Kim L. Powell

The P2X7 Receptor Drives Microglial Activation and Proliferation: A Trophic Role for P2X7R Pore

Microglial activation is an integral part of neuroinflammation associated with many neurodegenerative conditions. Interestingly, a number of neurodegenerative conditions exhibit enhanced P2X7 receptor (P2X7R) expression in the neuroinflammatory foci where activated microglia are a coexisting feature. Whether P2X7R overexpression is driving microglial activation or, conversely, P2X7R overexpression is a consequence of microglial activation is not known. We report that overexpression alone of a purinergic P2X7R, in the absence of pathological insults, is sufficient to drive the activation and proliferation of microglia in rat primary hippocampal cultures. The trophic responses observed in microglia were found to be P2X7R specific as the P2X7R antagonist, oxidized ATP (oxATP), was effective in markedly attenuating microgliosis. oxATP treatment of primary hippocampal cultures expressing exogenous P2X7Rs resulted in a significant decrease in the number of activated microglia. P2X7R is unusual in exhibiting two conductance states, a cation channel and a plasma membrane pore, and there are no pharmacological agents capable of cleanly discriminating between these two states. We used a point mutant of P2X7R (P2X7RG345Y) with intact channel function but ablated pore-forming capacity to establish that the trophic effects of increased P2X7R expression are exclusively mediated by the pore conductance. Collectively, and contrary to previous reports describing P2X7R as a “death receptor,” we provide evidence for a novel trophic role for P2X7R pore in microglia.

A Cav3.2 T-Type Calcium Channel Point Mutation Has Splice-Variant-Specific Effects on Function and Segregates with Seizure Expression in a Polygenic Rat Model of Absence Epilepsy

Low-voltage-activated, or T-type, calcium (Ca2+) channels are believed to play an essential role in the generation of absence seizures in the idiopathic generalized epilepsies (IGEs). We describe a homozygous, missense, single nucleotide (G to C) mutation in the Cav3.2 T-type Ca2+ channel gene (Cacna1h) in the genetic absence epilepsy rats from Strasbourg (GAERS) model of IGE. The GAERS Cav3.2 mutation (gcm) produces an arginine to proline (R1584P) substitution in exon 24 of Cacna1h, encoding a portion of the III–IV linker region in Cav3.2. gcm segregates codominantly with the number of seizures and time in seizure activity in progeny of an F1 intercross. We have further identified two major thalamic Cacna1h splice variants, either with or without exon 25. gcm introduced into the splice variants acts “epistatically,” requiring the presence of exon 25 to produce significantly faster recovery from channel inactivation and greater charge transference during high-frequency bursts. This gain-of-function mutation, the first reported in the GAERS polygenic animal model, has a novel mechanism of action, being dependent on exonic splicing for its functional consequences to be expressed.

T-Type Calcium Channel Blockers That Attenuate Thalamic Burst Firing and Suppress Absence Seizures

Two high-affinity T-type calcium channel blockers attenuate neural activity in the thalamus and suppress seizures in a genetic model of absence epilepsy. To Soothe a Seizure Some epileptic children and adolescents experience “absence” seizures hundreds of times a day. Although apparently mild, these seizures—so named because they involve a sudden, brief absence of consciousness—can be dangerous if they occur during swimming or driving, for example. Unfortunately, the drugs available for treating such seizures are not completely effective. Tringham et al. sought to address this problem by rational drug design. Although the root cause of such seizures is not known, they are associated with abnormal, highly synchronous neuronal activity in certain brain regions. Voltage-gated ion channels, which have crucial functions in generating and propagating neuronal signals, likely play a key role. Several lines of evidence link one type of ion channel, low voltage–activated T-type calcium channels, to absence seizures. Using the structure of an N-type calcium channel blocker as a starting point, the researchers designed and screened small, focused libraries of compounds in a high-throughput assay that monitored calcium influx via a recombinant T-type channel. Two high-affinity T-type calcium channel blockers, termed Z941 and Z944, were identified; Z944 was highly selective for T-type channels and exhibited a preference for inactivated channels (the likely configuration in hyperexcited neurons). In a rat model of absence epilepsy, both compounds markedly reduced the time spent in seizures and the number of seizures per hour. In contrast to current first-line drugs for treating absence seizures, Z941 and Z944 also reduced the average seizure duration and cycle frequency. Both compounds were well tolerated in rats. Given its in vitro and in vivo activities, Z944 will progress to phase 1 clinical studies to test its safety in humans. Further studies will be needed to determine whether its marked effects in the rat model of absence epilepsy translate to the more complicated human condition. Absence seizures are a common seizure type in children with genetic generalized epilepsy and are characterized by a temporary loss of awareness, arrest of physical activity, and accompanying spike-and-wave discharges on an electroencephalogram. They arise from abnormal, hypersynchronous neuronal firing in brain thalamocortical circuits. Currently available therapeutic agents are only partially effective and act on multiple molecular targets, including γ-aminobutyric acid (GABA) transaminase, sodium channels, and calcium (Ca2+) channels. We sought to develop high-affinity T-type specific Ca2+ channel antagonists and to assess their efficacy against absence seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model. Using a rational drug design strategy that used knowledge from a previous N-type Ca2+ channel pharmacophore and a high-throughput fluorometric Ca2+ influx assay, we identified the T-type Ca2+ channel blockers Z941 and Z944 as candidate agents and showed in thalamic slices that they attenuated burst firing of thalamic reticular nucleus neurons in GAERS. Upon administration to GAERS animals, Z941 and Z944 potently suppressed absence seizures by 85 to 90% via a mechanism distinct from the effects of ethosuximide and valproate, two first-line clinical drugs for absence seizures. The ability of the T-type Ca2+ channel antagonists to inhibit absence seizures and to reduce the duration and cycle frequency of spike-and-wave discharges suggests that these agents have a unique mechanism of action on pathological thalamocortical oscillatory activity distinct from current drugs used in clinical practice.

Glutamate is associated with a higher risk of seizures in patients with gliomas

Objective: To investigate the relationship of glutamate and glutamate transporter expression in human gliomas and surrounding peritumoral brain to the presence of tumor-associated seizures (TAS). Methods: We studied a retrospective (group 1: 190 patients) and then a prospective (group 2: 98 patients) cohort of patients who underwent a craniotomy for a supratentorial glioma. Tumor and peritumor tissue specimens were assayed for glutamate concentration and expression of glial glutamate transporters. Differences between the seizure (TAS) and seizure-free (non-TAS) groups were compared. Results: A total of 42% of patients had TAS, with 95% of seizures first occurring preoperatively. Clinical factors independently associated with risk of TAS were younger age, temporal lobe location, and tumors with oligodendroglial components. Molecular features in tumor specimens associated with TAS were higher glutamate concentrations, reduced EAAT2 expression, and increased system Xc− expression. In group 2, these results were also replicated in the peritumor tissue. Logistic regression analysis identified raised glutamate concentrations in tumor and peritumor tissue, increased expression of peritumor system Xc−, younger age, temporal lobe location, and tumors with oligodendroglial components as independently predictive of preoperative seizures. Conclusion: Relative increased glutamate concentration in gliomas, and altered glutamate transporter expression, are associated with the presence of TAS and may play a mechanistic role in their pathogenesis.

Regulators of synaptic transmission: Roles in the pathogenesis and treatment of epilepsy

Synaptic transmission is the communication between a presynaptic and a postsynaptic neuron, and the subsequent processing of the signal. These processes are complex and highly regulated, reflecting their importance in normal brain functioning and homeostasis. Sustaining synaptic transmission depends on the continuing cycle of synaptic vesicle formation, release, and endocytosis, which requires proteins such as dynamin, syndapin, synapsin, and synaptic vesicle protein 2A. Synaptic transmission is regulated by diverse mechanisms, including presynaptic modulators of synaptic vesicle formation and release, postsynaptic receptors and signaling, and modulators of neurotransmission. Neurotransmitters released presynaptically can bind to their postsynaptic receptors, the inhibitory γ‐aminobutyric acid (GABA)ergic receptors or the excitatory glutamate receptors. Once released, glutamate activates a variety of postsynaptic receptors including α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA), N‐methyl‐D‐aspartate (NMDA), kainate, and metabotropic receptors. The activation of the receptors triggers downstream signaling cascades generating a vast array of effects, which can be modulated by a numerous auxiliary regulatory subunits. Moreover, different neuropeptides such as neuropeptide Y, brain‐derived neurotrophic factor (BDNF), somatostatin, ghrelin, and galanin, act as regulators of diverse synaptic functions and along with the classic neurotransmitters. Abnormalities in the regulation of synaptic transmission play a critical role in the pathogenesis of numerous brain diseases, including epilepsy. This review focuses on the different mechanisms involved in the regulation of synaptic transmission, which may play a role in the pathogenesis of epilepsy: the presynaptic modulators of synaptic vesicle formation and release, postsynaptic receptors, and modulators of neurotransmission, including the mechanism by which drugs can modulate the frequency and severity of epileptic seizures.

Ethosuximide reduces epileptogenesis and behavioral comorbidity in the GAERS model of genetic generalized epilepsy

Ethosuximide (ESX) is a drug of choice for the symptomatic treatment of absence seizures. Chronic treatment with ESX has been reported to have disease‐modifying antiepileptogenic activity in the WAG/Rij rat model of genetic generalized epilepsy (GGE) with absence seizures. Here we examined whether chronic treatment with ESX (1) possesses antiepileptogenic effects in the genetic absence epilepsy rats from Strasbourg (GAERS) model of GGE, (2) is associated with a mitigation of behavioral comorbidities, and (3) influences gene expression in the somatosensory cortex region where seizures are thought to originate.

Decreases in HCN mRNA expression in the hippocampus after kindling and status epilepticus in adult rats

Purpose:  Studies in animal models and patients have implicated changes in hyperpolarization‐activated cyclic nucleotide‐gated cation channel (HCN) expression in the pathogenesis of temporal lobe epilepsy (TLE). However, the nature of HCN changes during the epileptogenic process and their commonality across different TLE models is unknown. Here HCN1 and HCN2 mRNA expression was quantitatively measured at different time points during epileptogenesis in two distinct animal models of TLE; the kainic acid (KA)‐induced status epilepticus (SE) and amygdala kindling models.

Low threshold T‐type calcium channels as targets for novel epilepsy treatments

Low voltage‐activated T‐type calcium channels were originally cloned in the 1990s and much research has since focused on identifying the physiological roles of these channels in health and disease states. T‐type calcium channels are expressed widely throughout the brain and peripheral tissues, and thus have been proposed as therapeutic targets for a variety of diseases such as epilepsy, insomnia, pain, cancer and hypertension. This review discusses the literature concerning the role of T‐type calcium channels in physiological and pathological processes related to epilepsy. T‐type calcium channels have been implicated in pathology of both the genetic and acquired epilepsies and several anti‐epileptic drugs (AEDs) in clinical use are known to suppress seizures via inhibition of T‐type calcium channels. Despite the fact that more than 15 new AEDs have become clinically available over the past 20 years at least 30% of epilepsy patients still fail to achieve seizure control, and many patients experience unwanted side effects. Furthermore there are no treatments that prevent the development of epilepsy or mitigate the epileptic state once established. Therefore there is an urgent need for the development of new AEDs that are effective in patients with drug resistant epilepsy, are anti‐epileptogenic and are better tolerated. We also review the mechanisms of action of the current AEDs with known effects on T‐type calcium channels and discuss novel compounds that are being investigated as new treatments for epilepsy.

Seizure expression, behavior, and brain morphology differences in colonies of Genetic Absence Epilepsy Rats from Strasbourg

Originally derived from a Wistar rat strain, a proportion of which displayed spontaneous absence‐type seizures, Genetic Absence Epilepsy Rats from Strasbourg (GAERS) represent the most widely utilized animal model of genetic generalized epilepsy. Here we compare the seizure, behavioral, and brain morphometric characteristics of four main GAERS colonies that are being actively studied internationally: two from Melbourne (MELB and STRAS‐MELB), one from Grenoble (GREN), and one from Istanbul (ISTAN).

Stargazin and AMPA receptor membrane expression is increased in the somatosensory cortex of Genetic Absence Epilepsy Rats from Strasbourg.

Absence-like seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model are believed to arise in hyperexcitable somatosensory cortical neurons, however the cellular basis of this increased excitability remains unknown. We have previously shown that expression of the Transmembrane AMPA receptor Regulatory Protein (TARP), stargazin, is elevated in the somatosensory cortex of GAERS. TARPs are critical regulators of the trafficking and function of AMPA receptors. Here we examine the developmental expression of stargazin and the impact this may have on AMPA receptor trafficking in the GAERS model. We show that elevated stargazin in GAERS is associated with an increase in AMPA receptor proteins, GluA1 and GluA2 in the somatosensory cortex plasma membrane of adult epileptic GAERS. Elevated stargazin expression is not seen in the epileptic WAG/Rij rat, which is a genetically distinct but phenotypically similar rat model also manifesting absence seizures, indicating that the changes seen in GAERS are unlikely to be a secondary consequence of the seizures. In juvenile (6 week old) GAERS, at the age when seizures are just starting to be expressed, there is elevated stargazin mRNA, but not protein expression for stargazin or the AMPA receptor subunits. In neonatal (7 day old) pre-epileptic GAERS there was no alteration in stargazin mRNA expression in any brain region examined. These data demonstrate that stargazin and AMPA receptor membrane targeting is altered in GAERS, potentially contributing to hyperexcitability in somatosensory cortex, with a developmental time course that would suggest a pathophysiological role in the epilepsy phenotype.