MyD88-mediated signaling is critical for the generation of seizure responses and cognitive impairment in a model of anti-N-methyl-D-aspartate receptor encephalitis.

OBJECTIVE: We previously demonstrated that interleukin-1 receptor-mediated immune activation contributes to seizure severity and memory loss in anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. In the present study, we assessed the role of the myeloid differentiation primary response gene 88 (MyD88), an adaptor protein in Toll-like receptor signaling, in the key phenotypic characteristics of anti-NMDAR encephalitis. METHODS: Monoclonal anti-NMDAR antibodies or control antibodies were infused into the lateral ventricle of MyD88 knockout mice (MyD88-/-) and control C56BL/6J mice (wild type [WT]) via osmotic minipumps for 2 weeks. Seizure responses were measured by electroencephalography. Upon completion of the infusion, the motor, anxiety, and memory functions of the mice were assessed. Astrocytic (glial fibrillary acidic protein [GFAP]) and microglial (ionized calcium-binding adaptor molecule 1 [Iba-1]) activation and transcriptional activation for the principal inflammatory mediators involved in seizures were determined using immunohistochemistry and quantitative real-time polymerase chain reaction, respectively. RESULTS: As shown before, 80% of WT mice infused with anti-NMDAR antibodies (n = 10) developed seizures (median = 11, interquartile range [IQR] = 3-25 in 2 weeks). In contrast, only three of 14 MyD88-/- mice (21.4%) had seizures (0, IQR = 0-.25, p = .01). The WT mice treated with antibodies also developed memory loss in the novel object recognition test, whereas such memory deficits were not apparent in MyD88-/- mice treated with anti-NMDAR antibodies (p = .03) or control antibodies (p = .04). Furthermore, in contrast to the WT mice exposed to anti-NMDAR antibodies, the MyD88-/- mice had a significantly lower induction of chemokine (C-C motif) ligand 2 (CCL2) in the hippocampus (p = .0001, Sidak tests). There were no significant changes in the expression of GFAP and Iba-1 in the MyD88-/- mice treated with anti-NMDAR or control antibodies. SIGNIFICANCE: These findings suggest that MyD88-mediated signaling contributes to the seizure and memory phenotype in anti-NMDAR encephalitis and that CCL2 activation may participate in the expression of these features. The removal of MyD88 inflammation may be protective and therapeutically relevant.

Profound seizure suppression and disease modification by targeting JAK1, a key driver of a pro-epileptogenic gene network.

Epilepsy is the 4th most prevalent neurological disorder with over 50 million cases worldwide. While a number of drugs exist to suppress seizures, approximately 1/3 of patients remain drug resistant, and no current treatments are disease modifying. Using network and systems-based approaches, we find that the histone methylase EZH2 suppresses epileptogenesis and slows disease progression, via repression of JAK1 and STAT3 signaling in hippocampal neurons. Pharmacological inhibition of JAK1 with the orally available, FDA-approved drug CP690550 (Tofacitinib) profoundly suppresses behavioral and electrographic seizures after the onset of epilepsy across preclinical rodent models of acquired epilepsy. This seizure suppression persists for weeks after drug withdrawal. Identification of an endogenous protective response to status epilepticus in the form of EZH2 induction has highlighted a critical role for the JAK1 kinase and STAT3 in both the initiation and propagation of epilepsy across preclinical rodent models and human disease. Overall, we find that STAT3 is transiently activated after insult, reactivates with spontaneous seizures, and remains targetable for disease modification in chronic epilepsy.

Temporal Expression of Neuroinflammatory and Oxidative Stress Markers and Prostaglandin E2 Receptor EP2 Antagonist Effect in a Rat Model of Epileptogenesis.

Traumatic brain injury (TBI) in patients results in a massive inflammatory reaction, disruption of blood-brain barrier, and oxidative stress in the brain, and these inciting features may culminate in the emergence of post-traumatic epilepsy (PTE). We hypothesize that targeting these pathways with pharmacological agents could be an effective therapeutic strategy to prevent epileptogenesis. To design therapeutic strategies targeting neuroinflammation and oxidative stress, we utilized a fluid percussion injury (FPI) rat model to study the temporal expression of neuroinflammatory and oxidative stress markers from 3 to 24 h following FPI. FPI results in increased mRNA expression of inflammatory mediators including cyclooxygenase-2 (COX-2) and prostanoid receptor EP2, marker of oxidative stress (NOX2), astrogliosis (GFAP), and microgliosis (CD11b) in ipsilateral cortex and hippocampus. The analysis of protein levels indicated a significant increase in the expression of COX-2 in ipsilateral hippocampus and cortex post-FPI. We tested FPI rats with an EP2 antagonist TG8-260 which produced a statistically significant reduction in the distribution of seizure duration post-FPI and trends toward a reduction in seizure incidence, seizure frequency, and duration, hinting a proof of concept that EP2 antagonism must be further optimized for therapeutic applications to prevent epileptogenesis.

Preclinical development of an EP2 antagonist for post-seizure cognitive deficits.

Cognitive comorbidities can substantially reduce quality of life in people with epilepsy. Inflammation is a component of all chronic diseases including epilepsy, as well as acute events like status epilepticus (SE). Neuroinflammation is the consequence of several broad signaling cascades including cyclooxygenase-2 (COX-2)-associated pathways. Activation of the EP2 receptor for prostaglandin E2 appears responsible for blood-brain barrier leakage and much of the inflammatory reaction, neuronal injury and cognitive deficit that follows seizure-provoked COX-2 induction in brain. Here we show that brief exposure of mice to TG11-77, a potent, selective, orally available and brain permeant EP2 antagonist, eliminates the profound cognitive deficit in Y-maze performance after SE and reduces delayed mortality and microgliosis, with a minimum effective i.p. dose (as free base) of 8.8 mg/kg. All in vitro studies required to submit an investigational new drug (IND) application for TG11-77 have been completed, and non-GLP dose range-finding toxicology in the rat identified no overt, organ or histopathology signs of toxicity after 7 days of oral administration at 1000 mg/kg/day. Plasma exposure in the rat was dose-linear between 15 and 1000 mg/kg dosing. TG11-77 thus appears poised to continue development towards the initial clinical test of the hypothesis that EP2 receptor modulation after SE can provide the first preventive treatment for one of the chief comorbidities of epilepsy.

Time-dependent neuropathology in rats following organophosphate-induced status epilepticus.

Exposure to high levels of a cholinesterase inhibiting organophosphorus (OP) agent often results in seizures that progress to status epilepticus (SE). Survivors of OP-induced SE often display neuropathological consequences the days following SE. In the current study, the temporal profile of neuropathology after SE was investigated in a rat model of diisopropylfluorophosphate (DFP)-induced SE. Adult Sprague-Dawley rats were injected with DFP to induce SE for one hour. Following termination of electrographic SE with urethane (0.8 g/kg, sc), cohorts of rats were euthanized 3, 24 and 48 h later and brain tissue was processed to determine immediate early gene and inflammatory mediator expression as well as blood-brain barrier changes and neurodegeneration. After SE rats displayed a time-dependent upregulation of immediate early genes such as cFos and ΔFosB as well as pro-inflammatory mediators COX-2, IL-1ß and IL-6. The profile of positive cFos staining, but not ΔFosB, coincided temporally with heightened brain activity measured by cortical electroencephalography (EEG). Neurodegeneration in limbic brain regions was absent 3 h after SE, but prominent 24 h later and continued to increase 48 h after SE. Serum albumin was detected in the cortex 3 h after SE suggesting early loss of blood brain barrier integrity. However, the blood-brain barrier appeared repaired 48 h after SE. This study demonstrates that following OP-poisoning in rats, immediate early gene expression in the brain precedes neuroinflammation followed by erosion of the blood-brain barrier and neurodegeneration. The study also demonstrates that seizure activity in brain nuclei coincides with cFos expression. Together, these studies give insight into the temporal molecular changes in the brain following organophosphate-induced status epilepticus.

Second-Generation Prostaglandin Receptor EP2 Antagonist, TG8-260, with High Potency, Selectivity, Oral Bioavailability, and Anti-Inflammatory Properties.

EP2, a G-protein-coupled prostaglandin-E2 receptor, has emerged as a seminal biological target for drug discovery. EP2 receptor activation is typically proinflammatory; therefore, the development of EP2 antagonists to mitigate the severity and disease pathology in a variety of inflammation-driven central nervous system and peripheral disorders would be a novel strategy. We have recently developed a second-generation EP2 antagonist TG8-260 and shown that it reduces hippocampal neuroinflammation and gliosis after pilocarpine-induced status epilepticus in rats. Here, we present details of synthesis, lead optimization on earlier leads that resulted in TG8-260, potency and selectivity evaluations using cAMP-driven time-resolved fluorescence resonance energy-transfer (TR-FRET) assays and [H3]-PGE2-binding assays, absorption, distribution, metabolism, and excretion (ADME), and pharmacokinetics. TG8-260 (2f) showed Schild K B = 13.2 nM (3.6-fold more potent than the previous lead TG8-69 (1c)) and 500-fold selectivity to EP2 against other prostanoid receptors. Pharmacokinetic data indicated that TG8-260 has a plasma half-life of 2.14 h (PO) and excellent oral bioavailability (77.3%). Extensive ADME tests indicated that TG8-260 is a potent inhibitor of CYP450 enzymes. Further, we show that TG8-260 displays antagonistic activity on the induction of EP2 receptor-mediated inflammatory gene expression in microglia BV2-hEP2 cells; therefore, it can serve as a tool for investigating anti-inflammatory pathways in peripheral inflammatory disease animal models.

Pharmacological antagonism of EP2 receptor does not modify basal cardiovascular and respiratory function, blood cell counts, and bone morphology in animal models.

The EP2 receptor has emerged as a therapeutic target with exacerbating role in disease pathology for a variety of peripheral and central nervous system disorders. We and others have recently demonstrated beneficial effects of EP2 antagonists in preclinical models of neuroinflammation and peripheral inflammation. However, it was earlier reported that mice with global EP2 knockout (KO) display adverse phenotypes on fertility and blood pressure. Other studies indicated that EP2 activation with an agonist has a beneficial effect of healing fractured bone in animal models. These results impeded the development of EP2 antagonists, and EP2 antagonism as therapeutic strategy. To determine whether treatment with EP2 antagonist mimics the adverse phenotypes of the EP2 global KO mouse, we tested two EP2 antagonists TG11-77. HCl and TG6-10-1 in mice and rats while they are on normal or high-salt diet, and by two different administration protocols (acute and chronic). There were no adverse effects of the antagonists on systolic and diastolic blood pressure, heart rate, respiratory function in mice and rats regardless of rodents being on a regular or high salt diet. Furthermore, chronic exposure to TG11-77. HCl produced no adverse effects on blood cell counts, bone-volume and bone-mineral density in mice. Our findings argue against adverse effects on cardiovascular and respiratory systems, blood counts and bone structure in healthy rodents from the use of small molecule reversible antagonists for EP2, in contrast to the genetic ablation model. This study paves the way for advancing therapeutic applications of EP2 antagonists against diseases involving EP2 dysfunction.

Prostaglandin EP2 receptor antagonist ameliorates neuroinflammation in a two-hit mouse model of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) causes substantial medical and societal burden with no therapies ameliorating cognitive deficits. Centralized pathologies involving amyloids, neurofibrillary tangles, and neuroinflammatory pathways are being investigated to identify disease-modifying targets for AD. Cyclooxygenase-2 (COX-2) is one of the potential neuroinflammatory agents involved in AD progression. However, chronic use of COX-2 inhibitors in patients produced adverse cardiovascular effects. We asked whether inhibition of EP2 receptors, downstream of the COX-2 signaling pathway, can ameliorate neuroinflammation in AD brains in presence or absence of a secondary inflammatory stimuli. METHODS: We treated 5xFAD mice and their non-transgenic (nTg) littermates in presence or absence of lipopolysaccharide (LPS) with an EP2 antagonist (TG11-77.HCl). In cohort 1, nTg (no-hit) or 5xFAD (single-hit-genetic) mice were treated with vehicle or TG11-77.HCl for 12 weeks. In cohort 2, nTg (single-hit-environmental) and 5xFAD mice (two-hit) were administered LPS (0.5 mg/kg/week) and treated with vehicle or TG11-77.HCl for 8 weeks. RESULTS: Complete blood count analysis showed that LPS induced anemia of inflammation in both groups in cohort 2. There was no adverse effect of LPS or EP2 antagonist on body weight throughout the treatment. In the neocortex isolated from the two-hit cohort of females, but not males, the elevated mRNA levels of proinflammatory mediators (IL-1ß, TNF, IL-6, CCL2, EP2), glial markers (IBA1, GFAP, CD11b, S110B), and glial proteins were significantly reduced by EP2 antagonist treatment. Intriguingly, the EP2 antagonist had no effect on either of the single-hit cohorts. There was a modest increase in amyloid-plaque deposition upon EP2 antagonist treatment in the two-hit female brains, but not in the single-hit genetic female cohort. CONCLUSION: These results reveal a potential neuroinflammatory role for EP2 in the two-hit 5xFAD mouse model. A selective EP2 antagonist reduces inflammation only in female AD mice subjected to a second inflammatory insult.

Monoclonal Antibodies From Anti-NMDA Receptor Encephalitis Patient as a Tool to Study Autoimmune Seizures.

Anti-N-methyl-D-aspartate (NMDA) receptor encephalitis manifests with precipitous cognitive decline, abnormal movements, and severe seizures that can be challenging to control with conventional anti-seizure medications. We previously demonstrated that intracerebroventricular (i.c.v.) administration of cerebrospinal fluid from affected patients, or purified NMDA receptor antibodies from encephalitis patients to mice precipitated seizures, thereby confirming that antibodies are directly pathogenic for seizures. Although different repertoires of anti-NMDA receptor antibodies could contribute to the distinct clinical manifestations in encephalitis patients, the role of specific antibodies in the expression of seizure, motor, and cognitive phenotypes remains unclear. Using three different patient-derived monoclonal antibodies with distinct epitopes within the N-terminal domain (NTD) of the NMDA receptor, we characterized the seizure burden, motor activity and anxiety-related behavior in mice. We found that continuous administration of 5F5, 2G6 or 3C11 antibodies for 2 weeks precipitated seizures, as measured with continuous EEG using cortical screw electrodes. The seizure burden was comparable in all three antibody-treated groups. The seizures were accompanied by increased hippocampal C-C chemokine ligand 2 (CCL2) mRNA expression 3 days after antibody infusion had stopped. Antibodies did not affect the motor performance or anxiety scores in mice. These findings suggest that neuronal antibodies targeting different epitopes within the NMDA receptor may result in a similar seizure phenotype.

A Glutamate N-Methyl-d-Aspartate (NMDA) Receptor Subunit 2B-Selective Inhibitor of NMDA Receptor Function with Enhanced Potency at Acidic pH and Oral Bioavailability for Clinical Use.

We describe a clinical candidate molecule from a new series of glutamate N-methyl-d-aspartate receptor subunit 2B-selective inhibitors that shows enhanced inhibition at extracellular acidic pH values relative to physiologic pH. This property should render these compounds more effective inhibitors of N-methyl-d-aspartate receptors at synapses responding to a high frequency of action potentials, since glutamate-containing vesicles are acidic within their lumen. In addition, acidification of penumbral regions around ischemic tissue should also enhance selective drug action for improved neuroprotection. The aryl piperazine we describe here shows strong neuroprotective actions with minimal side effects in preclinical studies. The clinical candidate molecule NP10679 has high oral bioavailability with good brain penetration and is suitable for both intravenous and oral dosing for therapeutic use in humans. SIGNIFICANCE STATEMENT: This study identifies a new series of glutamate N-methyl-d-aspartate (NMDA) receptor subunit 2B-selective negative allosteric modulators with properties appropriate for clinical advancement. The compounds are more potent at acidic pH, associated with ischemic tissue, and this property should increase the therapeutic safety of this class by improving efficacy in affected tissue while sparing NMDA receptor block in healthy brain.

Presynaptic Inhibitory Effects of Acetylcholine in the Hippocampus: A 40-Year Evolution of a Serendipitous Finding.

Cholinergic regulation of hippocampal circuit activity has been an active area of neurophysiological research for decades. The prominent cholinergic innervation of intrinsic hippocampal circuitry, potent effects of cholinomimetic drugs, and behavioral responses to cholinergic modulation of hippocampal circuitry have driven investigators to discover diverse cellular actions of acetylcholine in distinct sites within hippocampal circuitry. Further research has illuminated how these actions organize circuit activity to optimize encoding of new information, promote consolidation, and coordinate this with recall of prior memories. The development of the hippocampal slice preparation was a major advance that accelerated knowledge of how hippocampal circuits functioned and how acetylcholine modulated these circuits. Using this preparation in the early 1980s, we made a serendipitous finding of a novel presynaptic inhibitory effect of acetylcholine on Schaffer collaterals, the projections from CA3 pyramidal neurons to dendrites of CA1 pyramidal cells. We characterized this effect at cellular and pharmacological levels, published the findings in the first volume of the Journal of Neuroscience, and proceeded to pursue other scientific directions. We were surprised and thrilled to see that, nearly 40 years later, the paper is still being cited and downloaded because the data became an integral piece of the foundation of the science of cholinergic regulation of hippocampal function in learning and memory. This Progressions article is a story of how single laboratory findings evolve through time to be confirmed, challenged, and reinterpreted by other laboratories to eventually become part of the basis of fundamental concepts related to important brain functions.

Seizures and memory impairment induced by patient-derived anti-N-methyl-D-aspartate receptor antibodies in mice are attenuated by anakinra, an interleukin-1 receptor antagonist.

OBJECTIVE: Neuroinflammation associated with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis may facilitate seizures. We previously showed that intraventricular administration of cerebrospinal fluid from patients with anti-NMDAR encephalitis to mice precipitates seizures, thereby confirming that antibodies are directly pathogenic. To determine whether interleukin (IL)-1-mediated inflammation exacerbates autoimmune seizures, we asked whether blocking the effects of IL-1 by anakinra, a selective IL-1 receptor antagonist, blunts antibody-induced seizures. METHODS: We infused C57BL/6 mice intraventricularly with purified serum IgG from patients with anti-NMDAR encephalitis or monoclonal anti-NMDAR IgG; subdural electroencephalogram was continuously recorded. After a 6-day interval, mice received anakinra (25 mg/kg sc, twice daily) or vehicle for 5 days. Following a 4-day washout period, we performed behavioral tests to assess motor function, anxiety, and memory, followed by hippocampus tissue analysis to assess astrocytic (glial fibrillary acidic protein [GFAP]) and microglial (ionized calcium-binding adapter molecule [Iba]-1) activation. RESULTS: Of 31 mice infused with purified patient NMDAR-IgG (n = 17) or monoclonal NMDAR-IgG (n = 14), 81% developed seizures. Median baseline daily seizure count during exposure to antibodies was 3.9; most seizures were electrographic. Median duration of seizures during the baseline was 82.5 s. Anakinra administration attenuated daily seizure frequency by 60% (p = .02). Anakinra reduced seizure duration; however, the effect was delayed and became apparent only after the cessation of treatment (p = .04). Anakinra improved novel object recognition in mice with antibody-induced seizures (p = .03) but did not alter other behaviors. Anakinra reduced the expression of GFAP and Iba-1 in the hippocampus of mice with seizures, indicating decreased astrocytic and microglial activation. SIGNIFICANCE: Our evidence supports a role for IL-1 in the pathogenesis of seizures in anti-NMDAR encephalitis. These data are consistent with therapeutic effects of anakinra in other severe autoimmune and inflammatory seizure syndromes. Targeting inflammation via blocking IL-1 receptor-mediated signaling may be promising for developing novel treatments for refractory autoimmune seizures.

A Novel Second-Generation EP2 Receptor Antagonist Reduces Neuroinflammation and Gliosis After Status Epilepticus in Rats.

Prostaglandin-E2 (PGE2), an important mediator of inflammation, achieves its functions via four different G protein-coupled receptors (EP1, EP2, EP3, and EP4). We previously demonstrated that the EP2 receptor plays a proinflammatory and neurodegenerative role after status epilepticus (SE). We recently developed TG8-260 as a second-generation highly potent and selective EP2 antagonist. Here, we investigate whether TG8-260 is anti-inflammatory and combats neuropathology caused by pilocarpine-induced SE in rats. Adult male Sprague-Dawley rats were injected subcutaneously with pilocarpine (380-400 mg/kg) to induce SE. Following 60 min of SE, the rats were administered three doses of TG8-260 or vehicle and were allowed to recover. Neurodegeneration, neuroinflammation, gliosis, and blood-brain barrier (BBB) integrity were examined 4 days after SE. The results confirmed that pilocarpine-induced SE results in hippocampal neurodegeneration and a robust inflammatory response that persists days after SE. Furthermore, inhibition of the EP2 receptor by TG8-260 administered beginning 2 h after SE significantly reduced hippocampal neuroinflammation and gliosis but, in distinction to the earlier generation EP2 antagonist, did not mitigate neuronal injury or BBB breakdown. Thus, attenuation of neuroinflammation and gliosis is a common feature of EP2 inhibition following SE.

Comparison of neuropathology in rats following status epilepticus induced by diisopropylfluorophosphate and soman.

The increasing number of cases involving the use of nerve agents as deadly weapons has spurred investigation into the molecular mechanisms underlying nerve agent-induced pathology. The highly toxic nature of nerve agents restrict their use in academic research laboratories. Less toxic organophosphorus (OP) based agents including diisopropylfluorophosphate (DFP) are used as surrogates in academic research laboratories to mimic nerve agent poisoning. However, neuropathology resulting from DFP-induced status epilepticus (SE) has not been compared directly to neuropathology observed following nerve agent poisoning in the same study. Here, the hypothesis that neuropathology measured four days after SE is the same for rats exposed to DFP and soman was tested. Adult Sprague-Dawley rats were injected with soman or DFP to induce SE. Cortical electroencephalography (EEG) was recorded prior to and during soman-induced SE. EEG power analysis of rats administered soman revealed prolonged electrographic SE similar to that of rats that endure uninterrupted SE following injection of DFP. Rats that experienced soman-induced SE displayed less hippocampal neuroinflammation and gliosis compared to rats administered DFP. Seizure-induced weight change, blood-brain barrier (BBB) leakiness and neurodegeneration in most seizure sensitive limbic brain regions were similar for rats that endured SE following soman or DFP. The amalgamated pathology score calculated by combining pathological measures (weight loss, hippocampal neuroinflammation, gliosis, BBB integrity and neurodegeneration) was similar in rats administered the OP agents. These findings support use of the rat DFP model of SE as a suitable surrogate for investigating some, but not all delayed consequences produced by nerve agents.

Peripheral Myeloid Cell EP2 Activation Contributes to the Deleterious Consequences of Status Epilepticus.

A multidimensional inflammatory response ensues after status epilepticus (SE), driven partly by cyclooxygenase-2-mediated activation of prostaglandin EP2 receptors. The inflammatory response is typified by astrocytosis, microgliosis, erosion of the blood-brain barrier (BBB), formation of inflammatory cytokines, and brain infiltration of blood-borne monocytes. Our previous studies have shown that inhibition of monocyte brain invasion or systemic administration of an EP2 receptor antagonist relieves multiple deleterious consequences of SE. Here we identify those effects of EP2 antagonism that are reproduced by conditional ablation of EP2 receptors in immune myeloid cells and show that systemic EP2 antagonism blocks monocyte brain entry in male mice. The induction of hippocampal IL-6 after pilocarpine SE was nearly abolished in EP2 conditional KO mice. Serum albumin levels in the cortex, a measure of BBB breakdown, were significantly higher after SE in EP2-sufficient mice but not in EP2 conditional KOs. EP2 deficiency in innate immune cells accelerated the recovery from sickness behaviors following SE. Surprisingly, neurodegeneration was not alleviated in myeloid conditional KOs. Systemic EP2 antagonism prevented monocyte brain infiltration and provided broader rescue of SE-induced effects than myeloid EP2 ablation, including neuroprotection and broader suppression of inflammatory mediators. Reporter expression indicated that the cellular target of CD11b-driven Cre was circulating myeloid cells but, unexpectedly, not microglia. These findings indicate that activation of EP2 receptors on immune myeloid cells drives substantial deficits in behavior and disrupts the BBB after SE. The benefits of systemic EP2 antagonism can be attributed, in part, to blocking brain recruitment of blood-borne monocytes.SIGNIFICANCE STATEMENT Unabated seizures reduce quality of life, promote the development of epilepsy, and can be fatal. We previously identified activation of prostaglandin EP2 receptors as a driver of undesirable consequences of seizures. However, the relevant EP2-expressing cell types remain unclear. Here we identify peripheral innate immune cells as a driver of the EP2-related negative consequences of seizures. Removal of EP2 from peripheral immune cells was beneficial, abolishing production of a key inflammatory cytokine, accelerating weight regain, and limiting behavioral deficits. These findings provide evidence that EP2 engagement on peripheral immune and brain endothelia contributes to the deleterious effects of SE, and will assist in the development of beneficial therapies to enhance quality of life in individuals who suffer prolonged seizures.

Urethane attenuates early neuropathology of diisopropylfluorophosphate-induced status epilepticus in rats.

Seizures can be evident within minutes of exposure to an organophosphorus (OP) agent and often progress to status epilepticus (SE) resulting in a high mortality if left untreated. Effective medical countermeasures are necessary to sustain patients suffering from OP poisoning and to mitigate the ensuing brain injury. Here, the hypothesis was tested that a single subanesthetic dose of urethane prevents neuropathology measured 24 h following diisopropylfluorophosphate (DFP)-induced SE. Adult Sprague-Dawley rats were injected with DFP to induce SE. During SE rats displayed increased neuronal activity in the hippocampus and an upregulation of immediate early genes as well as pro-inflammatory mediators. In additional experiments rats were administered diazepam (10 mg/kg, ip) or urethane (0.8 g/kg, sc) 1 h after DFP-induced SE and compared to rats that experienced uninterrupted SE. Cortical electroencephalography (EEG) and power analysis strengthen the conclusion that urethane effectively terminates SE and prevents the overnight return of seizure activity. Neurodegeneration in limbic brain regions and the seizure-induced upregulation of key inflammatory mediators present 24 h after DFP-induced SE were strongly attenuated by administration of urethane. A trivial explanation for these beneficial effects, that urethane simply reactivates acetylcholinesterase, has been ruled out. These findings indicate that, by contrast to rats administered diazepam or rats that experience uninterrupted SE, the early neuropathology after SE is prevented by subanesthetic urethane, which terminates rather than interrupts, SE.

A rat model of organophosphate-induced status epilepticus and the beneficial effects of EP2 receptor inhibition.

This review describes an adult rat model of status epilepticus (SE) induced by diisopropyl fluorophosphate (DFP), and the beneficial outcomes of transient inhibition of the prostaglandin-E2 receptor EP2 with a small molecule antagonist, delayed by 2-4 h after SE onset. Administration of six doses of the selective EP2 antagonist TG6-10-1 over a 2-3 day period accelerates functional recovery, attenuates hippocampal neurodegeneration, neuroinflammation, gliosis and blood-brain barrier leakage, and prevents long-term cognitive deficits without blocking SE itself or altering acute seizure characteristics. This work has provided important information regarding organophosphate-induced seizure related pathologies in adults and revealed the effectiveness of delayed EP2 inhibition to combat these pathologies.

Novel Microglia Cell Line Expressing the Human EP2 Receptor.

Recently, EP2 signaling pathways were shown to regulate the classical activation and death of microglia in rat primary microglial culture. The study of microglial cells has been challenging because they are time-consuming to isolate in culture, they are demanding in their growth requirements, and they have a limited lifespan. To circumvent these difficulties, we created a murine BV2 microglial cell line stably expressing human EP2 receptors (BV2-hEP2) and further explored EP2 modulation of microglial functions. The BV2-hEP2 cells displayed cAMP elevation when exposed to the selective EP2 receptor agonists (ONO-AE1-259-1 and CP544326), and this response was competitively inhibited by TG4-155, a selective EP2 antagonist (Schild KB = 2.6 nM). By contrast, untransfected BV2 cells were unresponsive to selective EP2 agonists. Similar to the case of rat primary microglia, BV2-hEP2 microglia treated with lipopolysaccharide (LPS) (100 ng/mL) displayed rapid and robust induction of the inflammatory mediators COX-2, IL-1ß, TNFα, and IL-6. EP2 activation depressed TNFα induction but exacerbated that of the other inflammatory mediators. Like primary microglia, classically activated BV2 microglia phagocytose fluorescent-labeled latex microspheres. The presence of EP2, but not its activation by agonists, in BV2-hEP2 microglia reduced phagocytosis and proliferation by 65% and 32%, respectively, compared to BV2 microglia. Thus, BV2-hEP2 is the first microglial cell line that retains the EP2 modulation of immune regulation and phagocytic ability of native microglia. Suppression of phagocytosis by the EP2 protein appears unrelated to classical EP2 signaling pathways, which has implications for therapeutic development of EP2 antagonists.

5xFAD Mice Display Sex-Dependent Inflammatory Gene Induction During the Prodromal Stage of Alzheimer's Disease.

Alzheimer's disease (AD) pathology consists of extracellular deposits of amyloid-ß peptides (Aß) and intracellular neurofibrillary tangles. These pathological alterations are accompanied by a neuroinflammatory response consisting of increased expression of inflammatory mediators. An anti-inflammatory strategy designed to prevent or delay the development of AD would benefit from knowing when neuroinflammation appears in the transgenic models during prodromal disease stages relative to Aß pathology. We investigated the expression patterns of inflammatory mediators in the brain of 5xFAD mice in comparison to development of Aß deposition. Expression changes in inflammatory mediators and glial markers are more robust in female mice starting at three months of age, in contrast to males in which there is no clear trend through five months. Female and male 5xFAD mice also displayed an age-dependent increase in cortical Aß deposition congruent with neuroinflammation. Thus, in the 5xFAD mouse model of AD, administration of an anti-inflammatory agent would be most efficacious when administered before three months of age.