Antisense Knockdown of GLAST, a Glial Glutamate Transporter, Compromises Retinal Function

PURPOSE. TO elucidate the role of the glial glutamate transporter GLAST, in the regulation of retinal function. METHODS. Antisense oligonucleotides to GLAST were injected intravitreally into the left eye of Wistar rats. Sense oligonucleotides (control) were injected into the right eye over a period of ? days. Scotopic flash electroretinograms were recorded over a 20-day period. To assay whether the antisense oligonucleotides caused a reduction in the expression or the activity of GLAST, retinas were exposed to D-aspartate, a nonendogenous substrate of glutamate transporters. The retinas were immunolabeled with specific antibodies for D-aspartate. Retinal GLAST and glutamate distributions also were determined immunocytochemically. RESULTS. Antisense oligonucleotides markedly suppressed the electroretinogram b-wave, whereas sense oligonucleotides had no significant effect. Significant changes in the electroretinogram were apparent 5 days after injection of antisense oligonucleotide and were sustained for at least 20 days. A marked reduction of D-aspartate uptake into Muller cells of retinas that had been exposed to the antisense oligonucleotides 5 days previously suggests a reduction of GLAST activity. The retinas, however, displayed no evidence of excitotoxic neuronal degeneration, and the distribution of glutamate was unaffected by antisense treatment. CONCLUSIONS. The observed lack of neuronal degeneration suggests that reduced glutamate uptake into Muller cells does not cause excitotoxic tissue damage. A direct perturbation of glutamatergic signaling is more likely, because the rapid clearance of glutamate is necessary for light elicited signaling between photoreceptors and bipolar cells. This suggests that GLAST is essential for the maintenance of normal retinal transmission

Aberrant expression of the glutamate transporter excitatory amino acid transporter 1 (EAAT1) in Alzheimer's disease

Glutamate-mediated toxicity has been implicated in the neurodegeneration observed in Alzheimer's disease. In particular, glutamate transport dysfunction may increase susceptibility to glutamate toxicity, thereby contributing to neuronal cell injury and death. In this study, we examined the cellular localization of the glial glutamate transporter excitatory amino acid transporter 1 (EAAT1) in the cerebral cortex of control, Alzheimer's disease, and non-Alzheimer dementia cases. We found that EAAT1 was strongly expressed in a subset of cortical pyramidal neurons in dementia cases showing Alzheimer-type pathology. In addition, tau (which is a marker of neurofibrillary pathology) colocalized to those same pyramidal cells that expressed EAAT1. These findings suggest that EAAT1 changes are related to tau expression (and hence neurofibrillary tangle formation) in dementia cases showing Alzheimer-type pathology. This study implicates aberrant glutamate transporter expression as a mechanism involved in neurodegeneration in Alzheimer's disease

A novel splice variant of the Excitatory Amino Acid Transporter 5: cloning, immunolocalization and functional characterization of hEAAT5v in human retina

Excitatory Amino Acid Transporter 5 (EAAT5) is abundantly expressed by retinal photoreceptors and bipolar cells, where it acts as a slow glutamate transporter and a glutamate-gated chloride channel. The chloride conductance is large enough for EAAT5 to serve as an “inhibitory” glutamate receptor. Our recent work in rodents has shown that EAAT5 is differentially spliced and exists in many variant forms. The chief aim of the present study was to examine whether EAAT5 is also alternately spliced in human retina and, if so, what significance this might have for retinal function in health and disease. Retinal tissues from human donor eyes were used in RT-PCR to amplify the entire coding region of EAAT5. Amplicons of differing sizes were sub-cloned and analysis of sequenced data revealed the identification of wild-type human EAAT5 (hEAAT5) and an abundant alternately spliced form, referred to as hEAAT5v, where the open reading frame is expanded by insertion of an additional exon. hEAAT5v encodes a protein of 619 amino acids and when expressed in COS7 cells, the protein functioned as a glutamate transporter. We raised antibodies that selectively recognized the hEAAT5v protein and have performed immunocytochemistry to demonstrate expression in photoreceptors in human retina. We noted that in retinas afflicted by dry aged-related macular degeneration (AMD), there was a loss of hEAAT5v from the lesioned area and from photoreceptors adjacent to the lesion. We conclude that hEAAT5v protein expression may be perturbed in peri-lesional areas of AMD-afflicted retinas that do not otherwise exhibit evidence of damage. The loss of hEAAT5v could, therefore, represent an early pathological change in the development of AMD and might be involved in its aetiology

Reduction in Phencyclidine Induced Sensorimotor Gating Deficits in the Rat Following Increased System Xc − Activity in the Medial Prefrontal Cortex

Rationale: Aspects of schizophrenia, including deficits in sensorimotor gating, have been linked to glutamate dysfunction and/or oxidative stress in the prefrontal cortex. System xc −, a cystine–glutamate antiporter, is a poorly understood mechanism that contributes to both cellular antioxidant capacity and glutamate homeostasis. Objectives: Our goal was to determine whether increased system xc − activity within the prefrontal cortex would normalize a rodent measure of sensorimotor gating. Methods: In situ hybridization was used to map messenger RNA (mRNA) expression of xCT, the active subunit of system xc −, in the prefrontal cortex. Prepulse inhibition was used to measure sensorimotor gating; deficits in prepulse inhibition were produced using phencyclidine (0.3–3 mg/kg, sc). N-Acetylcysteine (10–100 μM) and the system xc − inhibitor (S)-4-carboxyphenylglycine (CPG, 0.5 μM) were used to increase and decrease system xc − activity, respectively. The uptake of 14C-cystine into tissue punches obtained from the prefrontal cortex was used to assay system xc − activity. Results: The expression of xCT mRNA in the prefrontal cortex was most prominent in a lateral band spanning primarily the prelimbic cortex. Although phencyclidine did not alter the uptake of 14C-cystine in prefrontal cortical tissue punches, intraprefrontal cortical infusion of N-acetylcysteine (10–100 μM) significantly reduced phencyclidine- (1.5 mg/kg, sc) induced deficits in prepulse inhibition. N-Acetylcysteine was without effect when coinfused with CPG (0.5 μM), indicating an involvement of system xc −. Conclusions: These results indicate that phencyclidine disrupts sensorimotor gating through system xc − independent mechanisms, but that increasing cystine–glutamate exchange in the prefrontal cortex is sufficient to reduce behavioral deficits produced by phencyclidine

Phase II study of induction chemotherapy with TPF followed by radioimmunotherapy with Cetuximab and intensity-modulated radiotherapy (IMRT) in combination with a carbon ion boost for locally advanced tumours of the oro-, hypopharynx and larynx - TPF-C-HIT

<p>Abstract</p> <p>Background</p> <p>Long-term locoregional control in locally advanced squamous cell carcinoma of the head and neck (SCCHN) remains challenging. While recent years have seen various approaches to improve outcome by intensification of treatment schedules through introduction of novel induction and combination chemotherapy regimen and altered fractionation regimen, patient tolerance to higher treatment intensities is limited by accompanying side-effects. Combined radioimmunotherapy with cetuximab as well as modern radiotherapy techniques such as intensity-modulated radiotherapy (IMRT) and carbon ion therapy (C12) are able to limit toxicity while maintaining treatment effects. In order to achieve maximum efficacy with yet acceptable toxicity, this sequential phase II trial combines induction chemotherapy with docetaxel, cisplatin, and 5-FU (TPF) followed by radioimmunotherapy with cetuximab as IMRT plus carbon ion boost. We expect this approach to result in increased cure rates with yet manageable accompanying toxicity.</p> <p>Methods/design</p> <p>The TPF-C-HIT trial is a prospective, mono-centric, open-label, non-randomized phase II trial evaluating efficacy and toxicity of the combined treatment with IMRT/carbon ion boost and weekly cetuximab in 50 patients with histologically proven locally advanced SCCHN following TPF induction chemotherapy. Patients receive 24 GyE carbon ions (8 fractions) and 50 Gy IMRT (2.0 Gy/fraction) in combination with weekly cetuximab throughout radiotherapy. Primary endpoint is locoregional control at 12 months, secondary endpoints are disease-free survival, progression-free survival, overall survival, acute and late radiation effects as well as any adverse events of the treatment as well as quality of life (QoL) analyses.</p> <p>Discussion</p> <p>The primary objective of TPF-C-HIT is to evaluate efficacy and toxicity of cetuximab in combination with combined IMRT/carbon ion therapy following TPF induction in locally advanced SCCHN.</p> <p>Trial Registration</p> <p>Clinical Trial Identifier: <a href="http://www.clinicaltrials.gov/ct2/show/NCT01245985">NCT01245985</a> (clinicaltrials.gov)</p> <p>EudraCT number: 2009 - 016489- 10</p

Disease control and functional outcome in three modern combined organ preserving regimens for locally advanced squamous cell carcinoma of the head and neck (SCCHN)

<p>Abstract</p> <p>Purpose</p> <p>To report our experience on disease control and functional outcome using three modern combined-modality approaches for definitive radiochemotherapy of locally advanced SCCHN with modern radiotherapy techniques: radiochemotherapy (RChT), radioimmunotherapy (RIT) with cetuximab, or induction chemotherapy with docetaxel, cisplatin, and 5-FU (TPF) combined with either RChT or RIT.</p> <p>Methods</p> <p>Toxicity and outcome was retrospectively analysed in patients receiving definitive RChT, RIT, or induction chemotherapy followed by RChT or RIT between 2006 and 2009. Outcome was estimated using Kaplan-Meier analyses, toxicity was analysed according to CTCAE v 3.0.</p> <p>Results</p> <p>Thirty-eight patients were treated with RChT, 38 patients with RIT, 16 patients received TPF followed by either RChT or RIT. Radiotherapy was mostly applied as IMRT (68%). Long-term toxicity was low, only one case of grad III dysphagia requiring oesophageal dilatation, no case of either xerostomia ≥ grade II or cervical plexopathy were observed. Median overall survival (OS) was 25.7 months (RChT) and 27.7 months (RIT), median locoregional progression-free survival (PFS) was not reached yet. Subgroup analysis showed no significant differences between TPF, RChT, and RIT despite higher age and co-morbidities in the RIT group. Results suggested improved OS, distant and overall PFS for the TPF regimen.</p> <p>Conclusion</p> <p>Late radiation effects in our cohort are rare. No significant differences in outcome between RChT and RIT were observed. Adding TPF suggests improved progression-free and overall survival, impact of TPF on locoregional PFS was marginal, therefore radiotherapeutic options for intensification of local treatment should be explored.</p

Neuropeptidomics of the Supraoptic Rat Nucleus

The mammalian supraoptic nucleus (SON) is a neuroendocrine center in the brain regulating a variety of physiological functions. Within the SON, peptidergic magnocellular neurons that project to the neurohypophysis (posterior pituitary) are involved in controlling osmotic balance, lactation, and parturition, partly through secretion of signaling peptides such as oxytocin and vasopressin into the blood. An improved understanding of SON activity and function requires identification and characteriza-tion of the peptides used by the SON. Here, small-volume sample preparation approaches are optimized for neuropeptidomic studies of isolated SON samples ranging from entire nuclei down to single magnocellular neurons. Unlike most previous mammalian peptidome studies, tissues are not im-mediately heated or microwaved. SON samples are obtained from ex vivo brain slice preparations via tissue punch and the samples processed through sequential steps of peptide extraction. Analyses of the samples via liquid chromatography mass spectrometry and tandem mass spectrometry result in the identification of 85 peptides, including 20 unique peptides from known prohormones. As the sample size is further reduced, the depth of peptide coverage decreases; however, even from individually isolated magnocellular neuroendocrine cells, vasopressin and several other peptides are detected

Cardiac Glycosides Ouabain and Digoxin Interfere with the Regulation of Glutamate Transporter GLAST in Astrocytes Cultured from Neonatal Rat Brain

Glutamate transport (GluT) in brain is mediated chiefly by two transporters GLT and GLAST, both driven by ionic gradients generated by (Na+, K+)-dependent ATPase (Na+/K+-ATPase). GLAST is located in astrocytes and its function is regulated by translocations from cytoplasm to plasma membrane in the presence of GluT substrates. The phenomenon is blocked by a naturally occurring toxin rottlerin. We have recently suggested that rottlerin acts by inhibiting Na+/K+-ATPase. We now report that Na+/K+-ATPase inhibitors digoxin and ouabain also blocked the redistribution of GLAST in cultured astrocytes, however, neither of the compounds caused detectable inhibition of ATPase activity in cell-free astrocyte homogenates (rottlerin inhibited app. 80% of Pi production from ATP in the astrocyte homogenates, IC50 = 25 μM). Therefore, while we may not have established a direct link between GLAST regulation and Na+/K+-ATPase activity we have shown that both ouabain and digoxin can interfere with GluT transport and therefore should be considered potentially neurotoxic

Enhanced tonic GABAA inhibition in typical absence epilepsy

The cellular mechanisms underlying typical absence seizures, which characterize various idiopathic generalized epilepsies, are not fully understood, but impaired GABAergic inhibition remains an attractive hypothesis. In contrast, we show here that extrasynaptic GABAA receptor–dependent ‘tonic’ inhibition is increased in thalamocortical neurons from diverse genetic and pharmacological models of absence seizures. Increased tonic inhibition is due to compromised GABA uptake by the GABA transporter GAT–1 in the genetic models tested, and GAT–1 is critical in governing seizure genesis. Extrasynaptic GABAA receptors are a requirement for seizures in two of the best characterized models of absence epilepsy, and the selective activation of thalamic extrasynaptic GABAA receptors is sufficient to elicit both electrographic and behavioural correlates of seizures in normal animals. These results identify an apparently common cellular pathology in typical absence seizures that may have epileptogenic significance, and highlight novel therapeutic targets for the treatment of absence epilepsy.peer-reviewe