e.g. pyriform cortex) is a sensitive and specific predictor (AUC=0.96, p<0.0001) for epilepsy. We further report focal BBB pathology (most common in the pyriform cortex) in 37% of dogs with epilepsy (n=46). Post-mortem analysis in 3 dogs confirmed uptake of serum albumin in astrocytes. Finally, in 25 patients with epilepsy, CE-MRI revealed interictal focal BBB pathology in >60% of patients, often co-localized with the suspected epileptic region.  We suggest CE- MRI as a diagnostic approach for epilepsy. Clinical trials are expected to confirm the localization and prevalence of microvascular pathology in different epilepsy syndromes, and test the potential of vascular-targeted therapeutics in the treatment of epilepsy.

Characterisation of an infantile rat model of epileptic encephalopathy

Teresa Ravizza, PhD

Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Milano, Italy

Paediatric status epilepticus (SE) results from acquired, metabolic, immune, genetic or unknown causes. We characterized an infantile rat model of de novo SE to study the pathological mechanisms ignited by unremitting seizures in the immature brain that lead to devastating sequelae including cognitive disorders and epilepsy.

Postnatal day (P)13 rats were electrode-implanted for EEG analysis and injected intra-amygdala with 2 µg kainic acid to evoke SE lasting for about 4 h. Immunohistochemical and RT-qPCR analyses showed astrocyte and microglia activation in forebrain and induction of the ictogenic molecules IL-1β and HMGB1 and the oxidative stress marker Nrf2 during one week post-SE. Degenerating neurons were detected.

A cohort of SE-exposed rats was longitudinally video-EEG monitored and exposed to MRI, and behavioral tests were done to evaluate cognition. Epilepsy developed in 60% of P65 rats 1 month post-SE which was similar in onset, severity and duration in all animals. MRI showed progressive brain atrophy before epilepsy onset likely representing a consequence of the SE at P13. Rats displayed impairment in the Morris Water Maze after epilepsy onset thus representing the encephalopathic effects of spontaneous seizures. This rat model can be exploited for mechanistic studies, to test novel antiepiletogenic or disease modifying drugs and for developing biomarkers of disease onset and progression.

Anti-inflammatory strategies for disease-modifications

Annamaria Vezzani

Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Milano, Italy

Neuroinflammation is a common hallmark of epileptogenic brain region in both human epilepsy and related animal models. Experimental evidence shows that neuroinflammation is involved in the pathogenesis of seizures. Notably, the initial clinical studies are supportive of the experimental evidence in animal models. The neuroinflammatory response in epilepsy has generated cellular and molecular targets for the development of potential new drugs, or for repurposing anti-inflammatory drugs which act on specific pathogenic mechanisms. This mechanistic approach aims at overcoming the mere symptomatic control of seizures while providing disease modification effects. In line with this intent, we demonstrated that a combination of anti-inflammatory drugs targeting the IL-1beta system and the Toll-like receptors (TLR4) prevents disease progression after transient administration either during epileptogenesis or after epilepsy onset. Similar therapeutic outcomes were attained by reducing oxidative stress, which is a phenomenon reinforcing neuroinflammation, with a combination of drugs in medical use, or by enhancing the endogenous resolution response. Finally, anti-inflammatory drugs may offer neuroprotection when timely applied during epileptogenesis and improve the neurological comorbidities. Since neuroinflammatory mediators are chiefly produced in glial cells, specific targeting of microglia and astrocytes is under investigation to elucidate the complexity and dynamics of the neuroinflammatory response in epilepsy.

Multi-targeted drug combinations for antiepileptogenesis

Wolfgang Löscher

University of Veterinary Medicine Hannover, Department of Pharmacology, Toxicology and Pharmacy

Epilepsy is a complex network phenomenon that, as yet, cannot be prevented or cured. We recently proposed multitargeted, network-based approaches to prevent epileptogenesis by combining clinically available drugs that were rationally chosen to impact diverse epileptogenic processes. In order to test this strategy preclinically, we developed an algorithm for testing such combinations of repurposed drugs in rodents, derived from human clinical drug development phases, including pharmacokinetics and tolerability testing (Phase I and Phase IIa) as well as evaluation of efficacy (Phase IIb) in mouse and rat models of acquired epilepsy. Overall, 18 drugs, including anti-inflammatory, anti-oxidant, neuroprotective, GABA-potentiating, and anti-glutamatergic drugs, were tested in 12 drug combinations in the intrahippocampal kainate mouse model. As yet, the most effective drug combination was to combine a presynaptically acting drug (levetiracetam) with a postsynaptically acting drug (topiramate), but significant antiepileptogenic or disease-modifying effects were also obtained with several other drug combinations. Next, the most promising drug combinations will be tested in a rat model, in which epilepsy develops after traumatic brain injury. The ultimate goal is to translate our findings to clinical epilepsy prevention trials. Overall, our data provide a rich collection of network-based combinatorial therapies as a basis for antiepileptogenic efficacy testing.

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Blood-Brain Barrier Leakage A New Biomarker in Transient Ischemic Attacks

In Stroke. 2019;50:00-00. doi: 10.1161/ STROKEAHA.119.025247

Yonatan Serlin, MD¹*; Jonathan Ofer, BSc²*; Gal Ben-Arie, MD²*; Ronel Veksler, MD, BSc²;  Gal Ifergane, MD⁴; Ilan Shelef, MD³; Jeffrey Minuk, MD⁵; Anat Horev, MD⁴†; Alon Friedman, MD, PhD^2,6†

1 Neurology Residency Training Program (Y.S.), McGill University, Montreal, QC, Canada

2 Department of Physiology and Cell Biology, Ben-Gurion University of the Negev, Beer-Sheva, Israel (J.O., R.V., A.F.), Soroka Medical Center, Beer-Sheva, Israel

3 Department of Medical Imaging (G.B.-A., I.S.), Soroka Medical Center, Beer-Sheva, Israel

4 Department of Neurology (G.I., A.H.), Soroka Medical Center, Beer-Sheva, Israel

5 Department of Neurology and Neurosurgery, Jewish General Hospital (J.M.), McGill University, Montreal, QC, Canada

6 Department of Medical Neuroscience, Dalhousie University, Halifax, NS, Canada (A.F.).

* These authors have contributed equally.

† Drs Horev and Friedman are joint co-senior authors.

Background and Purpose—The diagnosis of transient ischemic attack is challenging. Evidence of acute ischemia on MRI diffusion-weighted imaging is highly variable and confirmed in only about one-third of patients. This study investigated the significance of blood-brain barrier dysfunction (BBBD) mapping in patients with transient neurological deficits, as a diagnostic and prognostic biomarker required for risk stratification and stroke prevention.

Methods—We used dynamic contrast-enhanced MRI to quantitatively map BBBD in a prospective cohort study of 57 patients diagnosed with transient ischemic attack/minor stroke and 50 healthy controls.

Results—Brain volume with BBBD was significantly higher in patients compared with controls (P=0.002). BBBD localization corresponded with the clinical presentation in 41 patients (72%) and was more extensive in patients with acute infarct on diffusion-weighted imaging (P=0.05). Patients who developed new stroke during follow-up had a significantly greater BBBD at the initial presentation (P=0.03) with a risk ratio of 5.35 for recurrent stroke.

Conclusions—This is the first description of the extent and localization of BBBD in patients with transient ischemic attack/ minor stroke. We propose BBBD mapping as a valuable tool for detection of subtle brain ischemia and a promising predictive biomarker required for risk stratification and stroke prevention.

 

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