Nerve growth factor (NGF) binding to p75(NTR) influences TrkA signaling, yet the molecular mechanism is unknown. We observe that NGF stimulates TrkA polyubiquitination, which was attenuated in p75(-/-) mouse brain. TrkA is a substrate of tumor necrosis factor receptor-associated factor 6 (TRAF6), and expression of K63R mutant ubiquitin or an absence of TRAF6 abrogated TrkA polyubiquitination and internalization. NGF stimulated formation of a TrkA/p75(NTR) complex through the p62 scaffold, recruiting the E3/TRAF6 and E2/UbcH7. Peptide targeted to the TRAF6 binding site present in p62 blocked interaction with TRAF6 and inhibited ubiquitination of TrkA, signaling, internalization, and NGF-dependent neurite outgrowth. Mutation of K485 to R blocked TRAF6 and NGF-dependent polyubiquitination of TrkA, resulting in retention of the receptor on the membrane and an absence in activation of specific signaling pathways. These findings reveal that polyubiquitination serves as a common platform for the control of receptor internalization and signaling.

Prostaglandin E2 is now widely recognized to play critical roles in brain inflammation and injury, although the responsible prostaglandin receptors have not been fully identified. We developed a potent and selective antagonist for the prostaglandin E2 receptor subtype EP2, TG6-10-1, with a sufficient pharmacokinetic profile to be used in vivo. We found that in the mouse pilocarpine model of status epilepticus (SE), systemic administration of TG6-10-1 completely recapitulates the effects of conditional ablation of cyclooxygenase-2 from principal forebrain neurons, namely reduced delayed mortality, accelerated recovery from weight loss, reduced brain inflammation, prevention of blood-brain barrier opening, and neuroprotection in the hippocampus, without modifying seizures acutely. Prolonged SE in humans causes high mortality and morbidity that are associated with brain inflammation and injury, but currently the only effective treatment is to stop the seizures quickly enough with anticonvulsants to prevent brain damage. Our results suggest that the prostaglandin receptor EP2 is critically involved in neuroinflammation and neurodegeneration, and point to EP2 receptor antagonism as an adjunctive therapeutic strategy to treat SE.inflammatory cytokine | electroencephalography | epileptogenesis | gliosis | neuronal injury A s a dominant product of cyclooxygenase-2 (COX-2 or PTGS2) in the brain, prostaglandin E2 (PGE 2 ) is emerging as a crucial mediator of many COX-2-driven pathological events in the central nervous system (CNS) (1). PGE 2 acts on four G protein-coupled receptors named EP1, EP2, EP3, and EP4. Among these, the EP2 receptor is widely expressed in the brain and plays important physiologic functions, such as in neuronal plasticity (2, 3). However, recent studies have identified a possible link between EP2 signaling and secondary neurotoxicity in models of chronic inflammation and neurodegeneration (1,(4)(5)(6). In a rodent model of amyotrophic lateral sclerosis, for example, EP2 receptor knockout mice exhibit improved survival, down-regulation of proinflammatory enzymes, and reduced oxidative stress (6).Prolonged status epilepticus (SE) in humans is associated with brain injury and substantial morbidity. Mortality is high during refractory SE that requires general anesthesia (7,8), and the 30-d mortality is about 35-37% for adults who experience at least 60 min of SE (9). Outcome in humans is dependent upon age, etiology, and SE duration (8-10), and currently the only effective treatment is to stop the seizures quickly enough to prevent brain damage (10). Most deaths from nonrefractory SE occur in the 2-wk period after successful treatment rather than during the seizure episode itself (9), pointing to a delayed but cascading set of responsible events. In mice, prolonged SE induced by pilocarpine causes >25% delayed mortality (11), and is associated with a series of molecular and cellular events in the brain, including neurodegeneration, and selective inflammatory reactions involving reactive microglia and a...

With interest waning in the use of cyclooxygenase-2 (COX-2) inhibitors for inflammatory disease, prostaglandin receptors provide alternative targets for the treatment of COX-2-mediated pathological conditions in both the periphery and the central nervous system. Activation of prostaglandin E2 receptor (PGE 2 ) subtype EP2 promotes inflammation and is just beginning to be explored as a therapeutic target. To better understand physiological and pathological functions of the prostaglandin EP2 receptor, we developed a suite of small molecules with a 3-aryl-acrylamide scaffold as selective EP2 antagonists. The 12 most potent compounds displayed competitive antagonism of the human EP2 receptor with K B 2-20 nM in Schild regression analysis and 268-to 4,730-fold selectivity over the prostaglandin EP4 receptor. A brain-permeant compound completely suppressed the up-regulation of COX-2 mRNA in rat cultured microglia by EP2 activation and significantly reduced neuronal injury in hippocampus when administered in mice beginning 1 h after termination of pilocarpine-induced status epilepticus. The salutary actions of this novel group of antagonists raise the possibility that selective block of EP2 signaling via small molecules can be an innovative therapeutic strategy for inflammation-related brain injury.yclooxygenase-2 (COX-2), the inducible isoform of COX, is rapidly up-regulated in damaged tissue, for example in the central nervous system (CNS) after a seizure or cerebral ischemia (1-3). COX-2 induction in CNS overall contributes to inflammation and injury mainly by producing prostanoids (4-7). However, the deleterious cardio-and cerebrovascular side effects from sustained inhibition of COX-2 suggest that some COX-2 downstream prostanoid signaling might be beneficial (8), such that modulation of a specific prostanoid receptor or synthase could be a superior therapeutic strategy compared with generic block of the entire COX-2 cascade. Prostaglandin E2 (PGE 2 ), a dominant enzymatic product of COX-2 in the brain, can activate four Gprotein-coupled receptors (GPCRs): EP1, EP2, EP3, and EP4. Among these, EP2 and EP4 receptors are positively coupled through Gαs to cAMP production (9). In turn, cAMP can initiate multiple downstream events mediated by protein kinase A (PKA) or exchange protein activated by cAMP (Epac) (9).The EP2 receptor is widely expressed in both neurons and glia (3, 10). Neuronal EP2 activation appears to mediate some beneficial effects, such as PKA-dependent neuroprotection in acute models of ischemia and excitotoxicity (3,11,12), early neuroprotection following seizures (13), and promotion of spatial learning (14). Conversely, on the basis of the phenotype of EP2 knockout mice, EP2 activation is thought to promote inflammation and neurotoxicity in animal models of neurodegenerative diseases including Alzheimer's disease (15), Parkinson's disease (16), and amyotrophic lateral sclerosis (10). Glial, especially microglial EP2, is considered to play a major role in brain inflammation associated with chronic ne...

Modulation of a specific prostanoid synthase or receptor provides therapeutic alternatives to non-steroidal anti-inflammatory drugs (NSAIDs) for treating cyclooxygenase-2 (COX-2 or PTGS2)-governed pathological conditions. Among the COX-2 downstream signaling pathways, the prostaglandin E2 (PGE2) receptor EP2 subtype (PTGER2) is emerging as a crucial mediator of many physiological and pathological events. Genetic ablation strategies and recent advances in chemical biology provide tools for a better understanding of EP2 signaling. In the brain, the EP2 receptor modulates some beneficial effects including neuroprotection in acute models of excitotoxicity, neuroplasticity, and spatial learning via cAMP/PKA signaling. Conversely, EP2 activation accentuates chronic inflammation mainly through the cAMP/Epac pathway, likely contributing to delayed neurotoxicity. EP2 receptor activation also engages β-arrestin in a G protein-independent pathway that promotes tumor cell growth and migration. Understanding the conditions under which multiple EP2 signaling pathways are engaged might suggest novel therapeutic strategies targeting this key inflammatory prostaglandin receptor.

As a crucial component of brain innate immunity, neuroinflammation initially contributes to neuronal tissue repair and maintenance. However, chronic inflammatory processes within the brain and associated blood-brain barrier impairment often cause neurotoxicity and hyperexcitability. Mounting evidence points to a mutual facilitation between inflammation and epilepsy, suggesting that blocking the undesired inflammatory signaling within the brain might provide novel strategies to treat seizures and epilepsy. Neuroinflammation is primarily characterized by the upregulation of proinflammatory mediators in epileptogenic foci, among which, cyclooxygenase-2/prostaglandin E2, interleukin-1β, transforming growth factor-β, toll-like receptor 4, high-mobility group box 1, and tumor necrosis factor-α have been extensively studied. Small molecules that specifically target these key proinflammatory perpetrators have been evaluated for antiepileptic and antiepileptogenic effects in animal models. These important preclinical studies provide new insights into the regulation of inflammation in epileptic brains and guide drug discovery efforts aimed at developing novel anti-inflammatory therapies for seizures and epilepsy.

Summary Epilepsy is one of the more prevalent neurological disorders in the world, affecting approximately 50 million people of different ages and backgrounds. Epileptic seizures propagating through both lobes of the forebrain can have permanent debilitating effects on a patient's cognitive and somatosensory brain functions. Epilepsy, defined by the sporadic occurrence of recurrent seizures (SRS), is often accompanied by inflammation of the brain. Pronounced increases in the expression of key inflammatory mediators (e.g. IL-1β, TNFα, cyclooxygenase-2, CXCL10) after seizures may cause secondary damage in the brain and increase the likelihood of repetitive seizures. The cyclooxygenase-2 (COX-2) enzyme is induced rapidly during seizures. The increased level of COX-2 in specific areas of the epileptic brain can help to identify regions of seizure-induced brain inflammation. A good deal of effort has been expended to determine whether COX-2 inhibition might be neuroprotective and represent an adjunct therapeutic strategy along with antiepileptic drugs to treat epilepsy. However, the effectiveness of COX-2 inhibitors on epilepsy animal models appears to depend on the timing of administration. With all of the effort placed on making use of COX-2 inhibitors as therapeutic agents for the treatment of epilepsy, inflammation, and neurodegenerative diseases there has yet to be a selective and potent COX-2 inhibitor that has shown a clear therapeutic outcome with acceptable side effects.

As a prominent inflammatory effector of cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) mediates brain inflammation and injury in many chronic central nervous system (CNS) conditions including seizures and epilepsy, largely through its receptor subtype EP2. However, EP2 receptor activation might also be neuroprotective in models of excitotoxicity and ischemia. These seemingly incongruent observations expose the delicacy of immune and inflammatory signaling in the brain, thus the therapeutic window for quelling neuroinflammation might vary with injury type and target molecule. Here, we identify a therapeutic window for EP2 antagonism to reduce delayed mortality and functional morbidity after status epilepticus (SE) in mice. Importantly, treatment must be delayed relative to SE onset to be effective, a finding that could be explained by the time-course of COX-2 induction after SE and compound pharmacokinetics. A large number of inflammatory mediators were upregulated in hippocampus after SE with COX-2 and IL-1β temporally leading many others. Thus, EP2 antagonism represents a novel anti-inflammatory strategy to treat SE with a tightly-regulated therapeutic window.

Activation of the Gαs-coupled EP2 receptor for prostaglandin E2 (PGE 2 ) promotes cell survival in several models of tissue damage. To advance understanding of EP2 functions, we designed experiments to develop allosteric potentiators of this key prostaglandin receptor. Screens of 292,000 compounds identified 93 that at 20 μM (i) potentiated the cAMP response to a low concentration of PGE 2 by > 50%; (ii) had no effect on EP4 or β2 adrenergic receptors, the cAMP assay itself, or the parent cell line; and (iii) increased the potency of PGE 2 on EP2 receptors at least 3-fold. In aqueous solution, the active compounds are largely present as nanoparticles that appear to serve as active reservoirs for bioactive monomer. From 94 compounds synthesized or purchased, based on the modification of one hit compound, the most active increased the potency of PGE 2 on EP2 receptors 4-to 5-fold at 10 to 20 μM and showed substantial neuroprotection in an excitotoxicity model. These small molecules represent previously undescribed allosteric modulators of a PGE 2 receptor. Our results strongly reinforce the notion that activation of EP2 receptors by endogenous PGE 2 released in a cell-injury setting is neuroprotective.excitotoxicity | neuronal injury | prostaglandin E2 | time-resolved fluorescence resonance energy transfer | ultra high-throughput screening C yclooxygenase-2 (COX2), which is constitutively expressed at low to moderate levels in both neuronal cell bodies and dendritic spines in the hippocampus, is regulated by synaptic activity (1) and is rapidly induced in neurons after a seizure (2) or cerebral ischemia (3,4). Studies in rodents have demonstrated that COX2 activation by ischemia or status epilepticus generally contributes to neuronal injury (5-8), but multiple downstream COX2 signaling pathways suggest that the mechanisms promoting and opposing brain injury are complex. Among the enzymatic products of COX2 is prostaglandin E2 (PGE 2 ), which can activate four G protein-coupled receptors (GPCRs): EP1, EP2, EP3, and EP4. Activation of the EP2 receptor by PGE 2 appears to be neuroprotective after ischemia (3, 4), whereas EP1 activation promotes neurodegeneration (8). The yin-yang nature of the PGE 2 receptor family demonstrates the value of a neuroprotection strategy involving modulation of a specific prostanoid receptor rather than generic block of the entire COX2 signaling cascade.EP2 is a Gαs-coupled receptor that, when activated by PGE 2 , stimulates adenylate cyclase, resulting in elevation of cytoplasmic cAMP (cAMP) level. In addition to its suspected neuroprotective role, EP2 activation has also been shown to promote spatial learning (9), to improve survival of epithelial cells after radiation injury (10), to accelerate bone healing after fracture (11), and to improve renal function in a HgCl 2 model of chronic renal failure (12). On the other hand, EP2 activation in microglia could promote inflammation and neurotoxicity in some neurodegenerative disease models, such as Alzheimer's disease and amyotrophic late...