Federica Frigerio

Misplaced NMDA receptors in epileptogenesis contribute to excitotoxicity

Pharmacological blockade of NR2B-containing N-methyl-d-aspartate receptors (NMDARs) during epileptogenesis reduces neurodegeneration provoked in the rodent hippocampus by status epilepticus. The functional consequences of NMDAR activation are crucially influenced by their synaptic vs extrasynaptic localization, and both NMDAR function and localization are dependent on the presence of the NR2B subunit and its phosphorylation state. We investigated whether changes in NR2B subunit phosphorylation, and alterations in its neuronal membrane localization and cellular expression occur during epileptogenesis, and if these changes are involved in neuronal cell loss. We also explored NR2B subunit changes both in the acute phase of status epilepticus and in the chronic phase of spontaneous seizures which encompass the epileptogenesis phase. Levels of Tyr1472 phosphorylated NR2B subunit decreased in the post-synaptic membranes from rat hippocampus during epileptogenesis induced by electrical status epilepticus. This effect was concomitant with a reduced interaction between NR2B and post-synaptic density (PSD)-95 protein, and was associated with decreased CREB phosphorylation. This evidence suggests an extra-synaptic localization of NR2B subunit in epileptogenesis. Accordingly, electron microscopy showed increased NR2B both in extra-synaptic and pre-synaptic neuronal compartments, and a concomitant decrease of this subunit in PSD, thus indicating a shift in NR2B membrane localization. De novo expression of NR2B in activated astrocytes was also found in epileptogenesis indicating ectopic receptor expression in glia. The NR2B phosphorylation changes detected at completion of status epilepticus, and interictally in the chronic phase of spontaneous seizures, are predictive of receptor translocation from synaptic to extrasynaptic sites. Pharmacological blockade of NR2B-containing NMDARs by ifenprodil administration during epileptogenesis significantly reduced pyramidal cell loss in the hippocampus, showing that the observed post-translational and cellular changes of NR2B subunit contribute to excitotoxicity. Therefore, pharmacological targeting of misplaced NR2B-containing NMDARs, or prevention of these NMDAR changes, should be considered to block excitotoxicity which develops after various pro-epileptogenic brain injuries.

Albumin induces excitatory synaptogenesis through astrocytic TGF-β/ALK5 signaling in a model of acquired epilepsy following blood–brain barrier dysfunction

Post-injury epilepsy (PIE) is a common complication following brain insults, including ischemic, and traumatic brain injuries. At present, there are no means to identify the patients at risk to develop PIE or to prevent its development. Seizures can occur months or years after the insult, do not respond to anti-seizure medications in over third of the patients, and are often associated with significant neuropsychiatric morbidities. We have previously established the critical role of blood-brain barrier dysfunction in PIE, demonstrating that exposure of brain tissue to extravasated serum albumin induces activation of inflammatory transforming growth factor beta (TGF-β) signaling in astrocytes and eventually seizures. However, the link between the acute astrocytic inflammatory responses and reorganization of neural networks that underlie recurrent spontaneous seizures remains unknown. Here we demonstrate in vitro and in vivo that activation of the astrocytic ALK5/TGF-β-pathway induces excitatory, but not inhibitory, synaptogenesis that precedes the appearance of seizures. Moreover, we show that treatment with SJN2511, a specific ALK5/TGF-β inhibitor, prevents synaptogenesis and epilepsy. Our findings point to astrocyte-mediated synaptogenesis as a key epileptogenic process and highlight the manipulation of the TGF-β-pathway as a potential strategy for the prevention of PIE.

Long-lasting pro-ictogenic effects induced in vivo by rat brain exposure to serum albumin in the absence of concomitant pathology.

Purpose:  Dysfunction of the blood–brain barrier (BBB) is a common finding during seizures or following epileptogenic brain injuries, and experimentally induced BBB opening promotes seizures both in naive and epileptic animals. Brain albumin extravasation was reported to promote hyperexcitability by inducing astrocytes dysfunction. To provide in vivo evidence for a direct role of extravasated serum albumin in seizures independently on the pathologic context, we did the following: (1) quantified the amount of serum albumin extravasated in the rat brain parenchyma during status epilepticus (SE); (2) reproduced a similar concentration in the hippocampus by intracerebroventricular (i.c.v.) albumin injection in naive rats; (3) measured electroencephalography (EEG) activity in these rats, their susceptibility to kainic acid (KA)–induced seizures, and their hippocampal afterdischarge threshold (ADT).

Modulation of neuronal excitability by immune mediators in epilepsy.

A complex set of inflammatory molecules and their receptors has been described in epileptogenic foci in different forms of pharmacoresistant epilepsies. By activating receptor-mediated pathways in neurons, these molecules have profound neuromodulatory effects that are distinct from their canonical activation of immune functions. Importantly, the neuromodulatory actions of some inflammatory molecules contribute to hyperexcitability in neural networks that underlie seizures. This review summarizes recent findings related to the role of cytokines (IL-1beta and TNF-alpha) and danger signals (HMGB1) in decreasing seizure threshold, thereby contributing to seizure generation and the associated neuropathology. We will discuss preclinical studies suggesting that pharmacological inhibition of specific inflammatory signals may be useful to treat drug-resistant seizures in human epilepsy, and possibly arrest epileptogenesis in individuals at risk of developing the disease.

Molecular isoforms of high-mobility group box 1 are mechanistic biomarkers for epilepsy

Approximately 30% of epilepsy patients do not respond to antiepileptic drugs, representing an unmet medical need. There is evidence that neuroinflammation plays a pathogenic role in drug-resistant epilepsy. The high-mobility group box 1 (HMGB1)/TLR4 axis is a key initiator of neuroinflammation following epileptogenic injuries, and its activation contributes to seizure generation in animal models. However, further work is required to understand the role of HMGB1 and its isoforms in epileptogenesis and drug resistance. Using a combination of animal models and sera from clinically well-characterized patients, we have demonstrated that there are dynamic changes in HMGB1 isoforms in the brain and blood of animals undergoing epileptogenesis. The pathologic disulfide HMGB1 isoform progressively increased in blood before epilepsy onset and prospectively identified animals that developed the disease. Consistent with animal data, we observed early expression of disulfide HMGB1 in patients with newly diagnosed epilepsy, and its persistence was associated with subsequent seizures. In contrast with patients with well-controlled epilepsy, patients with chronic, drug-refractory epilepsy persistently expressed the acetylated, disulfide HMGB1 isoforms. Moreover, treatment of animals with antiinflammatory drugs during epileptogenesis prevented both disease progression and blood increase in HMGB1 isoforms. Our data suggest that HMGB1 isoforms are mechanistic biomarkers for epileptogenesis and drug-resistant epilepsy in humans, necessitating evaluation in larger-scale prospective studies.

Cognitive deficits and brain myo-Inositol are early biomarkers of epileptogenesis in a rat model of epilepsy

One major unmet clinical need in epilepsy is the identification of therapies to prevent or arrest epilepsy development in patients exposed to a potential epileptogenic insult. The development of such treatments has been hampered by the lack of non-invasive biomarkers that could be used to identify the patients at-risk, thereby allowing to design affordable clinical studies. Our goal was to test the predictive value of cognitive deficits and brain astrocyte activation for the development of epilepsy following a potential epileptogenic injury. We used a model of epilepsy induced by pilocarpine-evoked status epilepticus (SE) in 21-day old rats where 60-70% of animals develop spontaneous seizures after around 70days, although SE is similar in all rats. Learning was evaluated in the Morris water-maze at days 15 and 65 post-SE, each time followed by proton magnetic resonance spectroscopy for measuring hippocampal myo-Inositol levels, a marker of astrocyte activation. Rats were video-EEG monitored for two weeks at seven months post-SE to detect spontaneous seizures, then brain histology was done. Behavioral and imaging data were retrospectively analysed in epileptic rats and compared with non-epileptic and control animals. Rats displayed spatial learning deficits within three weeks from SE. However, only epilepsy-prone rats showed accelerated forgetting and reduced learning rate compared to both rats not developing epilepsy and controls. These deficits were associated with reduced hippocampal neurogenesis. myo-Inositol levels increased transiently in the hippocampus of SE-rats not developing epilepsy while this increase persisted until spontaneous seizures onset in epilepsy-prone rats, being associated with a local increase in S100β-positive astrocytes. Neuronal cell loss was similar in all SE-rats. Our data show that behavioral deficits, together with a non-invasive marker of astrocyte activation, predict which rats develop epilepsy after an acute injury. These measures have potential clinical relevance for identifying individuals at-risk for developing epilepsy following exposure to epileptogenic insults, and consequently, for designing adequately powered antiepileptogenesis trials.

Electrocorticographic dynamics as a novel biomarker in five models of epileptogenesis

Postinjury epilepsy (PIE) is a devastating sequela of various brain insults. While recent studies offer novel insights into the mechanisms underlying epileptogenesis and discover potential preventive treatments, the lack of PIE biomarkers hinders the clinical implementation of such treatments. Here we explored the biomarker potential of different electrographic features in five models of PIE. Electrocorticographic or intrahippocampal recordings of epileptogenesis (from the insult to the first spontaneous seizure) from two laboratories were analyzed in three mouse and two rat PIE models. Time, frequency, and fractal and nonlinear properties of the signals were examined, in addition to the daily rate of epileptiform spikes, the relative power of five frequency bands (theta, alpha, beta, low gamma, and high gamma) and the dynamics of these features over time. During the latent pre-seizure period, epileptiform spikes were more frequent in epileptic compared with nonepileptic rodents; however, this feature showed limited predictive power due to high inter- and intra-animal variability. While nondynamic rhythmic representation failed to predict epilepsy, the dynamics of the theta band were found to predict PIE with a sensitivity and specificity of >90%. Moreover, theta dynamics were found to be inversely correlated with the latency period (and thus predict the onset of seizures) and with the power change of the high-gamma rhythm. In addition, changes in theta band power during epileptogenesis were associated with altered locomotor activity and distorted circadian rhythm. These results suggest that changes in theta band during the epileptogenic period may serve as a diagnostic biomarker for epileptogenesis, able to predict the future onset of spontaneous seizures. SIGNIFICANCE STATEMENT Postinjury epilepsy is an unpreventable and devastating disorder that develops following brain injuries, such as traumatic brain injury and stroke, and is often associated with neuropsychiatric comorbidities. As PIE affects as many as 20% of brain-injured patients, reliable biomarkers are imperative before any preclinical therapeutics can find clinical translation. We demonstrate the capacity to predict the epileptic outcome in five different models of PIE, highlighting theta rhythm dynamics as a promising biomarker for epilepsy. Our findings prompt the exploration of theta dynamics (using repeated electroencephalographic recordings) as an epilepsy biomarker in brain injury patients.

High Mobility Group Box 1 is a novel pathogenic factor and a mechanistic biomarker for epilepsy

Approximately 30% of epilepsy patients experience seizures that are not controlled by the available drugs. Moreover, these drugs provide mainly a symptomatic treatment since they do not interfere with the diseases mechanisms. A mechanistic approach to the discovery of key pathogenic brain modifications causing seizure onset, recurrence and progression is instrumental for designing novel and rationale therapeutic interventions that could modify the disease course or prevent its development. In this regard, increasing evidence shows that neuroinflammation is a pathogenic factor in drug-resistant epilepsies. The High Mobility Group Box 1 (HMGB1)/Toll-like receptor 4 axis is a key initiator of neuroinflammation following brain injuries leading to epilepsy, and its activation contributes to seizure mechanisms in animal models. Recent findings have shown dynamic changes in HMGB1 and its isoforms in the brain and blood of animals exposed to acute brain injuries and undergoing epileptogenesis, and in surgically resected epileptic foci in humans. HMGB1 isoforms reflect different pathophysiological processes, and the disulfide isoform, which is generated in the brain during oxidative stress, is implicated in seizures, cell loss and cognitive dysfunctions. Interfering with disulfide HMGB1-activated cell signaling mediates significant therapeutic effects in epilepsy models. Moreover, both clinical and experimental data suggest that HMGB1 isoforms may serve as mechanistic biomarkers for epileptogenesis and drug-resistant epilepsy. These novel findings suggest that the HMGB1 system could be targeted to prevent seizure generation and may provide clinically useful prognostic biomarkers which may also predict the patients response to therapy.

The Early Phases of Epileptogenesis Induced by Status Epilepticus Are Characterized by Persistent Dynamical Regime of Intermittency Type

Pre-clinical studies aimed to test potential anti-epileptogenic therapies by using the animal models of epileptogenesis induced by status epilepticus (SE), highlighted that the early days following the end of this primary insult represent a crucial temporal window for the subsequent development of epilepsy. In this study, we characterized the EEG dynamics during such crucial period of epileptogenesis, according to the conceptual framework of nonlinear dynamical systems. To this aim, we analyzed by recurrence quantification analysis (RQA) the EEG signals associated to the early days of epileptogenesis induced by SE in rodents according to two well-known experimental protocols, i.e., (i) SE induced by electrical stimulation of the hippocampus in rats (n = 7) and (ii) SE induced by the intra-amygdala administration of kainic-acid in mice (n = 6). We show that the EEG signals during the early 1–2 days post-SE are characterized by an enhanced and persistent rate of occurrence of dynamical regimes of intermittency type. This finding is common to both models of SE, hence it could represent the dynamical hallmark of pro-epileptogenic insults and could correlate with the efficacy of such insults to promote functional changes leading to the development of epilepsy. Future works aimed to deepen our findings could lead to the identification of a potential prognostic factor of the development of epilepsy as well as improve the portability of pre-clinical studies aimed to target new potential therapeutics designed to prevent the development of epilepsy.

n-3 Docosapentaenoic acid-derived protectin D1 promotes resolution of neuroinflammation and arrests epileptogenesis

Frigerio et al. report that pro-resolving mechanisms of neuroinflammation are dysregulated during epileptogenesis, thereby promoting a persistent neuroinflammatory response that contributes to seizure generation and cognitive deficits. Boosting endogenous resolution responses by administering a specific lipid mediator improves disease outcomes in a murine epilepsy model, suggesting a novel treatment avenue.