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Interleukin-1beta is a proinflammatory cytokine implicated under pathological conditions involving NMDA receptor activation, including the AIDS dementia complex (HAD). No information is available on the molecular mechanisms recruited by native interleukin-1beta produced under this type of condition. Using a sandwich co-culture of primary hippocampal neurons and glia, we investigated whether native interleukin-1beta released by HIV-gp120-activated glia (i) affects NMDAR functions and (ii) the relevance on neuronal spine density and survival, two specific traits of HAD. Increased phosphorylation of NR2B Tyr-1472 was observed after 24 h of exposure of neurons to 600 pm gp120. This effect occurred only when neurons were treated in the presence of glial cells and was abolished by the interleukin-1 receptor antagonist (IL-1ra). Gp120-induced phosphorylation of NR2B resulted in a sustained elevation of intracellular Ca(2+) in neurons and in a significant increase of NR2B binding to PSD95. Increased intracellular Ca(2+) was prevented by 10 mum ifenprodil, that selectively inhibits receptors containing the NR2B, by interleukin-1ra and by Ca-pYEEIE, a Src family SH2 inhibitor peptide. These last two inhibitors, prevented also NR2B binding to PSD95. Finally, gp120 reduced by 35% of the total PSD95 positive spine density after 48 h of treatment and induced by 30% of the neuronal death. Again, both of these effects were blocked by Ca-pYEEIE. Altogether, our data show that gp120 releasing interleukin-1beta from glia increases tyrosine phosphorylation of NMDAR. Thus, tyrosine phosphorylation may contribute to the sensitization of the receptor increasing its function and synaptic localization. Both of these effects are relevant for neurodegeneration.

Ischemic brain injury resulting from stroke arises from primary neuronal losses and by inflammatory responses. Previous studies suggest that erythropoietin (EPO) attenuates both processes. Although EPO is clearly antiapoptotic for neurons after experimental stroke, it is unknown whether EPO also directly modulates EPO receptor (EPO-R)-expressing glia, microglia, and other inflammatory cells. In these experiments, we show that recombinant human EPO (rhEPO; 5,000 U/kg body weight, i.p.) markedly reduces astrocyte activation and the recruitment of leukocytes and microglia into an infarction produced by middle cerebral artery occlusion in rats. In addition, ischemia-induced production of the proinflammatory cytokines tumor necrosis factor, interleukin 6, and monocyte chemoattractant protein 1 concentration is reduced by >50% after rhEPO administration. Similar results were also observed in mixed neuronal-glial cocultures exposed to the neuronal-selective toxin trimethyl tin. In contrast, rhEPO did not inhibit cytokine production by astrocyte cultures exposed to neuronal homogenates or modulate the response of human peripheral blood mononuclear cells, rat glial cells, or the brain to lipopolysaccharide. These findings suggest that rhEPO attenuates ischemia-induced inflammation by reducing neuronal death rather than by direct effects upon EPO-R-expressing inflammatory cells.

The thymotoxic organotin compounds di-n-butyltin dichloride (DBTC) and tri-n-butyltin chloride (TBTC) are known to induce apoptosis in vitro in rat thymocytes. They also affect macromolecular synthesis, inhibiting DNA synthesis and increasing RNA synthesis. Since these RNA molecules, likely to be involved in the initiation of the apoptotic process, have not been identified yet, the purpose of this research was to characterize by a cDNA macroarray the expression of genes involved in DBTC-induced apoptosis. We found that nur77 was rapidly transcripted in vitro following exposure of freshly isolated rat thymocytes to 3 microM DBTC. nur77 induction has also been observed in vivo after treatment of rats with apoptotic doses (60 mg/kg body wt) of DBTC. The products of nur77 are known to be involved in the apoptotic process, as nur77 is a transcription factor expressed in response to T-cell receptor-mediated apoptosis in immature T cells. Antisense oligonucleotide inhibition of nur77 expression prevented apoptosis induced by DBTC, supporting a role for nur77 in organotin-induced apoptotic cell death., ((c) 2002 Elsevier Science (USA).)

Di-n-butyltin dichloride (DBTC) and tri-n-butyltin chloride (TBTC) cause thymus atrophy in rodents. At low doses, antiproliferative modes of action have been shown to be involved, whereas at higher doses apoptosis seems to be the mechanism of thymotoxicity by these chemicals. In vitro, a similar concentration-dependency has been observed. The purpose of the present research was to investigate the mechanisms underlying DNA fragmentation induced by these organotin compounds in freshly isolated rat thymocytes. As previously shown for TBTC, DBTC is also able to significantly increase intracellular Ca(2+) level ([Ca(2+)](i)). The rise in [Ca(2+)](i), already evident 5 min after treatment, was followed by a dose- and time-dependent generation of reactive oxygen species (ROS) at the mitochondrial level. Simultaneously, organotins induced the release of cytochrome c from the mitochondrial membrane into the cytosol. ROS production and the release of cytochrome c were reduced by BAPTA, an intracellular Ca(2+) chelator, or rotenone, an inhibitor of the electron entry from complex I to ubiquinone, indicating the important role of Ca(2+) and mitochondria during these early intracellular events. Furthermore, we demonstrated that rotenone prevents apoptosis induced by 3 microM DBTC or TBTC and, in addition, that both BAPTA and Z-DEVD FMK (mainly a caspase-3 inhibitor) decreased apoptosis by DBTC (already shown for TBTC). Taken together these data show the apoptotic pathway followed by organotin compounds starts with an increase of [Ca(2+)](i), then continues with release of ROS and cytochrome c from mitochondria, activation of caspases, and finally results in DNA fragmentation., (Copyright 2000 Academic Press.)

Trisubstituted organotin pesticides are lethal for different cell types. In this study we investigated whether triphenyltin chloride (TPT) causes apoptosis in HL-60 promyelocytic cells and, if so, by what mechanisms. We report that 5 microM TPT increased intracellular Ca2+ in HL-60 cells within seconds; concomitantly actin depolymerization was detected 30 s and 1 min after the treatment. This was followed 15 min later by NF-kappaB activation, and apoptotic bodies and DNA fragmentation were evident after 3 and 6 h, respectively. At these times TPT also induced the release of tumor necrosis factor-alpha (TNF-alpha). Prior treatment of the cells with a polyclonal antibody to human TNF-alpha abolished TPT-induced DNA fragmentation, which suggests that the ultimate effect of TPT may be mediated by TNF-alpha. Prior treatment of the cells with 100 microM pyrrolidine dithiocarbamate, an antioxidant and potent inhibitor of NF-kappaB activation, prevented actin depolymerization, NF-kappaB activation, and DNA fragmentation, although it did not affect TPT-induced Ca2+ mobilization. These findings suggest that TPT increases intracellular Ca2+, alters actin polymerization and the cytoskeleton, and induces NF-kappaB activation, TNF-alpha synthesis, DNA degradation, and apoptosis. Reactive oxygen species seem to be essential to NF-kappaB activation, TNF-alpha synthesis, and the subsequent steps.

We examined the effects of four tri-substituted organotin compounds on intracellular Ca2+ and survival in PC12 cells. Treatment with micromolar concentrations of tributyltin and triphenyltin caused a rapid increase in the cytosolic free Ca2+ concentration ([Ca2+]i). This was due to enhanced Ca2+ influx and release from intracellular stores. When the [Ca2+]i elevation was maintained for over 30 min, internucleosomal DNA cleavage typical of apoptotic cell death followed. In contrast, when the increase in [Ca2+]i was transient, cells did not show internucleosomal DNA cleavage and retained their viability. Triethyltin modified [Ca2+]i only slightly, whereas trimethyltin had no effect. The latter two agents did not modify cell viability. Our data suggest that the toxicity of organotins in PC12 cells is linked to their ability to promote intracellular Ca2+ overload, which triggers apoptosis.