Tocotrienol prevents AAPH-induced neurite degeneration in neuro2a cells.

OBJECTIVES: Reactive oxygen species induce neurite degeneration before inducing cell death. However, the degenerative mechanisms have not yet been elucidated. While tocotrienols have a known neuroprotective function, the underlying mechanism remains unclear and may or may not involve antioxidant action. In this study, we hypothesize that free radical-derived membrane injury is one possible mechanism for inducing neurite degeneration. Therefore, we examined the potential neuroprotective effect of tocotrienols mediated through its antioxidant activity. METHODS: Mouse neuroblastoma neuro2a cells were used to examine the effect of the water-soluble free radical generator 2,2'-azobis(2-methylpropionamide) dihydrochloride (AAPH) on neurite dynamics. After 24 hours of AAPH treatment, cell viability, neurite number, and the number of altered neurites were measured in the presence or absence of α-tocotrienol. RESULTS: Treatment of neuro2a cells with a low concentration of AAPH induces neurite degeneration, but not cell death. Treatment with 5 µM α-tocotrienol significantly inhibited neurite degeneration in AAPH-treated neuro2a cells. Furthermore, morphological changes in AAPH-treated neuro2a cells were similar to those observed with colchicine treatment. CONCLUSIONS: α-Tocotrienol may scavenge AAPH-derived free radicals and alkoxyl radicals that are generated from AAPH-derived peroxyl radicals on cell membranes. Therefore, α-tocotrienol may have a neuroprotective effect mediated by its antioxidant activity.

Calcium leak through ryanodine receptor is involved in neuronal death induced by mutant huntingtin.

Huntington's disease (HD) is a neurodegenerative disorder caused by an abnormal expansion of polyglutamine (polyQ) tract in huntingtin (htt) protein. Although altered calcium (Ca(2+)) homeostasis is suggested in HD, its molecular mechanisms have remained poorly understood despite their important role in the pathogenesis. In this study, we examined involvement of ryanodine receptor (RyR), an endoplasmic reticulum-resident Ca(2+) channel, in mutant htt-induced neuronal death. Inhibitors of RyR attenuated cell death induced by mutant htt, while co-expression of RyR enhanced htt toxicity. Intracellular Ca(2+) imaging revealed that mutant htt caused excessive basal Ca(2+) release (Ca(2+) leak) through RyR leading to depletion of internal Ca(2+) store. Ca(2+) leak was also observed in striatal and cortical neurons from R6/2 HD model mice. Moreover, expression of FK506-binding protein 12 (FKBP12), a RyR stabilizer, suppressed both Ca(2+) leak and cell death. These results provide novel evidence suggesting altered RyR function is involved in neuronal cell death, and its stabilization might be beneficial for treatment of HD.

Vitamin E deficiency induces axonal degeneration in mouse hippocampal neurons.

Several lines of evidence demonstrate the relationship between vitamin E deficiency and cognitive dysfunction in rodent models, but little is known about the underlying mechanisms. In this study, we found axonal injury in the hippocampal CA1 region of vitamin E-deficient and normal old mice using immunohistochemical assay. The number of cells in the hippocampal CA1 region of vitamin E-deficient mice and normal old mice was significantly lower than in normal young mice. It is well known that collapsin response mediator protein (CRMP)-2 plays a crucial role in the maintenance of axonal conditions. The expressions of CRMP-2 in the cerebral cortex and hippocampus of vitamin E-deficient mice were significantly lower than both the regions of normal ones. In normal old mice, the expression of CRMP-2 in the cerebral cortex was significantly lower than in the normal ones. In addition, the appearance of microtubule-associated protein (MAP)-light chain 3 (LC3), a major index of autophagy, was higher in the cerebral cortex and hippocampus of vitamin E-deficient mice than in normal young and old mice. These results indicate that axonal degeneration is induced in living tissues, but not cultured cells, and that changes in CRMP-2 and MAP-LC3 may underlie vitamin E-deficiency-related axonal degeneration.

Tocotrienols prevent hydrogen peroxide-induced axon and dendrite degeneration in cerebellar granule cells.

It is well known that reactive oxygen species (ROS) attack several living tissues and increase the risk of development and progression of serious diseases. In neuronal level, ROS induce cell death in concentration-dependent fashion. However, little is known about the mechanisms of neuronal changes by ROS prior to induction of cell death. Here we found that treatment of cerebellar granule neurons (CGCs) with 0.5 µM hydrogen peroxide induced axonal injury, but not cell death. The number of dendrites remarkably decreased in hydrogen peroxide-treated CGCs, and extensive beading was observed on survival dendrites. In addition, an abnormal band of the original collapsin response mediator protein (CRMP)-2 was detected by Western blotting in hydrogen peroxide-treated CGCs. Treatment with each tocotrienol isoform prevented axonal and dendrite degeneration and induction of the abnormal band of the original band of CRMP-2 in hydrogen peroxide-treated CGCs. These results indicate that treatment with tocotrienols may therefore be neuroprotective in the presence of hydrogen peroxide by preventing changes to the CRMP-2 that occur before neuron death.

Oleate-induced beta cell dysfunction and apoptosis: a proteomic approach to glucolipotoxicity by an unsaturated fatty acid.

High levels of fatty acids contribute to loss of functional beta cell mass in type 2 diabetes, in particular in combination with high glucose levels. The aim of this study was to elucidate the role of the unsaturated free fatty acid oleate in glucolipotoxicity and to unravel the molecular pathways involved. INS-1E cells were exposed to 0.5 mM oleate, combined or not with 25 mM glucose, for 24 h. Protein profiling of INS-1E cells was done by 2D-DIGE, covering pH ranges 4-7 and 6-9 (n = 4). Identification of differentially expressed proteins (P < 0.05) was based on MALDI-TOF analysis using Peptide Mass Fingerprint (PMF) and fragmentation (MS/MS) of the most intense peaks of PMF and proteomic results were confirmed by functional assays. Oleate impaired glucose-stimulated insulin secretion and decreased insulin content. 2D-DIGE analysis revealed 53 and 54 differentially expressed proteins for oleate and the combination of oleate and high glucose, respectively. Exposure to oleate down-regulated chaperones, hampered insulin processing and ubiquitin-related proteasomal degradation, and induced perturbations in vesicle transport and budding. In combination with high glucose, shunting of excess amounts of glucose toward reactive oxygen species production worsened beta cell death. The present findings provide new insights in oleate-induced beta cell dysfunction and identify target proteins for preservation of functional beta cell mass in type 2 diabetes.

Hydrogen peroxide induces neurite degeneration: Prevention by tocotrienols.

Reactive oxygen species (ROS) may attack several types of tissues and chronic exposure to ROS may attenuate various biological functions and increase the risk of several types of serious disorders. It is known that treatments with ROS attack neurons and induce cell death. However, the mechanisms of neuronal change by ROS prior to induction of cell death are not yet understood. Here, it was found that treatment of neurons with low concentrations of hydrogen peroxide induced neurite injury, but not cell death. Unusual bands located above the original collapsin response mediator protein (CRMP)-2 protein were detected by western blotting. Treatment with tocopherol or tocotrienols significantly inhibited these changes in neuro2a cells and cerebellar granule neurons (CGCs). Furthermore, prevention by tocotrienols of hydrogen peroxide-induced neurite degeneration was stronger than that by tocopherol. These findings indicate that neurite beading is one of the early events of neuronal degeneration prior to induction of death of hydrogen peroxide-treated neurons. Treatment with tocotrienols may protect neurite function through its neuroprotective function.

Releasing factors from mature neurons modulate microglial survival via purinergic receptor activation.

Activated microglia release many types of substances to neurons. However, little is known concerning how information from neurons is received by microglia prior to the induction of these substances. Here, we examined whether neurons modulate microglial function. Treatment with conditioned medium of mature cerebellar granule neurons (CGCs) and cortical neurons significantly induced the death of lipopolysaccharide (LPS)-stimulated microglia. On the other hand, treatment with conditioned medium of mature superior ganglion neurons induced microglial cell death in neither the presence nor absence of LPS. Conditioned medium of mature CGCs induced nuclear condensation. In contrast, treatment with heat-treated conditioned medium or low-calcium ion medium prevented the death of LPS-stimulated microglia. Pretreatment with P2X7 agonist enhanced microglial cell death in neither the presence nor absence of LPS. These findings suggest that unknown pyrolytic releasing factors of brain-derived mature neurons influence microglial survival.

GRP78 expression inhibits insulin and ER stress-induced SREBP-1c activation and reduces hepatic steatosis in mice.

Hepatic steatosis is present in insulin-resistant obese rodents and is concomitant with active lipogenesis. Hepatic lipogenesis depends on the insulin-induced activation of the transcription factor SREBP-1c. Despite prevailing insulin resistance, SREBP-1c is activated in the livers of genetically and diet-induced obese rodents. Recent studies have reported the presence of an ER stress response in the livers of obese ob/ob mice. To assess whether ER stress promotes SREBP-1c activation and thus contributes to lipogenesis, we overexpressed the chaperone glucose-regulated protein 78 (GRP78) in the livers of ob/ob mice using an adenoviral vector. GRP78 overexpression reduced ER stress markers and inhibited SREBP-1c cleavage and the expression of SREBP-1c and SREBP-2 target genes. Furthermore, hepatic triglyceride and cholesterol contents were reduced, and insulin sensitivity improved, in GRP78-injected mice. These metabolic improvements were likely mediated by restoration of IRS-2 expression and tyrosine phosphorylation. Interestingly, GRP78 overexpression also inhibited insulin-induced SREBP-1c cleavage in cultured primary hepatocytes. These findings demonstrate that GRP78 inhibits both insulin-dependent and ER stress-dependent SREBP-1c proteolytic cleavage and explain the role of ER stress in hepatic steatosis in obese rodents.

Neuronal maturation-associated resistance of neurite degeneration caused by trophic factor deprivation or microtubule-disrupting agents.

Neurite (axon and dendrite) degeneration requires self-destructive programs independent of cell death programs to segregate neurite degeneration from cell soma demise. We have here addressed the question of whether neuritic degeneration is delayed or occurs normally under conditions in which sympathetic neurons acquire resistance to somal apoptosis upon maturation. For this purpose, we have examined both beading formation and fragmentation, two hall-marks of neurite degeneration, caused by three experimental paradigms including NGF deprivation, treatment with microtubule-disrupting agents, and in vitro Wallerian degeneration. Sympathetic neurons from 1-day-old mice or newborn rats were grown for 5-6 days (young) or 3 weeks (mature). Mature neurons acquired resistance to apoptosis caused by colchicine as well as NGF withdrawal. Neither cytochrome c release nor DNA fragmentation occurred. Both beading formation and subsequent fragmentation were delayed in mature neurons following NGF deprivation, treatment with colchicine, or in vitro Wallerian degeneration. Neuritic ATP levels of young ganglia decreased rapidly, while those of mature ganglia did so slowly during degeneration, although the basal levels of neuritic ATP of both ganglia were similar. Notably, mature neurites were resistant to fragmentation caused by NGF deprivation and capable of growing again after replenishment of NGF. This development of resistance to neurite degeneration in mature neurons may be thought as an important protective mechanism for the maintenance of the adult nervous system.

Transport of autophagosomes in neurites of PC12 cells during serum deprivation.

Autophagy has been linked to various human diseases, including many neurodegenerative disorders. The induction of autophagy has been detected in degenerating neurites initiated by different experimental paradigms and hence is of major interest. Axonal and dendritic degeneration was significantly delayed either by the application of the autophagy inhibitor 3-methyladenine (3-MA) or by knocking down the key autophagy-related genes Atg7 and Beclin 1. In addition, Tomato-LC3-labelled autophagosomes accumulate in neuritic beadings of PC12 cells during nerve growth factor (NGF) deprivation, which might be due to the failure of neurite transport. However, little is known about routes and dynamics of autophagosomes in the neurites of living cells. Here, we further demonstrate that LC3-labelled small autophagosomes are motile and move along the neurites of PC12 cells in both anterograde and retrograde directions after serum deprivation. The autophagosomes paused, re-started, and sometimes changed directions. These results provide valuable insight into neuritic transport of autophagosomes and imply a close relationship between the autophagic process and neurite degeneration.

Axon & dendrite degeneration: its mechanisms and protective experimental paradigms.

Accumulating evidence suggests that axon and dendrite (or neurite) degeneration both in vivo and in vitro requires self-destructive programs independent of cell death programs to segregate neurite degeneration from cell soma demise. This review will deal with the mechanisms of neurite degeneration caused by several experimental paradigms including trophic factor deprivation and Wallerian degeneration as well as those under pathological conditions. The involvement of autophagy and mitochondrial dysfunction is emphasized in these mechanisms. The mechanisms through which protective agents including the Wld(s) protein rescue neurites from degeneration or fail to do so will be discussed.

Calpain-mediated cleavage of collapsin response mediator protein(CRMP)-2 during neurite degeneration in mice.

Axon or dendrite degeneration involves activation of the ubiquitin-proteasome system, failure to maintain neuritic ATP levels, microtubule fragmentation and a mitochondrial permeability transition that occur independently of the somal death programs. To gain further insight into the neurite degeneration mechanims we have compared two-dimensional gel electrophoresis patterns of neurite proteins from suprior cervical ganglia during degeneration caused by nerve growth factor (NGF) deprivation. We show here that collapsin response mediator protein (CRMP)-2 and CMRP-4 protein patterns were altered during beading formation, an early hallmark of neurite degeneration, prior to neurite fragmentation, the final stage of degeneration. Western blotting using a monoclonal antibody against CRMP-2 shows that the native form (64 kDa) was cleaved to generate a truncated form (58 kDa). No cleavage of CRMP-2 or -4 occurred in NGF-deprived neurites from Wld(s) (Wallerian degeneration slow) mutant mice in which neurite degeneration is markedly delayed. Using different protease inhibitors, purified calpain 1 protein and calpain 1-specific siRNA, we have demonstrated that CRMP-2 is a substrate for calpain 1. Indeed, caplain activity was activated at an early phase of neuronal degeneration in cerebellar granule neurons, and down-regulation of caplain 1 expression suppressed CRMP-2 cleavage. Furthermore, this cleavage occurred after vinblastine treatment or in vitro Wallerian degeneration, suggesting that it represents a common step in the process of dying neurites. CRMP-2 and -4 play a pivotal role in axonal growth and transport, and the C-terminus region of CRMP-2 is essential for its binding to kinesin-1. Hence, this cleavage will render them dysfunctional and subject to autophagic processing associated with beading formation, as evidenced by the finding that the truncated form was localized in the beadings.

Induction of autophagy in neurite degeneration of mouse superior cervical ganglion neurons.

Emerging lines of evidence show that the mechanisms of neurite degeneration are convergent, with poor neuritic transport, mitochondrial dysfunction and an increase in intra-axonal calcium being the principal convergence points. Nevertheless, the details are unclear. Here, we revealed the induction of autophagy in degenerating neurites of sympathetic neuron initiated by three different experimental paradigms. Autophagosomes were colocalized with collapsed cytoskeletal proteins in neuritic beadings during degeneration. Accumulation of microtubule-associated protein light chain 3-II, which is the most reliable marker for autophagy, was observed in the early stage of neurite degeneration. The autophagy inhibitor 3-methyladenine efficiently suppressed neurite degeneration by protecting neurites from the loss of viability and mitochondrial function. Furthermore, knocking down the key autophagy-related genes Atg7 and Beclin1 significantly delayed axonal and dendritic degeneration after nerve growth factor deprivation. Reduced expression of Atg7 also suppressed neurite fragmentation after transection. Therefore, our present data suggest the critical role of autophagy in neurite degeneration and may provide a valuable clue in understanding the mechanism of axonal and dendritic degeneration.

Cellular Zn2+ chelators cause "dying-back" neurite degeneration associated with energy impairment.

Most cellular zinc is tightly associated with metalloproteins and other Zn2+-dependent proteins, which along with cellular Zn2+ compartments may coordinately regulate cytoplasmic free Zn2+ levels in the picomolar range. Moreover, Zn2+-containing endosomes or protein complexes appear to move along axons or dendrites, suggesting a dynamic mechanism for trafficking, exchanging, or scavenging Zn2+ and/or Zn2+ protein complexes in neurons. It is therefore interesting to examine whether cellular Zn2+ levels might alter neurite integrity and dynamics. Here we show that membrane-permeable zinc chelators, including 1,10-phenanthroline, N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN), and zinquin, selectively elicit axon and dendrite degeneration but leave the cell body intact in sympathetic neurons. The process begins distally and then moves retrogradely, with a distinct "dying-back" pattern. An inactive isomer of 1,10-phenanthroline failed to cause neuite degeneration, and these chelators mediated their effects by selectively chelating Zn2+, but not other metals. Moreover, neurite degeneration was associated with a decrease in neuritic ATP levels and was caused by energy failure, because an exogenous supply of nicotinamide adenine dinucleotide (NAD) or its precursor nicotinamide suppressed the degeneration by delaying axonal ATP reduction caused by Zn2+ depletion. Blockage of autophagy by 3-methyladenine provided partial protection against degeneration of terminal axons or dendrites; there was, however, no obvious alteration in that of medial portions. Collectively, our results show that cellular Zn2+ depletion induces a "dying-back" degeneration characterized by an NAD- and autophagy-dependent process, independently of neurite elongation dynamics.

Resveratrol abolishes resistance to axonal degeneration in slow Wallerian degeneration (WldS) mice: activation of SIRT2, an NAD-dependent tubulin deacetylase.

Resveratrol is a natural polyphenol having a wide range of biological and pharmacological activities. Here we have investigated the effect of resveratrol on neurodegeneration in cultured cerebellar granule cells from slow Wallerian degeneration (Wld(S)) mice, characteristic of substantial delay of degeneration in the distal stump of transected axons. Resveratrol diminished resistance of Wld(S) neurons to axonal degeneration induced by colchicine, a microtubule depolymerizing drug. Resveratrol also decreased the level of tubulin acetylation in Wld(S) neurons and their homogenates. This promoting effect on tubulin deacetylation was mimicked by NAD, suggesting the involvement of SIRT2, an NAD-dependent tubulin deacetylase. Indeed, resveratrol promoted tubulin deacetylation in the presence of GFP-SIRT2 but not GFP-SIRT2 N168A, a catalytically inactive mutant. Moreover, SIRT2 silencing restored the resistance to axonal degeneration in resveratrol-treated Wld(S) neurons. These results suggest that resveratrol abolishes the resistance of Wld(S) mice to axonal degeneration by enhancing SIRT2-mediated tubulin deacetylation.

Early apoptotic and late necrotic components associated with altered Ca2+ homeostasis in a peptide-delivery model of polyglutamine-induced neuronal death.

The mechanisms by which polyglutamine expansion causes common features of neuronal death remain unclear. Here we describe an approach for delivering polyglutamine expansions directly into cultured sympathetic neurons. Glutamine (Q) residues (n = 10, 22, 30) were conjugated with a peptide possessing translocation properties across plasma membranes (PDP) and a nuclear localization signal (NLS). These peptides were rapidly incorporated into sympathetic neurons and showed neurotoxicity in a length- and dose-dependent manner. A robust induction of c-jun and cyclin D1 occurred following treatment with PDP-Q22-NLS. Enhanced c-Jun phosphorylation showed c-Jun N-terminal kinase (JNK) activation. Coincidentally, TrkA tyrosine phosphorylation was decreased in association with loss of phospho-Akt, the downstream target of PI-3 kinase. Despite such proapoptotic signals, neither release of cytochrome c from mitochondria nor caspase-3/7 activation was detected. TdT-mediated dUTP nick-end labeling-positive nuclear condensation, but no fragmentation, occurred. At 24 hr of treatment, cytoplasmic Ca2+ levels began to become elevated, and the cellular level of ATP was decreased. Cytoplasmic Ca2+ responses to KCl depolarization displayed a delayed recovery, providing evidence for lack of Ca2+ homeostasis. The neurons became committed to death at about 36 hr when mitochondrial Ca2+ uptake declined concurrently with loss of mitochondrial membrane potential. Collectively, these results show that, despite induction of early apoptotic signals, nonapoptotic neuronal cell death occurred via perturbed Ca2+ homeostasis and suggest that mitochondrial permeability transition may play important roles in this model of neuronal death.

N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) suppresses neuritic degeneration caused by different experimental paradigms including in vitro Wallerian degeneration.

Accumulating evidence indicates that neurite degeneration occurs via a distinct mechanism from somal death programs. We have previously shown that neuritic ATP level in sympathetic neurons decreases, whereas somal ATP level remains unaltered during degeneration caused by the microtubule-disrupting agent, vinblastine. Moreover, caspase activation occurs only in cell soma, supporting the view of somal apoptosis and neuritic necrosis. Therefore, the ATP level of neurites is crucial for their degeneration; it appears to correlate with membrane blebbing or beading which precedes late whole fragmentation of neurites under these conditions. Based on these metabolic and morphological criteria, we have tested the effects of various protease inhibitors on vinblastine-induced neurite degeneration in superior cervical ganglia from neonatal mice. Among agents tested, N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), the trypsin-like serine protease inhibitor, but not N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), the chymotrypsin-like serine protease inhibitor, protected sympathetic neurites from beading formation, neuritic fragmentation and a decrease in their ATP level. The commitment time for the saving effect of TLCK occurred around 7 h following treatment with vinblastine, at a time point after microtubule degradation (2 h) and before massive beading formation (later than 12 h). Moreover, TLCK was also capable of suppressing Wallerian degeneration in culture and neuritic degeneration following withdrawal of NGF in a dose-dependent manner. These results strongly suggest that TLCK intervenes in a common step in the cascade of neuritic degeneration caused by these different experimental paradigms and provides a helpful clue for identifying such a molecular step.

Magnetic field exposure saves rat cerebellar granule neurons from apoptosis in vitro.

Accumulating evidence demonstrates that extremely low frequency magnetic fields (ELF-MFs) are capable of modifying neuronal function. Here we examine the effect of ELF-MF exposure on neuronal apoptosis. For this purpose cerebellar granule neurons (CGNs) from postnatal rats were employed, which are known to undergo apoptosis under normal condition (5.4 mM K+) in vitro. Exposure to a rotating (50 Hz) ELF-MF for 5 days saved immature CGNs from apoptosis and promoted survival at the flux density of 300 mT, whereas virtually no neuronal survival was observed without exposure (sham). The survival-promoting effect of ELF-MFs occurred in a manner that depended on the size of culture flasks, suggesting that induced current plays a role in this phenomenon. A maximal survival-promoting effect was comparable to that of membrane depolarization (25 mM K+) and greater than that of brain-derived neurotrophic factor (BDNF). These results imply that ELF-MFs may serve as a potential tool for manipulating neuronal death and/or survival.

Neutral amino acid transporter ASCT1 is preferentially expressed in L-Ser-synthetic/storing glial cells in the mouse brain with transient expression in developing capillaries.

Nonessential amino acid L-Ser plays an essential role in neuronal survival and differentiation, through preferential expression of the L-Ser biosynthetic enzyme 3-phosphoglycerate dehydrogenase (3PGDH), in particular in glial cells but not in neurons. To seek the molecular candidates responsible for glia-borne L-Ser transport, we performed histochemical analyses on amino acid transporter ASCT1, which prefers small neutral amino acids, such as Ala, Ser, Cys, and Thr, and mediates their obligatory exchange. At early developmental stages, neuroepithelial cells constituting the ventricular zone expressed ASCT1 mRNA and protein ubiquitously. Thereafter, ASCT1 expression was gradually downregulated in neuronal populations during the late embryonic and neonatal periods, whereas its high expression was transmitted to radial glial cells and then to astrocytes. High levels of ASCT1 were also detected in the olfactory ensheathing glia. The preferential glial expression of ASCT1 was consistent with that of 3PGDH, and their extensive colocalization was demonstrated at the cellular level. Moreover, high cellular contents of L-Ser were revealed in these glial cells by using a specific antibody to L-Ser. These results strongly suggest that a large amount of L-Ser is synthesized and stored in these glial cells and is released through ASCT1 in exchange for other extracellular substrates. In addition, we observed prominent expression of ASCT1 in capillary endothelial cells of embryonic and neonatal brains. Therefore, ASCT1 appears to be regulated to meet metabolic demands by differentiating and mature neurons through the transport of glia- and blood-borne small neutral amino acids.

Selective inflammatory stimulations enhance release of microglial response factor (MRF)-1 from cultured microglia.

The mrf-1 gene has been isolated from microglia exposed to cultured cerebellar granule neurons undergoing apoptosis. We have shown that mrf-1 is upregulated in response to neuronal death and degeneration both in vitro and in vivo. However, the exact role of MRF-1 remains unknown. Here we show that MRF-1 is released from cultured rat microglia, and its release is greatly enhanced under inflammatory conditions. When microglia were treated with ATP, the amount of MRF-1 that was released increased 10-fold compared to the basal level of release. Enhanced MRF-1 release was induced within 10 min and peaked within 1 h; after approximately 4 h, the MRF-1 release had returned to normal. MRF-1 release was stimulated by 2-methyl-thio-ATP (five-fold) and a P2X(7) selective agonist, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (ten-fold). Moreover, the ATP-stimulated MRF-1 release was inhibited by a P2X(7) selective antagonist, oxidized ATP (oATP), and also under a Ca(2+)-free condition. These results indicate that the effects of ATP are dependent on Ca(2+) influx through P2X(7) receptors. MRF-1 release was enhanced by Ca(2+)-ionophore A23187 (sixfold), thapsigargin (threefold); however, it was not enhanced by glutamate or lipopolysaccharide. Moreover, a platelet-activating factor enhanced microglial MRF-1 release in a dose-dependent manner. We also showed that a conditioned medium from cerebellar granule neurons undergoing apoptosis markedly increased MRF-1 release from microglia; that effect was significantly inhibited by oATP. These results indicate that selective inflammatory stimulations, including ATP and PAF, enhance MRF-1 release from microglia through a Ca(2+)-dependent mechanism and suggest that MRF-1 may play a role in cell-cell interactions under inflammatory conditions.