Corticotropin-releasing factor (CRF) and related peptides confer neuroprotection via type 1 CRF receptors.

Corticotropin-releasing factor (CRF) receptors are members of the superfamily of G-protein coupled receptors that utilise adenylate cyclase and subsequent production of cAMP for signal transduction in many tissues. Activation of cAMP-dependent pathways, through elevation of intracellular cAMP levels is known to promote survival of a large variety of central and peripheral neuronal populations. Utilising cultured primary rat central nervous system neurons, we show that stimulation of endogenous cAMP signalling pathways by forskolin confers neuroprotection, whilst inhibition of this pathway triggers neuronal death. CRF and the related CRF family peptides urotensin I, urocortin, and sauvagine, which also induced cAMP production, prevented the apoptotic death of cerebellar granule neurons triggered by inhibition of phosphatidylinositol kinase-3 pathway activity with LY294002. These effects were negated by the highly selective CRF-R1 antagonist CP154,526. CRF even conferred neuroprotection when its application was delayed by up to 8 h following LY294002 addition. The CRF peptides also protected cortical and hippocampal neurons against death induced by beta-amyloid peptide (1-42), in a CRF-R1 dependent manner. In separate experiments, LY294002 reduced neuronal protein kinase B activity while increasing glycogen synthase kinase-3, whilst CRF (and related peptides) promoted phosphorylation of glycogen synthase kinase-3 without protein kinase B activation. Taken together, these results suggest that the neuroprotective activity of CRF may involve cAMP-dependent phosphorylation of glycogen synthase kinase-3.

Mast cells differentially express and release active high molecular weight neurotrophins.

Nerve growth factor (NGF), a target-derived factor for survival and maintenance of peripheral and central neurons, has been implicated in inflammatory processes. Mast cells are the principal effector cells in IgE-dependent hypersensitivity reactions, and also play a role in diseases characterised by inflammation, including those of the nervous system like multiple sclerosis. Mast cells are capable of synthesising and responding to NGF, although the occurrence of other members of the NGF family of neurotrophins and their protein forms have not been described. Immunoblot analysis with highly selective neurotrophin antibodies has now been used to show that rat peritoneal mast cells express a higher molecular weight form (73 kDa) of NGF, but not the monomeric (13 kDa) NGF polypeptide. Mast cells also expressed 73 kDa forms of neurotrophin-4 and neurotrophin-3; brain-derived neurotrophic factor was not detected. Medium conditioned by degranulating peritoneal mast cells contained similar high molecular weight forms of NGF and neurotrophin-4 on Western blots, but no neurotrophin-3. Mast cell-derived neurotrophin immunoreactivities were inhibited by the respective peptide antigen, further demonstrating the specificity of the mast cell-derived neurotrophic protein. Mast cell-released proteins supported the survival of cultured chicken embryonic neural crest- and placode-derived sensory neurons; neurotrophic activities were inhibited by neutralising antibodies for NGF and neurotrophin-4, respectively. High molecular isoforms of neurotrophins have been reported to occur in experimental colitis and in the inflamed gut of patients with Crohn's disease and ulcerative colitis, tissue sites rich in mast cells. The data suggest an important role for neurotrophins in the pathophysiology of inflammatory disease.

Neuronal protein kinase signaling cascades and excitotoxic cell death.

Perturbation of normal survival mechanisms may play a role in a large number of disease processes. Glutamate neurotoxicity, particularly when mediated by the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, has been hypothesized to underlie several types of acute brain injury, including stroke. Several neurological insults linked to excessive release of glutamate and neuronal death result in tyrosine kinase activation, including p44/42 mitogen activated protein (MAP) kinase. To further explore a role for MAP kinase activation in excitotoxicity, we used a novel tissue culture model to induce neurotoxicity. Removal of the endogenous blockade by Mg2+ of the NMDA receptor in cultured hippocampal neurons triggers a self perpetuating cycle of excitotoxicity, which has relatively slow onset, and is critically dependent on NMDA receptors and activation of voltage gated Na+ channels. These injury conditions led to a rapid phosphorylation of p44/42 that was blocked by MAP kinase kinase (MEK) inhibitors. MEK inhibition was associated with protection against synaptically mediated excitotoxicity. Interestingly, hippocampal neurons preconditioned by a sublethal exposure to Mg(2+)-free conditions were rendered resistant to injury induced by a subsequently longer exposure to this insult; the preconditioning effect was MAP kinase dependent. The MAP kinase signaling pathway can also promote polypeptide growth factor mediated neuronal survival. MAP kinase regulated pathways may act to promote survival or death, depending upon the cellular context in which they are activated.

Upregulation of death pathway molecules in rat cerebellar granule neurons undergoing apoptosis.

Cerebellar granule neurons can be maintained in culture in a medium containing high serum and depolarising levels of KCl. When serum is removed and the KCl levels lowered from 25 to 5 mM, the cells undergo apoptosis. Apoptosis can be prevented by inhibitors of transcription or translation, suggesting a need for macromolecular synthesis in the apoptotic process. Using quantitative reverse transcription-polymerase chain reaction the levels of mRNA for a range of genes postulated to be important in apoptosis have been examined. Elevated levels of caspase 3, c-Jun, and Fas ligand were found, in addition to a corresponding increase in c-Jun protein and activation of caspase-3. These results suggest that cerebellar granule neurons upregulate components of both death receptor-mediated and the mitochondrial-mediated death pathways.

Myelin-associated glycoprotein interacts with ganglioside GT1b. A mechanism for neurite outgrowth inhibition.

Myelin-associated glycoprotein (MAG) is expressed on myelinating glia and inhibits neurite outgrowth from post-natal neurons. MAG has a sialic acid binding site in its N-terminal domain and binds to specific sialylated glycans and gangliosides present on the surface of neurons, but the significance of these interactions in the effect of MAG on neurite outgrowth is unclear. Here we present evidence to suggest that recognition of sialylated glycans is essential for inhibition of neurite outgrowth by MAG. Arginine 118 on MAG is known to make a key contact with sialic acid. We show that mutation of this residue reduces the potency of MAG inhibitory activity but that residual activity is also a result of carbohydrate recognition. We then go on to investigate gangliosides GT1b and GD1a as candidate MAG receptors. We show that MAG specifically binds both gangliosides and that both are expressed on the surface of MAG-responsive neurons. Furthermore, antibody cross-linking of cell surface GT1b, but not GD1a, mimics the effect of MAG, in that neurite outgrowth is inhibited through activation of Rho kinase. These data strongly suggest that interaction with GT1b on the neuronal cell surface is a potential mechanism for inhibition of neurite outgrowth by MAG.

Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death.

The phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (PKB; also known as Akt) signalling pathway is recognized as playing a central role in the survival of diverse cell types. Glycogen synthase kinase-3 (GSK-3) is a ubiquitously expressed serine/threonine protein kinase that is one of several known substrates of PKB. PKB phosphorylates GSK-3 in response to insulin and growth factors, which inhibits GSK-3 activity and leads to the modulation of multiple GSK-3 regulated cellular processes. We show that the novel potent and selective small-molecule inhibitors of GSK-3; SB-415286 and SB-216763, protect both central and peripheral nervous system neurones in culture from death induced by reduced PI 3-kinase pathway activity. The inhibition of neuronal death mediated by these compounds correlated with inhibition of GSK-3 activity and modulation of GSK-3 substrates tau and beta-catenin. Thus, in addition to the previously assigned roles of GSK-3, our data provide clear pharmacological and biochemical evidence that selective inhibition of the endogenous pool of GSK-3 activity in primary neurones is sufficient to prevent death, implicating GSK-3 as a physiologically relevant principal regulatory target of the PI 3-kinase/PKB neuronal survival pathway.

Potentiation by histamine of synaptically mediated excitotoxicity in cultured hippocampal neurones: a possible role for mast cells.

Excessive glutamatergic neurotransmission, particularly when mediated by the N:-methyl-D-aspartate (NMDA) subtype of glutamate receptor, is thought to underlie neuronal death in a number of neurological disorders. Histamine has been reported to potentiate NMDA receptor-mediated events under a variety of conditions. In the present study we have utilized primary hippocampal neurone cultures to investigate the effect of mast cell-derived, as well as exogenously applied, histamine on neurotoxicity evoked by excessive synaptic activity. Exposure of mature cultures for 15 min to an Mg(2+)-free/glycine-containing buffer to trigger synaptic transmission through NMDA receptors, caused a 30-35% neuronal loss over 24 h. When co-cultured with hippocampal neurones, activated mast cells increased excitotoxic injury to 60%, an effect that was abolished in the presence of histaminase. Similarly, addition of histamine during magnesium deprivation produced a concentration-dependent potentiation (+ 60%; EC(50) : 5 microM) of neuronal death which was inhibited by sodium channel blockers and NMDA receptor antagonists, although this effect did not involve known histamine receptors. The histamine effect was further potentiated by acidification of the culture medium. Cultures 'preconditioned' by sublethal (5 min) Mg(2+) deprivation exhibited less neuronal death than controls when exposed to a more severe insult. NMDA receptor activation and the extracellular regulated kinase cascade were required for preconditioning neuroprotection. The finding that histamine potentiates NMDA receptor-mediated excitotoxicity may have important implications for our understanding of conditions where enhanced glutamatergic neurotransmission is observed in conjunction with tissue acidification, such as cerebral ischaemia and epilepsy.

The FGFR1 inhibitor PD 173074 selectively and potently antagonizes FGF-2 neurotrophic and neurotropic effects.

Basic fibroblast growth factor (FGF-2) promotes survival and/or neurite outgrowth from a variety of neurons in cell culture and regenerative processes in vivo. FGFs exert their effects by activating cell surface receptor tyrosine kinases. FGF receptor (FGFR) inhibitors have not been characterized on neuronal cell behaviors to date. In the present study, we show that the FGFR1 inhibitor PD 173074 potently and selectively antagonized the neurotrophic and neurotropic actions of FGF-2. Nanomolar concentrations of PD 173074 prevented FGF-2, but not insulin-like growth factor-1, support of cerebellar granule neuron survival under conditions of serum/K(+) deprivation; another FGF-2 inhibitor, SU 5402, was effective only at a 1,000-fold greater concentration. Neither PD 173074 nor SU 5402, at 100 times their IC(50) values, interfered with the survival of dorsal root ganglion neurons promoted by nerve growth factor, ciliary neurotrophic factor, or glial cell line-derived neurotrophic factor. PD 173074 and SU 5402 displayed 1,000-fold differential IC(50) values for inhibition of FGF-2-stimulated neurite outgrowth in PC12 cells and in granule neurons, and FGF-2-induced mitogen-activated protein kinase (p44/42) phosphorylation. The two inhibitors failed to disturb downstream signalling stimuli of FGF-2. PD 173074 represents a valuable tool for dissecting the role of FGF-2 in normal and pathological nervous system function without compromising the actions of other neurotrophic factors.

The immunosuppressant steroid cholesterylphosphoserine inhibits tumour necrosis factor-alpha secretion in vitro and in vivo.

The synthetic steroid cholesterylphosphoserine (CPHS) inhibited the secretion of TNF-alpha in lipopolysaccharide-challenged human monocytes. CPHS (5-20 microM) was effective when added together with the endotoxin, or after an interval (1-2 h) sufficient to have allowed for initiation of TNF-alpha synthesis. Consistently, CPHS did not alter TNF-alpha gene transcription. In contrast to its action on TNF-alpha, CPHS showed only marginal effects on interleukin 1beta secretion. Given intraperitoneally to mice 2 h before lipopolysaccharide CPHS prevented the rise in plasma TNF-alpha (IC(50): 5 mg/kg). The inhibition of TNF-alpha secretion by CPHS may contribute to the immunosuppressive activity of this steroid.

Intracellular glutathione levels determine cerebellar granule neuron sensitivity to excitotoxic injury by kainic acid.

Glutathione (GSH) is a key component of the cellular defence cascade against injury caused by reactive oxygen species. Kainic acid (KA) is a potent central nervous system excitotoxin. KA-elicited neuronal death may result from the generation of ROS. The present study was undertaken to characterize the role of GSH in KA-induced neurotoxicity. Cultures of cerebellar granule neurons were prepared from 8-day-old rats, and used at 8, 14 and 20 days in vitro (DIV). Granule neurons displayed a developmental increase in their sensitivity to KA injury, as quantified by an ELISA-based assay with the tetrazolium salt MTT. At DIV 14 and 20, a 30-min challenge with KA (500 microM) reduced cell viability by 45% after 24 h, significantly greater (P<0.01) than the 22% cell loss with DIV 8 cultures. Moreover acute (30 min) KA exposure concentration-dependently reduced intracellular GSH and enhanced reactive oxygen species generation (evaluated by 2', 7'-dichlorofluorescein diacetate). In comparison to control, KA (500 microM) lowered GSH levels in DIV 8 granule neurons by 16% (P=0. 0388), and by 36% (P=0.0001) in both DIV 14 and DIV 20 neurons, after 30 min. Preincubation of granule neurons with the membrane permeant GSH delivery agent, GSH ethyl ester (5 mM), for 30 min significantly increased intracellular GSH content. Importantly, GSH ethyl ester reduced the toxic effects of KA, becoming significant at 1 mM (P=0.007 vs. KA-treated group), and was maximal at >/=2.5 mM (P<0.0001). GSH ethyl ester displayed a similar dose-dependence in its ability to counteract KA-induced depletion of cellular GSH. The data strengthen the notion that cellular GSH levels have a fundamental role in KA-induced neurotoxicity.

Excitotoxicity, oxidative stress, and the neuroprotective potential of melatonin.

The brain consumes large quantities of oxygen relative to its contribution to total body mass. This, together with its paucity of oxidative defense mechanisms, places this organ at risk for damage mediated by reactive oxygen species. The pineal secretory product melatonin possesses broad-spectrum free radical scavenging and antioxidant activities, and prevents kainic acid-induced neuronal lesions, glutathione depletion, and reactive oxygen species-mediated apoptotic nerve cell death. Melatonin's action is thought to involve electron donation to directly detoxify free radicals such as the highly toxic hydroxyl radical, which is a probable end-product of the reaction between NO. and peroxynitrite. Moreover, melatonin limits NO.-induced lipid peroxidation, inhibits cerebellar NO. synthase, scavenges peroxynitrite, and alters the activities of enzymes that improve the total antioxidative defense capacity of the organism. Melatonin function as a free radical scavenger and antioxidant is likely facilitated by the ease with which it crosses morphophysiological barriers, e.g., the blood-brain barrier, and enters cells and subcellular compartments. Pinealectomy, which eliminates the nighttime rise in circulating and tissue melatonin levels, worsens both reactive oxygen species-mediated tissue damage and brain damage after focal cerebral ischemia and excitotoxic seizures. That melatonin protects against hippocampal neurodegeneration linked to excitatory synaptic transmission is fully consistent with the last study. Conceivably, the decreased melatonin secretion that is documented to accompany the aging process may be exaggerated in populations with dementia.

Human immunodeficiency virus type 1 envelope glycoprotein gp120 induces tumor necrosis factor-alpha in astrocytes.

gp120 induction of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) was studied in cultures of purified astrocytes. Incubation of pure mouse cortical astrocytes with gp120 IIIB induced the expression of TNF-alpha mRNA, assessed by in situ hybridization. Anti- TNF-alpha immunocytochemical staining of gp120 IIIB stimulated astrocytes indicated the presence of TNF-alpha. gp120 IIIB treatment also stimulated secretion of bioactive TNF-alpha from astrocytes, which was prevented by inhibitors of transcription and translation. Hippocampal and cerebellar astrocytes displayed similar behaviors. Further, gp120 displayed cytotoxicity for astrocytes that depended on macromolecular synthesis. The data are the first to show gp120 IIIB induction of de novo TNF-alpha production by pure astrocytes. Because TNF-alpha exerts a wide array of effects in the brain of infected individuals and has HIV-1 inducing activity as well, induction of this cytokine by gp120 IIIB in astrocytes may contribute importantly to the pathogenesis of AIDS dementia complex. Since TNF-alpha can stimulate astrocyte reactivity and proliferation by an autocrine mechanism, the extent of the gp120 effect could conceivably increase with HIV-1 disease progression in a self-amplifying loop, involving other cell types, thus favoring both virus persistence and a chronic disease state.

Melatonin prevents the delayed death of hippocampal neurons induced by enhanced excitatory neurotransmission and the nitridergic pathway.

The mechanisms by which neurons die after stroke and status epilepticus and related neuropathological conditions are unclear, but may involve voltage-dependent Na+ channels, glutamate receptors, and nitric oxide (NO.). These questions were investigated using an in vitro primary cell culture model in which hippocampal pyramidal neurons undergo a gradual and delayed neurodegeneration induced by enhanced excitatory neurotransmission. When cells were treated with Mg2+-free, glycine-supplemented medium for a brief period (15 min) and examined 24 h later, approximately 30-40% of the neurons had died. Cell death could be inhibited by blockers of voltage-sensitive Na+ channels and by N-methyl-D-aspartate receptor antagonists. Application of either the endogenous antioxidant melatonin (EC50: 19.2+/-2.8 microM) or the NO. synthase inhibitor Nomega-nitro-L-arginine after, but not during, Mg2+-free exposure protected against delayed neuronal death; significant neuroprotection was observed when the addition was delayed for up to 4 h. This operational time window suggests that an enduring production of NO. and reactive oxygen species from neuronal sources is responsible for delayed cell death. A role for reactive oxygen species in this injury process was strengthened by the finding that, whereas neurons cocultured with astroglia were more resistant to killing, agents capable of lowering intracellular glutathione negated this protection. Because secretion levels of melatonin are decreased with aging, reductions in this pineal hormone may place neurons at a heightened risk for damage by excitatory synaptic transmission.

Neurotrophins rescue cerebellar granule neurons from oxidative stress-mediated apoptotic death: selective involvement of phosphatidylinositol 3-kinase and the mitogen-activated protein kinase pathway.

Cerebellar granule neurons maintained in medium containing serum and 25 mM K+ reliably undergo an apoptotic death when switched to serum-free medium with 5 mM K+. New mRNA and protein synthesis and formation of reactive oxygen intermediates are required steps in K+ deprivation-induced apoptosis of these neurons. Here we show that neurotrophins, members of the nerve growth factor gene family, protect from K+/serum deprivation-induced apoptotic death of cerebellar granule neurons in a temporally distinct manner. Switching granule neurons, on day in vitro (DIV) 4, 10, 20, 30, or 40, from high-K+ to low-K+/serum-free medium decreased viability by >50% when measured after 30 h. Treatment of low-K+ granule neurons at DIV 4 with nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3, or neurotrophin-4/5 (NT-4/5) demonstrated concentration-dependent (1-100 ng/ml) protective effects only for BDNF and NT-4/5. Between DIV 10 and 20, K+-deprived granule neurons showed decreasing sensitivity to BDNF and no response to NT-4/5. Cerebellar granule neuron death induced by K+ withdrawal at DIV 30 and 40 was blocked only by neurotrophin-3. BDNF and NT-4/5 also circumvented glutamate-induced oxidative death in DIV 1-2 granule neurons. Granule neuron death caused by K+ withdrawal or glutamate-triggered oxidative stress was, moreover, limited by free radical scavengers like melatonin. Neurotrophin-protective effects, but not those of antioxidants, were blocked by selective inhibitors of phosphatidylinositol 3-kinase or the mitogen-activated protein kinase pathway, depending on the nature of the oxidant stress. These observations indicate that the survival-promoting effects of neurotrophins for central neurons, whose cellular antioxidant defenses are challenged, require activation of distinct signal transduction pathways.

Melatonin maintains glutathione homeostasis in kainic acid-exposed rat brain tissues.

Reduced glutathione (GSH) is a key component of the cellular defense cascade against injury caused by reactive oxygen species. Because kainic acid (KA) neurotoxicity is probably mediated at least in part by oxidative stress, we examined the influence of KA treatment on GSH content and GSH-related enzyme activities in adult rats. A single injection of KA (10 mg/kg i.p.) time-dependently decreased forebrain GSH (maximal reduction at 48 h). KA also markedly lowered GSH levels in amygdala and hippocampus, but not in the corpus striatum, which is resistant to KA injury. The pineal secretory product melatonin has been shown to exert neuroprotective effects against KA-induced excitotoxicity in rats. Melatonin (2.5 mg/kg i.p., administered four times) partially prevented all decreases in GSH of KA-treated rats. These neuroprotective effects of melatonin may result from a sparing of glutathione reductase, which decreased in KA-treated but not in KA/melatonin-treated animals. Moreover, KA caused a rapid decrease in the GSH content of cultured cerebellar granule neurons but not astrocytes. These cell types both express functional KA receptors, but only the former are sensitive to reactive oxygen species-dependent KA injury. Melatonin counteracted the changes in GSH induced by KA in cultured cerebellar granule neurons. Our results suggest that melatonin prevents the neurotoxic effects of reactive oxygen species linked to KA receptor activation by maintaining cellular GSH homeostasis.

Mast cells contain large quantities of secretagogue-sensitive N-acetylaspartate.

Mast cells play a central role in both immediate allergic reactions and inflammation. A functional nerve-mast cell interaction has been proposed, given the morphological association between mast cells and neuropeptide-containing peripheral nerves. We now show that purified rat peritoneal mast cells contain large quantities of N-acetylaspartate (NAA; 747.50 nmol/mg of protein). Mast cell levels of NAA were rapidly reduced, by 64.0 and 86.4%, following treatment with compound 48/80 and mastoparan, respectively. These secretagogues strongly decreased mast cell histamine content over the same time period, suggesting also that NAA is stored in secretory granules. The data are the first to show that NAA is present in an immune effector cell type. Because NAA may be involved in myelin synthesis and glutamyl peptide metabolism, NAA released from mast cells following nervous or other stimuli could participate in neuroimmune interactions. Mast cells in multiple sclerosis plaques may contribute to the reported elevations in brain NAA in this disease.

The ALIAmide palmitoylethanolamide and cannabinoids, but not anandamide, are protective in a delayed postglutamate paradigm of excitotoxic death in cerebellar granule neurons.

The amino acid L-glutamate is a neurotransmitter that mediates fast neuronal excitation in a majority of synapses in the central nervous system. Glutamate stimulates both N-methyl-D-aspartate (NMDA) and non-NMDA receptors. While activation of NMDA receptors has been implicated in a variety of neurophysiologic processes, excessive NMDA receptor stimulation (excitotoxicity) is thought to be primarily responsible for neuronal injury in a wide variety of acute neurological disorders including hypoxia-ischemia, seizures, and trauma. Very little is known about endogenous molecules and mechanisms capable of modulating excitotoxic neuronal death. Saturated N-acylethanolamides like palmitoylethanolamide accumulate in ischemic tissues and are synthesized by neurons upon excitatory amino acid receptor activation. Here we report that palmitoylethanolamide, but not the cognate N-acylamide anandamide (the ethanolamide of arachidonic acid), protects cultured mouse cerebellar granule cells against glutamate toxicity in a delayed postagonist paradigm. Palmitoylethanolamide reduced this injury in a concentration-dependent manner and was maximally effective when added 15-min postglutamate. Cannabinoids, which like palmitoylethanolamide are functionally active at the peripheral cannabinoid receptor CB2 on mast cells, also prevented neuron loss in this delayed postglutamate model. Furthermore, the neuroprotective effects of palmitoylethanolamide, as well as that of the active cannabinoids, were efficiently antagonized by the candidate central cannabinoid receptor (CB1) agonist anandamide. Analogous pharmacological behaviors have been observed for palmitoylethanolamide (ALI-Amides) in downmodulating mast cell activation. Cerebellar granule cells expressed mRNA for CB1 and CB2 by in situ hybridization, while two cannabinoid binding sites were detected in cerebellar membranes. The results suggest that (i) non-CB1 cannabinoid receptors control, upon agonist binding, the downstream consequences of an excitotoxic stimulus; (ii) palmitoylethanolamide, unlike anandamide, behaves as an endogenous agonist for CB2-like receptors on granule cells; and (iii) activation of such receptors may serve to downmodulate deleterious cellular processes following pathological events or noxious stimuli in both the nervous and immune systems.

Mast cell activation causes delayed neurodegeneration in mixed hippocampal cultures via the nitric oxide pathway.

Mast cells are pleiotropic bone marrow-derived cells found in mucosal and connective tissues and in close apposition to neurons, where they play important roles in tissue inflammation and in neuroimmune interactions. Connective tissue mast cells, with which intracranial mast cells share many characteristics, contain cytokines that can cause inflammation. Here, we report that myelin basic protein, a major suspected immunogen in multiple sclerosis, as well as an antigenic stimulus, provokes mast cells to trigger a delayed cytotoxicity for neurons in mixed neuron-gila cultures from hippocampus. Neurotoxicity required a prolonged period (12 h) of mast cell incubation, and appeared to depend largely on elaboration of the free radical nitric oxide by astrocytes. Activation of astrocytes was mediated, in part, by mast cell-secreted tumor necrosis factor-alpha. Myelin basic protein and 17 beta-estradiol had a synergistic action on the induction of mast cell-associated neuronal injury. The cognate mast cell line RBL-2H3, when subjected to an antigenic stimulus, released tumor necrosis factor-alpha which, together with exogenous interleukin-1 beta (or interferon-gamma), induced astroglia to produce neurotoxic quantities of nitric oxide. A small but significant proportion of mast cell-derived neurotoxicity under the above conditions occurred independently of glial nitric oxide synthase induction. Further, palmitoylethanolamide, which has been reported to reduce mast cell activation by a local autacoid mechanism, decreased neuron loss resulting from mast cell stimulation in the mixed cultures but not that caused by direct cytokine induction of astrocytic nitric oxide synthase. These results support the notion that brain mast cells could participate in the pathophysiology of chronic neurodegenerative and inflammatory diseases of the nervous system, and suggest that down-modulation of mast cell activation in such conditions could be of therapeutic benefit.