Susan L. Campbell

Dnmt1 and Dnmt3a maintain DNA methylation and regulate synaptic function in adult forebrain neurons

Dnmt1 and Dnmt3a are important DNA methyltransferases that are expressed in postmitotic neurons, but their function in the CNS is unclear. We generated conditional mutant mice that lack Dnmt1, Dnmt3a or both exclusively in forebrain excitatory neurons and found that only double knockout (DKO) mice showed abnormal long-term plasticity in the hippocampal CA1 region together with deficits in learning and memory. Although we found no neuronal loss, hippocampal neurons in DKO mice were smaller than in the wild type; furthermore, DKO neurons showed deregulated expression of genes, including the class I MHC genes and Stat1, that are known to contribute to synaptic plasticity. In addition, we observed a significant decrease in DNA methylation in DKO neurons. We conclude that Dnmt1 and Dnmt3a are required for synaptic plasticity, learning and memory through their overlapping roles in maintaining DNA methylation and modulating neuronal gene expression in adult CNS neurons.

Glutamate release by primary brain tumors induces epileptic activity

Epileptic seizures are a common and poorly understood comorbidity for individuals with primary brain tumors. To investigate peritumoral seizure etiology, we implanted human-derived glioma cells into severe combined immunodeficient mice. Within 14–18 d, glioma-bearing mice developed spontaneous and recurring abnormal electroencephalogram events consistent with progressive epileptic activity. Acute brain slices from these mice showed marked glutamate release from the tumor mediated by the system xc− cystine-glutamate transporter (encoded by Slc7a11). Biophysical and optical recordings showed glutamatergic epileptiform hyperexcitability that spread into adjacent brain tissue. We inhibited glutamate release from the tumor and the ensuing hyperexcitability by sulfasalazine (SAS), a US Food and Drug Administration–approved drug that blocks system xc−. We found that acute administration of SAS at concentrations equivalent to those used to treat Crohns disease in humans reduced epileptic event frequency in tumor-bearing mice compared with untreated controls. SAS should be considered as an adjuvant treatment to ameliorate peritumoral seizures associated with glioma in humans.

Functional Expression of Kir4.1 Channels in Spinal Cord Astrocytes

Spinal cord astrocytes (SCA) have a high permeability to K+ and hence have hyperpolarized resting membrane potentials. The underlying K+ channels are believed to participate in the uptake of neuronally released K+. These K+ channels have been studied extensively with regard to their biophysics and pharmacology, but their molecular identity in spinal cord is currently unknown. Using a combination of approaches, we demonstrate that channels composed of the Kir4.1 subunit are responsible for mediating the resting K+ conductance in SCA. Biophysical analysis demonstrates astrocytic Kir currents as weakly rectifying, potentiated by increasing [K+]o, and inhibited by micromolar concentrations of Ba2+. These currents were insensitive to tolbutemide, a selective blocker of Kir6.x channels, and to tertiapin, a blocker for Kir1.1 and Kir3.1/3.4 channels. PCR and Western blot analysis show prominent expression of Kir4.1 in SCA, and immunocytochemistry shows localization Kir4.1 channels to the plasma membrane. Kir4.1 protein levels show a developmental upregulation in vivo that parallels an increase in currents recorded over the same time period. Kir4.1 is highly expressed throughout most areas of the gray matter in spinal cord in vivo and recordings from spinal cord slices show prominent Kir currents. Electrophysiological recordings comparing SCA of wild‐type mice with those of homozygote Kir4.1 knockout mice confirm a complete and selective absence of Kir channels in the knockout mice, suggesting that Kir4.1 is the principle channel mediating the resting K+ conductance in SCA in vitro and in situ.

Reactive Astrogliosis Causes the Development of Spontaneous Seizures

Epilepsy is one of the most common chronic neurologic diseases, yet approximately one-third of affected patients do not respond to anticonvulsive drugs that target neurons or neuronal circuits. Reactive astrocytes are commonly found in putative epileptic foci and have been hypothesized to be disease contributors because they lose essential homeostatic capabilities. However, since brain pathology induces astrocytes to become reactive, it is difficult to distinguish whether astrogliosis is a cause or a consequence of epileptogenesis. We now present a mouse model of genetically induced, widespread chronic astrogliosis after conditional deletion of β1-integrin (Itgβ1). In these mice, astrogliosis occurs in the absence of other pathologies and without BBB breach or significant inflammation. Electroencephalography with simultaneous video recording revealed that these mice develop spontaneous seizures during the first six postnatal weeks of life and brain slices show neuronal hyperexcitability. This was not observed in mice with neuronal-targeted β1-integrin deletion, supporting the hypothesis that astrogliosis is sufficient to induce epileptic seizures. Whole-cell patch-clamp recordings from astrocytes further suggest that the heightened excitability was associated with impaired astrocytic glutamate uptake. Moreover, the relative expression of the cation-chloride cotransporters (CCC) NKCC1 (Slc12a2) and KCC2 (Slc12a5), which are responsible for establishing the neuronal Cl− gradient that governs GABAergic inhibition were altered and the NKCC1 inhibitor bumetanide eliminated seizures in a subgroup of mice. These data suggest that a shift in the relative expression of neuronal NKCC1 and KCC2, similar to that observed in immature neurons during development, may contribute to astrogliosis-associated seizures.

Differential effects of Usp14 and Uch-L1 on the ubiquitin proteasome system and synaptic activity.

The ubiquitin proteasome pathway has been implicated in the pathogenesis of many neurodegenerative diseases, and alterations in two different deubiquitinating enzymes, Uch-L1 and Usp14, result in neurological phenotypes in mice. We identified a new mutation in Uch-L1 and compared the roles of Uch-L1 and Usp14 in the ubiquitin proteasome system. Deficiencies in either Uch-L1 or Usp14 result in decreased levels of ubiquitin, suggesting that they both regulate ubiquitin stability in the nervous system. However, the effect of ubiquitin depletion on viability and onset of symptoms is more severe in the Usp14-deficient mice, and changes in hippocampal synaptic transmission were only observed in Usp14-deficient mice. In addition, while Usp14 appears to function at the proteasome, Uch-L1 deficiency resulted in up-regulation of lysosomal components, indicating that Uch-L1 and Usp14 may differentially affect the ubiquitin proteasome system and synaptic activity by regulating different pools of ubiquitin in the cell.

Glutamate transporters regulate excitability in local networks in rat neocortex.

Excitatory postsynaptic currents (EPSCs) in the neocortex are principally mediated by glutamate receptors. Termination of excitation requires rapid removal of glutamate from the synaptic cleft following release. Glutamate transporters are involved in EPSC termination but the effect of uptake inhibition on excitatory neurotransmission varies by brain region. Epileptiform activity is largely mediated by a synchronous synaptic activation of cells in local cortical circuits, presumably associated with a large release of glutamate. The role of glutamate transporters in regulating epileptiform activity has not been addressed. Here we examine the effect of glutamate transport inhibition on EPSCs and epileptiform events in layer II/III pyramidal cells in rat neocortex. Inhibiting glutamate transporters with DL-threo-beta-benzyloxyaspartic acid (TBOA; 30 microM) had no effect on the amplitude or decay time of evoked, presumably alpha-amino-3-hydroxyl-5-methyl-isoxazolepropionic acid-mediated, EPSCs. In contrast, the amplitude and duration of epileptiform discharges were significantly enhanced. TBOA resulted also in a decreased threshold for evoking epileptiform activity and an increased probability of occurrence of spontaneous epileptiform discharges. TBOAs effects were not inhibited by the group I and II metabotropic glutamate receptors antagonist (S)-alpha-methyl-4-carboxyphenylglycine or the kainate receptor antagonist [(3S,4aR, 6S, 8aR)-6-((4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid]. D-(-)-2-amino-5-phosphonovaleric acid could both prevent excitability changes by TBOA and block already induced changes. Dihydrokainate (300 microM) had effects similar to TBOA suggesting involvement of the glial transporter GLT-1. Inhibiting glutamate transport increases local network excitability under conditions where there is an enhanced release of glutamate. Our results indicate that uptake inhibition produces an elevation of extracellular glutamate levels and activation of N-methyl-D-aspartate receptors.

SLC7A11 expression is associated with seizures and predicts poor survival in patients with malignant glioma

SLC7A11, the catalytic subunit of the cystine/glutamate antiporter, System xc− (SXC), is up-regulated in a subpopulation of patient gliomas, where it is responsible for excitotoxic glutamate release, accelerated tumor growth, and tumor-associated seizures. Seizing an opportunity to study glioma Gliomas are the most common type of malignant brain tumors, and they frequently cause seizures. A new study by Robert et al. uncovers some of the mechanisms involved in this process, showing how a specific cystine/glutamate transporter contributes to excitotoxic glutamate release, causing the death of surrounding cells and inducing seizures. The authors also showed that tumors expressing this transporter were more aggressive and grew more quickly, possibly because the destruction of surrounding normal cells allowed the tumors to expand more rapidly. These findings suggest that the expression of this cystine/glutamate transporter may be useful as a predictor of outcome and a potential therapeutic target in glioma. Glioma is the most common malignant primary brain tumor. Its rapid growth is aided by tumor-mediated glutamate release, creating peritumoral excitotoxic cell death and vacating space for tumor expansion. Glioma glutamate release may also be responsible for seizures, which complicate the clinical course for many patients and are often the presenting symptom. A hypothesized glutamate release pathway is the cystine/glutamate transporter System xc− (SXC), responsible for the cellular synthesis of glutathione (GSH). However, the relationship of SXC-mediated glutamate release, seizures, and tumor growth remains unclear. Probing expression of SLC7A11/xCT, the catalytic subunit of SXC, in patient and mouse-propagated tissues, we found that ~50% of patient tumors have elevated SLC7A11 expression. Compared with tumors lacking this transporter, in vivo propagated and intracranially implanted SLC7A11-expressing tumors grew faster, produced pronounced peritumoral glutamate excitotoxicity, induced seizures, and shortened overall survival. In agreement with animal data, increased SLC7A11 expression predicted shorter patient survival according to genomic data in the REMBRANDT (National Institutes of Health Repository for Molecular Brain Neoplasia Data) database. In a clinical pilot study, we used magnetic resonance spectroscopy to determine SXC-mediated glutamate release by measuring acute changes in glutamate after administration of the U.S. Food and Drug Administration–approved SXC inhibitor, sulfasalazine (SAS). In nine glioma patients with biopsy-confirmed SXC expression, we found that expression positively correlates with glutamate release, which is acutely inhibited with oral SAS. These data suggest that SXC is the major pathway for glutamate release from gliomas and that SLC7A11 expression predicts accelerated growth and tumor-associated seizures.

Decreased glutamate transport enhances excitability in a rat model of cortical dysplasia

Glutamate transporters function to maintain low levels of extracellular glutamate and play an important role in synaptic transmission at many synapses. Disruption of glutamate transporter function or expression can result in increased extracellular glutamate levels. Alterations in glutamate transporter expression have been reported in human epilepsy and animal seizure models. Functional electrophysiological changes that occur when transporter expression is disrupted in chronic epilepsy models have not been examined. Here, we used a freeze-induced model of cortical dysplasia to test the role of glutamate transporters in synaptic hyperexcitability. We report that inhibiting glutamate transporters with the non-selective antagonist, DL-threo-beta-benzylozyaspartic acid (TBOA) preferentially prolongs postsynaptic currents (PSCs) and decreases the threshold for evoking epileptiform activity in lesioned compared to control cortex. The effect of inhibiting uptake is mediated primarily by the glia glutamate transporter (GLT-1) since the selective antagonist dihydrokainate (DHK) mimicked the effects of TBOA. The effect of uptake inhibition is mediated by activation of N-methyl-D-aspartate (NMDA) receptors since D-(-)-2-amino-5-phosphonovaleric acid (APV) prevents TBOA-induced effects. Neurons in lesioned cortex also have a larger tonic NMDA current. These results indicate that chronic changes in glutamate transporters and NMDA receptors contribute to hyperexcitability in cortical dysplasia.

Pre- and postsynaptic effects of kainate on layer II/III pyramidal cells in rat neocortex.

Kainate receptors mediate both direct excitatory and indirect modulatory actions in the CNS. We report here that kainate has both pre- and postsynaptic actions in layer II/III pyramidal neurons of rat prefrontal cortex. Application of low concentration of kainate (50-500 nM) increased the amplitude of evoked excitatory postsynaptic currents (EPSCs) whereas higher concentrations (3 microM) caused a decrease. The frequency of spontaneous and miniature (action potential-independent) EPSCs was increased by low concentrations of kainate without affecting their amplitudes, suggesting a presynaptic mechanism of action. The facilitatory and inhibitory effects of kainate were mimicked by the GluR5 subunit selective agonist ATPA. In addition to decreasing EPSC amplitudes, high concentrations of kainate and ATPA induced an inward current which was not blocked by AMPA- or NMDA-receptor antagonists GYKI52466 and D-APV, respectively. The inward currents were blocked by the AMPA/KA receptor antagonist CNQX, indicating the presence of postsynaptic kainate receptors. Single shock stimulation in the presence of GYKI52466 and D-APV evoked an EPSC which was blocked by CNQX. The GluR5 antagonist LY382884 changed paired-pulse facilitation to paired pulse depression, indicating that synaptically released glutamate can activate presynaptic kainate receptors. These results suggest that kainate receptors containing GluR5 subunits play a major role in glutamatergic transmission in rat neocortex, having both presynaptic modulatory and direct postsynaptic excitatory actions.

GABAergic disinhibition and impaired KCC2 cotransporter activity underlie tumor-associated epilepsy

Seizures frequently accompany gliomas and often escalate to peritumoral epilepsy. Previous work revealed the importance of tumor‐derived excitatory glutamate (Glu) release mediated by the cystine‐glutamate transporter (SXC) in epileptogenesis. We now show a novel contribution of GABAergic disinhibition to disease pathophysiology. In a validated mouse glioma model, we found that peritumoral parvalbumin‐positive GABAergic inhibitory interneurons are significantly reduced, corresponding with deficits in spontaneous and evoked inhibitory neurotransmission. Most remaining peritumoral neurons exhibit elevated intracellular Cl− concentration ([Cl−]i) and consequently depolarizing, excitatory gamma‐aminobutyric acid (GABA) responses. In these neurons, the plasmalemmal expression of KCC2, which establishes the low [Cl−]i required for GABAAR‐mediated inhibition, is significantly decreased. Interestingly, reductions in inhibition are independent of Glu release, but the presence of both decreased inhibition and decreased SXC expression is required for epileptogenesis. We suggest GABAergic disinhibition renders peritumoral neuronal networks hyper‐excitable and susceptible to seizures triggered by excitatory stimuli, and propose KCC2 as a therapeutic target. GLIA 2015;63:23–36