Role of Nrf2, HO-1 and GSH in Neuroblastoma Cell Resistance to Bortezomib.

The activation of Nrf2 has been demonstrated to play a crucial role in cancer cell resistance to different anticancer therapies. The inhibition of proteasome activity has been proposed as a chemosensitizing therapy but the activation of Nrf2 could reduce its efficacy. Using the highly chemoresistant neuroblastoma cells HTLA-230, here we show that the strong reduction in proteasome activity, obtained by using low concentration of bortezomib (BTZ, 2.5 nM), fails in reducing cell viability. BTZ treatment favours the binding of Nrf2 to the ARE sequences in the promoter regions of target genes such as heme oxygenase 1 (HO-1), the modulatory subunit of γ-glutamylcysteine ligase (GCLM) and the transporter for cysteine (x-CT), enabling their transcription. GSH level is also increased after BTZ treatment. The up-regulation of Nrf2 target genes is responsible for cell resistance since HO-1 silencing and GSH depletion synergistically decrease BTZ-treated cell viability. Moreover, cell exposure to all-trans-Retinoic acid (ATRA, 3 µM) reduces the binding of Nrf2 to the ARE sequences, decreases HO-1 induction and lowers GSH level increasing the efficacy of bortezomib. These data suggest the role of Nrf2, HO-1 and GSH as molecular targets to improve the efficacy of low doses of bortezomib in the treatment of malignant neuroblastoma.

Evaluation of quinacrine treatment for prion diseases.

Based on in vitro observations in scrapie-infected neuroblastoma cells, quinacrine has recently been proposed as a treatment for Creutzfeldt-Jakob disease (CJD), including a new variant CJD which is linked to contamination of food by the bovine spongiform encephalopathy (BSE) agent. The present study investigated possible mechanisms of action of quinacrine on prions. The ability of quinacrine to interact with and to reduce the protease resistance of PrP peptide aggregates and PrPres of human and animal origin were analyzed, together with its ability to inhibit the in vitro conversion of the normal prion protein (PrPc) to the abnormal form (PrPres). Furthermore, the efficiencies of quinacrine and chlorpromazine, another tricyclic compound, were examined in different in vitro models and in an experimental murine model of BSE. Quinacrine efficiently hampered de novo generation of fibrillogenic prion protein and PrPres accumulation in ScN2a cells. However, it was unable to affect the protease resistance of preexisting PrP fibrils and PrPres from brain homogenates, and a "curing" effect was obtained in ScGT1 cells only after lengthy treatment. In vivo, no detectable effect was observed in the animal model used, consistent with other recent studies and preliminary observations in humans. Despite its ability to cross the blood-brain barrier, the use of quinacrine for the treatment of CJD is questionable, at least as a monotherapy. The multistep experimental approach employed here could be used to test new therapeutic regimes before their use in human trials.

Peripheral inflammatory response in Alzheimer's disease and multiinfarct dementia.

Whether peripheral inflammatory molecules can be considered markers of dementia is still an open issue. We have investigated the presence of circulating cytokines and the ability of blood cells to release them in response to an inflammatory stimulus in patients with different types of dementia and in age-matched controls. A significant increase in circulating interleukin-1beta in moderate Alzheimer and in multiinfarct (145 and 224 times control concentration, respectively) dementia and in circulating tumor necrosis factor-alpha concentration in multiinfarct dementia patient group (156%) were found. Tumor necrosis factor-alpha and interleukin-6 released from blood cells after exposure to lipopolysaccharide were significantly reduced in moderate Alzheimer (60%, both cytokines) and multiinfarct patients (71 and 50%, respectively), while interleukin-10 was decreased only in multiinfarct patients (61%). The results show that patients with Alzheimer disease or multiinfarct dementia have an upregulation of circulating cytokines and a downregulation of cytokines released by blood cells.

Impaired central stress-induced release of noradrenaline in rats with heart failure: a microdialysis study.

Sympathetic hyperactivity in rats with heart failure is associated with increased extracellular noradrenaline in the hypothalamic paraventricular nucleus at rest. However, it is unknown how this nucleus responds to stressful stimuli. In the present study we therefore examined the basal and stress-induced release of noradrenaline in the paraventricular nucleus of conscious Sprague-Dawley rats with heart failure measured by in vivo microdialysis. Basal noradrenaline concentration in the paraventricular nucleus of rats with heart failure was more than double that in sham-operated controls. Immobilization stress decreases noradrenaline levels in the paraventricular nucleus of rats with heart failure to 57% of baseline, while it increased in sham-operated controls to 228%. However, serum corticosterone was similarly elevated at 30 and 90 min post-stress in both experimental groups. We have shown that heart failure causes an impairment of the central noradrenergic system's response to acute sympatho-excitation but does not affect the hypothalamo-pituitary-adrenocortical response.

Inflammatory markers in Alzheimer's disease and multi-infarct dementia.

Inflammation has been involved in the pathogenesis of dementia. The study evaluates the presence and the source of pro- and anti- inflammatory cytokines in the blood of patients with Alzheimer's disease (AD), multi-infarct dementia (MID) or in non-demented elderly people (controls). Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-10, IL-1 receptor antagonist (IL-1Ra) and soluble TNF receptor I (sTNF-RI) plasma concentrations and release from blood cells stimulated with lipopolysaccharide (LPS, 1 microg/ml) were determined. The results show that TNF-alpha released from blood cells is significantly decreased (27%) in all demented patients compared to controls. Circulating TNF-alpha is increased (400%) only in MID patients. In these patients plasma levels of sTNF-RI are increased (53%) and IL-10 from stimulated blood cells decreased (47%) compared to non-demented subjects. The results show that: (1) peripheral production of TNF-alpha is blunted in demented (both AD and MID) patients compared to non-demented age-matched subjects; (2) AD patients have a selective disregulation of the peripheral TNF-alpha system; (3) different cytokines are up- or down- regulated in MID patients showing that in this condition the pro- and anti-inflammatory peripheral cytokine system is more widely affected.

Powerful anticonvulsant action of IL-1 receptor antagonist on intracerebral injection and astrocytic overexpression in mice.

IL-1beta and its endogenous receptor antagonist (IL-1Ra) are rapidly induced by seizures in the rodent hippocampus. Exogenously applied IL-1beta prolongs seizures in an IL-1R type I-mediated manner. This effect depends on N-methyl-d-aspartate receptor activation. We report here that intrahippocampal application of recombinant IL-1Ra or its selective endogenous overexpression in astrocytes under the control of glial acidic fibrillary protein promoter potently inhibits motor and electroencephalographic seizures induced by bicuculline methiodide in mice. Accordingly, transgenic mice show a reduced seizure-related c-fos mRNA expression in various forebrain areas compared with their wild-type littermates. Recombinant IL-1Ra was ineffective in mice deficient in IL-1R type I, having per se a delayed onset to generalized convulsions. These results demonstrate that IL-1Ra mediates potent anticonvulsant effects acting on IL-1R type I and suggest that the balance between brain IL-1beta and IL-1Ra represents a crucial mechanism to control seizure generalization.

Inflammatory cytokines and related genes are induced in the rat hippocampus by limbic status epilepticus.

Limbic status epilepticus was induced in rats by unilateral 60-min electrical stimulation of the CA3 region of the ventral hippocampus. As assessed by RT-PCR followed by Southern blot analysis, transcripts of interleukin-1beta, interleukin-6, interleukin-1 receptor antagonist and inducible nitric oxide synthase were significantly increased 2 h after status epilepticus in the stimulated hippocampus. Induction was maximal at 6 h for interleukin-1beta (445%), interleukin-6 (405%) and tumour necrosis factor-alpha (264%) and at 24 h for interleukin-1 receptor antagonist (494%) and inducible nitric oxide synthase (432%). In rats with spontaneous seizures (60 days after status epilepticus), interleukin-1beta mRNA was still higher than controls (241%). Immunocytochemical staining of interleukin-1beta, interleukin-6 and tumour necrosis factor-alpha was enhanced in glia with a time-course similar to that of the respective transcripts. Sixty days after status epilepticus, interleukin-1beta immunoreactivity was increased exclusively in neurons in one third of the animals. Multiple intracerebroventricular injections of interleukin-1 receptor antagonist (0.5 microg/3 microL) significantly decreased the severity of behavioural convulsions during electrical stimulation and selectively reduced tumour necrosis factor-alpha content in the hippocampus measured 18 h after status epilepticus. Thus, the induction of spontaneously recurring seizures in rats involves the activation of inflammatory cytokines and related pro- and anti-inflammatory genes in the hippocampus. These changes may play an active role in hyperexcitability of the epileptic tissue.

Leukocyte recruitment in the cerebrospinal fluid of mice with experimental meningitis is inhibited by an antibody to junctional adhesion molecule (JAM).

The mechanisms that govern leukocyte transmigration through the endothelium are not yet fully defined. Junctional adhesion molecule (JAM) is a newly cloned member of the immunoglobulin superfamily which is selectively concentrated at tight junctions of endothelial and epithelial cells. A blocking monoclonal antibody (BV11 mAb) directed to JAM was able to inhibit monocyte transmigration through endothelial cells in in vitro and in vivo chemotaxis assays. In this study, we report that BV11 administration was able to attenuate cytokine-induced meningitis in mice. The intravenous injection of BV11 mAb significantly inhibited leukocyte accumulation in the cerebrospinal fluid and infiltration in the brain parenchyma. Blood-brain barrier permeability was also reduced by the mAb. We conclude that JAM may be a new target in limiting the inflammatory response that accompanies meningitis.

Interleukin-1beta immunoreactivity and microglia are enhanced in the rat hippocampus by focal kainate application: functional evidence for enhancement of electrographic seizures.

Using immunocytochemistry and ELISA, we investigated the production of interleukin (IL)-1beta in the rat hippocampus after focal application of kainic acid inducing electroencephalographic (EEG) seizures and CA3 neuronal cell loss. Next, we studied whether EEG seizures per se induced IL-1beta and microglia changes in the hippocampus using bicuculline as a nonexcitotoxic convulsant agent. Finally, to address the functional role of this cytokine, we measured the effect of human recombinant (hr)IL-1beta on seizure activity as one marker of the response to kainate. Three and 24 hr after unilateral intrahippocampal application of 0.19 nmol of kainate, IL-1beta immunoreactivity was enhanced in glia in the injected and the contralateral hippocampi. At 24 hr, IL-1beta concentration increased by 16-fold (p < 0.01) in the injected hippocampus. Reactive microglia was enhanced with a pattern similar to IL-1beta immunoreactivity. Intrahippocampal application of 0.77 nmol of bicuculline methiodide, which induces EEG seizures but not cell loss, enhanced IL-1beta immunoreactivity and microglia, although to a less extent and for a shorter time compared with kainate. One nanogram of (hr)IL-1beta intrahippocampally injected 10 min before kainate enhanced by 226% the time spent in seizures (p < 0.01). This effect was blocked by coinjection of 1 microgram (hr)IL-1beta receptor antagonist or 0.1 ng of 3-((+)-2-carboxypiperazin-4-yl)-propyl-1-phosphonate, selective antagonists of IL-1beta and NMDA receptors, respectively. Thus, convulsant and/or excitotoxic stimuli increase the production of IL-1beta in microglia-like cells in the hippocampus. In addition, exogenous application of IL-1beta prolongs kainate-induced hippocampal EEG seizures by enhancing glutamatergic neurotransmission.

The sympathetic nervous system tonically inhibits peripheral interleukin-1beta and interleukin-6 induction by central lipopolysaccharide.

To study the role of the sympathetic nervous system in the induction of inflammatory cytokines elicited by central lipopolysaccharide, sympathetic chemical denervation was performed by intraperitoneal injection of 6-hydroxydopamine. Rats received the neurotoxin according to the following schedule: 50 mg/kg on days 1 and 2, 100 mg/kg on days 3, 4 and 7. On day 8, lipopolysaccharide (2.5 microg/6 microl/rat) was injected intracerebroventricularly and rats were killed 2 h later. 6-Hydroxydopamine reduced noradrenaline and dopamine content in the spleen by 88.7% and 88.8% respectively, without affecting striatal contents indicating that the chemical sympathectomy had been effective and selective. In sympathectomized rats, lipopolysaccharide raised interleukin-1beta and interleukin-6 serum levels more than in control rats given the vehicle. Tumour necrosis factor-alpha serum levels in sympathectomized rats were no different from those in vehicle-treated rats. Interleukin-1beta and interleukin-6 messenger RNA expression, measured by northern blot analysis, was clearly detectable in adrenals and spleen of rats given lipopolysaccharide. Sympathectomy increased lipopolysaccharide-induced interleukin-1beta and interleukin-6 messenger RNA in adrenals and spleen. Corticosterone basal levels were raised by central lipopolysaccharide and not further changed by sympathectomy. The present study shows that sympathetic nervous system denervation enhances the synthesis and production of peripheral interleukin-1beta and interleukin-6 in rats given central lipopolysaccharide and suggests a tonic inhibitory control of the sympathetic nervous system on these inflammatory cytokines.

Interleukin-6, tumor necrosis factor and corticosterone induction by central lipopolysaccharide in aged rats.

Age-related changes of the immune-adrenal axis were studied in rats treated intracerebroventricularly with lipopolysaccharide (LPS, 2.5 microg/5 microl). Serum interleukin-6 (IL-6), tumor necrosis factor (TNF) and corticosterone levels were evaluated in young (3 months) and old (24 months) Sprague-Dawley rats at different time-points. Old rats showed higher IL-6 levels compared to young rats while no change was observed on TNF levels in the two age groups. Corticosterone increase induced by LPS was lower in old than in young rats. The results show that heterogeneous modifications of the immune-adrenal axis occur that could have a pathophysiological role in the altered response to brain infections during aging.

Central endotoxin induces different patterns of interleukin (IL)-1 beta and IL-6 messenger ribonucleic acid expression and IL-6 secretion in the brain and periphery.

Centrally injected endotoxin induced high levels of interleukin (IL)-6 in serum, but the mechanisms of this induction and the signal conveying the information from the brain to the periphery are not yet known. To help characterize the pathway of centrally mediated induction of IL-6 in periphery, the cytokine levels were measured in rat serum and cerebrospinal fluid at different times after intracerebro-ventricular endotoxin (LPS, 2.5 micrograms/rat). In the same experiments, IL-6 and IL-1 beta messenger RNA (mRNA) expression, measured by Northern blot analysis, were evaluated in the periphery (adrenals, lymph nodes, and mononuclear cells) and brain (hypothalamus, hippocampus and striatum). In serum, IL-6 levels were highest after 2h; then they rapidly decreased. IL-6 mRNA showed the same time-course in adrenals and lymph nodes. The pattern in the central nervous system was different: in the cerebrospinal fluid, IL-6 was detectable starting from 2h, reaching a plateaux at 4-8h and remaining detectable until 16 h. IL-6 mRNA expression in the brain areas showed a similar time-course, reaching a maximum at 4-8 h. IL-1 beta mRNA induction started at the same time in brain and periphery, i.e. 1 h after LPS, but the maximal effect was reached at 2 h in mononuclear cells, adrenals, and lymph nodes, and at 8 h in brain regions. The results indicate that circulating IL-6 induced by central LPS is produced mainly peripherally and that synthesis of IL-6 and IL-1 beta are regulated differently in the brain and periphery.

Permissive role of glucocorticoids on interleukin-1 activation of the hypothalamic serotonergic system.

The present study analyses the effect of IL-1 beta (10 ng i.c.v.) on the hypothalamic serotonergic system and the modulatory role of glucocorticoids. Changes in the serotonin metabolite 5-hydroxyindolacetic acid (5-HIAA) were recorded in freely moving rats by in vivo voltammetry using chronically implanted carbon fiber electrodes (8 microns) in the medial preoptic area. IL-1 beta induced a dual increase in 5HIAA levels: a rapid, short-term rise was followed by a lasting increase possibly due to newly synthesized IL-1. The synthetic glucocorticoid dexamethasone (DEX, 3 mg/kg i.p., 30 min before IL-1 beta), prevented the effect of IL-1 beta starting from 150 min, suggesting that it only inhibited the second increase. In adrenalectomized rats IL-1 beta had no effect but when these rats were given DEX (40 micrograms/kg a day for 3 days) the short-term increase was restored. The glucocorticoid receptor antagonist RU38486 (25 mg/kg s.c., 60 min before IL-1 beta) completely prevented IL-1 beta activation of the serotonergic system. The results indicate that the glucocorticoids are effective inhibitors of IL-1 synthesis but that they play a permissive role on IL-1 beta induced activation of the serotonergic system.

Modulation of systemic interleukin-6 induction by central interleukin-1.

Centrally administered interleukin (IL)-1 [both alpha and beta forms, 200 ng/rat intracerebroventricularly (icv)] results in a larger increase in serum IL-6 than after systemic injection, indicating the brain's role in the acute phase response. This action was prevented by the IL-1-receptor antagonist IL-1Ra (20 micrograms/rat icv). Neither antiserum against corticotropin-releasing factor (CRF) nor the alpha-helical-CRF antagonist (25 micrograms/rat icv) affected IL-6 induction by central IL-1 beta, which, however, was significantly prevented by the synthetic glucocorticoid dexamethasone [3 mg/kg intraperitoneally (ip)]. Naloxone, the opiate antagonist, but not naloxone methiodide, its quaternary salt that does not penetrate the blood-brain barrier (both administered at 10 mg/kg ip), antagonized this action of IL-1 beta. After intracerebroventricular IL-1 beta, IL-6 levels in brain areas (striatum, hippocampus, hypothalamus) were extremely low, suggesting that the brain does not significantly contribute to IL-6 synthesis in this condition. The results show that induction of high serum IL-6 levels by central IL-1 beta is mediated by brain IL-1 receptors and is sensitive to inhibition by corticosteroids. The inhibitory effect of naloxone suggests that central opiates are required for this action of IL-1 beta.

In vivo studies on the enhancement of serotonin reuptake by tianeptine.

The present study investigates the in vivo effects of the serotonin uptake enhancer tianeptine. The serotonin metabolite, 5-hydroxy-indolacetic acid (5-HIAA) was measured by in vivo voltammetry and carbon fiber electrodes chronically implanted in different brain areas of freely moving rats. Tianeptine (10 mg/kg i.p.) increased extracellular 5-HIAA in the hippocampus and hypothalamus. The interaction between tianeptine and drugs known to interfere with the uptake or release of serotonin (sertraline, buspirone, D-norfenfluramine) was then studied and, to ascertain the in vivo pharmacological relevance of tianeptine's effects, its ability to reduce the serotoninergic syndrome was evaluated. Both the biochemical and behavioral data indicate that in vivo tianeptine's effects on the serotoninergic system are likely to be due to serotonin uptake enhancement.

Miniaturized optoelectronic system for telemetry of in vivo voltammetric signals.

In vivo voltammetry is an electrochemical technique that uses carbon fiber microelectrodes stereotaxically implanted in brain areas to monitor monoamine metabolism and release continuously, in freely moving animals. Electric wires connect the polarograph to the animal. A wire-less transmission system (optoelectronic transmission, OPT) of voltammetric signals is described here. It uses infrared diffused light, exploiting the diffusion of the transmitted light over walls and ceiling towards a receiver. The transmission system consists of a main unit and a satellite unit (40 x 30 x 5 mm) positioned on the animal's back. Voltammetric recordings obtained by the classical system (with wires) and by OPT are well defined and almost identical in shape. The power supply is provided by two thin lithium batteries (+/- 3V) that can record for up to 20 h. OPT permits detailed behavioral observations since the animal can be left free to move in a spacious environment. Voltammetry using OPT allows simultaneous recording of neuronal firing activity as well as electroencephalographic recordings (EEG) since there is no cross-talk between the circuits used. The results illustrate the reliability and usefulness of this wire-less transmission system for studying relationships between neurochemical, behavioral and electrophysiological activities.

Intracerebroventricular injection of interleukin 1 induces high circulating levels of interleukin 6.

IL-1 is known to have a central role in the induction of acute-phase response, and some of its activities (including induction of some acute-phase proteins) were reported to be mediated by an induction of IL-6. Administration to rats of 200 ng of human rIL-1 by intracerebroventricular injection resulted in a more marked induction of circulating IL-6 than the same dose of IL-1 administered systemically (intravenously or intraperitoneally). Induction of serum IL-6 by centrally administered IL-1 was also observed in hypophysectomized or adrenalectomized rats, suggesting that activation of the hypothalamus-pituitary-adrenal axis is not essential for this effect of IL-1. IL-6 induction was also observed after pretreatment with indomethacin, indicating that the effect was dissociated from the pyrogenic activity of IL-1. Induction of IL-6 by a central action could represent a novel pathway in IL-1-induced acute-phase response.

Effect of buspirone and its metabolite 1-(2-pyrimidinyl)-piperazine on hippocampal serotoninergic system, studied in freely moving rats.

The effects of the anxiolytic drug buspirone and its metabolite 1-(2-pyrimidinyl)-piperazine (1PP) were studied on the serotoninergic system in the hippocampus of freely moving rats. Pulse voltammetry was used in association with chronically implanted carbon fiber microelectrodes to record 5HIAA, the serotonin metabolite in the extracellular space, almost continuously. Buspirone, 2.5 mg/kg i.p. was ineffective, but the dose of 10 mg/kg lowered 5HIAA between about 45 and 150 min; the same decrease was obtained with 40 mg/kg. This effect can be explained by an agonistic action on 5HT1 A receptors. The metabolite 1PP, which displays alpha 2 adrenoceptor blocking properties, either had no effect or raised extracellular 5HIAA, depending on the dose (1.5 or 6 mg/kg). The rapid metabolization of buspirone to 1PP can thus explain the short time course of the drug effect. Pretreatment with 1PP could only partially prevent buspirone's effect on the serotoninergic system.

Different effects of fenfluramine isomers and metabolites on extracellular 5-HIAA in nucleus accumbens and hippocampus of freely moving rats.

The effects of optical isomers of fenfluramine and their metabolites, d- and l-norfenfluramine on the serotonergic system were studied in the nucleus accumbens and hippocampus of freely moving rats by in vivo voltammetry. Both isomers and the metabolites induced a slow, sustained decrease in 5HIAA but only d-fenfluramine and its metabolite, d-norfenfluramine, increased the 5HIAA levels in nucleus accumbens shortly after injection, the increase being greater after the metabolite. No effect could be detected in the hippocampus after the higher dose of d-fenfluramine.

Multiple and complex effects of buspirone on central dopaminergic system.

The effects of the anxiolytic drug buspirone and its metabolite 1-PP on the dopaminergic system were investigated. A single buspirone administration was found to decrease DA levels and increase its metabolite DOPAC in striatal samples. The levels of the other DA metabolite, 3MT, were unaffected; however its formation rate after inhibition of its metabolism, was found to be increased by buspirone. 1-PP did not affect either DOPAC or 3MT levels and formation. Striatal microdialysis showed that buspirone enhances DA release. In vivo voltammetry indicates that the increase of DA metabolism is identical in the two sampled dopaminergic areas, striatum and nucleus accumbens. On the basis of the results obtained ex vivo and in vivo the multiple effect of buspirone on dopaminergic system is discussed.