Retinoic acid inhibits expression of TNF-alpha and iNOS in activated rat microglia

The release of proinflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha) and nitric oxide by microglia has been implicated in neurotoxicity in chronic neurodegenerative diseases such as Alzheimer's disease. As all-trans-retinoic acid (RA) has been reported to exert anti-inflammatory actions in various cell types, we have examined its effects on the expression of TNF-alpha and inducible nitric oxide synthase (iNOS) in microglia activated by beta-amyloid peptide (Abeta) and lipopolysaccharide (LPS). Exposure of primary cultures of rat microglial cells to Abeta or LPS stimulated the mRNA expression level of TNF-alpha (6-116-fold) and iNOS (8-500-fold) significantly. RA acted in a dose-dependent manner (0.1-10 microM) by attenuating both TNF-alpha (29-97%) and iNOS (61-96%) mRNA expression in microglia exposed to Abeta or LPS. RA-induced inhibition of TNF-alpha and iNOS mRNA expression in activated microglia was accompanied by the concomitant reduction in release of iNOS and TNF-alpha proteins as revealed by nitrite assay and ELISA, respectively. The anti-inflammatory effects of RA were correlated with the enhanced expression of retinoic acid receptor-beta, and transforming growth factor-beta1 as well as the inhibition of NF-kappaB translocation. These results suggest that RA may inhibit the neurotoxic effect of activated microglia by suppressing the production of inflammatory cytokines and cytotoxic molecules.     

Neurotoxicant-induced elevation of adrenomedullin expression in hippocampus and glia cultures.

Adrenomedullin (AM), a vasoactive peptide first isolated from pheochromocytoma, has been reported to be present in neurons in the central nervous system and in tumors of neural and glial origin. In this study, we investigated AM expression both in the hippocampus and in glial cell cultures using a chemical-induced model of injury. An acute intraperitoneal injection of the organometal trimethyltin (TMT) results in neurodegeneration of the hippocampal CA3-4 pyramidal cell layer. Within 4 days of injection, sparse, punctate staining for AM and lectin was evident in the CA3-4 region; by 10 days, a minimal level of CA3-4 neuronal degeneration was evident, with an increase in glial fibrillary acidic protein (GFAP)-positive astrocytes throughout the hippocampus. Degeneration progressed in severity until 30 days post-TMT, with distinct positive immunoreactivity for AM in the CA4 region. mRNA levels for tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha, GFAP, and AM in the hippocampus were increased over control levels within 4 days following TMT. In cultured glial cells, a 6 hr exposure to TMT (10 microM) produced a morphological response of the cells and increased immunoreactivity for vimentin, GFAP, and AM. mRNA levels for TNFalpha, IL-1alpha, GFAP, vimentin, and AM were elevated within 3-6 hr of exposure. In culture, neutralizing antibodies to IL-1alpha and TNFalpha were effective in inhibiting the TMT-induced elevation of AM mRNA. These data suggest an interaction between the proinflammatory cytokines and glia response in the regulation of AM in response to injury.     

Thyroid hormone stimulates gamma-glutamyl transpeptidase in the developing rat cerebra and in astroglial cultures

Hypothyroidism in the developing rat brain is associated with enhanced oxidative stress, one of the earliest manifestations of which is a decline in the level of glutathione (GSH). To investigate the role of thyroid hormone (TH) on GSH homeostasis, the effect of TH on gamma-glutamyl transpeptidase (gammaGT), the key enzyme involved in the catalysis of GSH, was studied. Hypothyroidism declined the specific activity of cerebral gammaGT at all postnatal ages examined (postnatal day 1-20) with a maximum inhibition of 42% at postnatal day 10. Intraperitoneal injection of TH to 15-day-old rat pups increased the specific activity of gammaGT by 25-30% within 4-6 hr. Treatment of primary cultures of astrocytes by TH also enhanced the specific activity of gammaGT by 30-40% within 4-6 hr. The induction of gammaGT by TH was blocked by actinomycin D or cycloheximide. gammaGT is an ectoenzyme that is normally involved in the catabolism of GSH released by astrocytes. In the presence of the gammaGT-inhibitor, acivicin, GSH released in the culture medium of astrocytes increased linearly for at least 6 hr and TH had no effect on this accumulation pattern. In the absence of acivicin, GSH content of the medium from TH-treated cells was significantly lower than that of untreated controls due to activation of gammaGT by TH and a faster processing of GSH. Because the products of gammaGT reaction are putative precursors for neuronal GSH, the activation of gammaGT by TH may be conducive to GSH synthesis in neurons and their protection from oxidative stress.     

Alpha 1 and alpha 2 Adrenergic receptors in mouse brain astrocytes from primary cultures

Mouse brain astrocytes from primary cultures were found to contain both alpha 1 and alpha 2 adrenergic receptors. 3H WB 4101 labeled one category of binding site (KD = 1.5 +/- 0.39 nM, Bmax = 64 +/- 7.9 fmoles/mg protein) with typical alpha 1 adrenergic specificity (WB 4101 greater than prazosin greater than yohimbine). The density of alpha 1 adrenergic receptors was 2-3 times higher in mouse cerebral cortex than in glial cells. Like rat brain [U'Pritchard et al, 1979; Rouot et al, 1980], mouse glial cells were found to contain two categories of 3H clonidine binding sites: high affinity sites, which were identical to the high but not to the low affinity sites found in rat brain, since 1) they displayed the same affinity for 3H clonidine (KD = 1.2 +/- 0.13 nM, n = 4) and the same typical alpha 2 adrenergic specificity (yohimbine greater than WB 4101 greater than prazosin); 2) the dissociation rate constant for clonidine binding was equal to 0.06 min-1, a value close to that found previously for the high affinity 3H clonidine binding sites in rat brain (0.05 min-1); and 3) divalent cations augmented and guanyl nucleotides reduced 3H clonidine binding as in rat brain. Na+ decreased 3H clonidine binding in a complex manner. The number of high affinity sites in glial cells (52 +/- 9.4 fmoles/mg protein, n = 4) was half the number found in mouse cerebral cortex (98 fmoles/mg protein). Low affinity 3H clonidine binding sites (KD = 81 +/- 18 nM, Bmax = 96 +/- 5.8 fmoles/mg protein, n = 3) were not fully characterized. In conclusion, glial cells contained the same alpha adrenergic receptors as those described in brain, but their physiological function is not yet known.     

Pigment epithelium-derived factor induces the production of chemokines by rat microglia

Many studies have shown that pigment epithelium-derived factor (PEDF) has neurotrophic effects on retinal cells and hippocampal, spinal cord, and cerebellar granule cell neurons, but much less work has examined the effects of PEDF on glia. In this study, we show that PEDF changes microglial morphology within 1 h of exposure, to a more deactivated form, while having no effect on the expression of such activation markers as OX-42 and ED-1. In contrast, urea activates acid phosphatase, and PEDF blocks that activation. PEDF also activates NFkappaB, accompanied by the induction of mRNAs and proteins for the chemokines macrophage inflammatory protein-1alpha (MIP-1alpha, MIP-2, and MIP-3alpha. All the chemokines stimulate acid phosphatase activity, and high doses of MIP-2 and MIP-3alpha), alter the morphology of the microglia at 1 h after treatment. These results suggest that the use of PEDF for clinical treatments, such as for retinal neovascularization, brain injury, or ischemia, should be undertaken with caution because of the possibility of induction of inflammation caused by microglial or other immune cell migration in response to the chemokines induced by PEDF.     

Effect of anandamide uptake inhibition in the production of nitric oxide and in the release of cytokines in astrocyte cultures

Astrocytes play a key role regulating aspects of inflammation in the central nervous system (CNS). Several enzymes, such as the inducible nitric oxide synthase (iNOS) or the cyclooxygenase-2 (COX-2), along with different inflammatory mediators such as the free radical nitric oxide (NO) or proinflammatory cytokines, have been proposed to be involved in the cell damage associated with neuroinflammation. Recent studies suggest that the endogenous cannabinoid system (ECS) may be involved in the regulation of neuroinflammation. Cannabinoid agonists decrease neurotoxicity and release of proinflammatory factors from activated glial cells and anandamide itself is able to promote antiinflammatory responses in astrocytes via CB1 cannabinoid receptors. The present study is aimed at studying whether UCM707, a potent and selective anandamide uptake inhibitor, is able to inhibit the production of proinflammatory mediators by LPS-stimulated astrocytes. Our findings indicate that UCM707 is able to reduce NO release, iNOS expression, and the production of the proinflammatory cytokines tumoral necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in a significant manner, while producing a slight increase in IL-6 levels. These effects can be reproduced by administration of the synthetic agonist HU210 and partially or totally blocked by administration of CB1 or CB2 selective antagonists, further supporting the involvement of the ECS. These results confirm the ability of UCM707 to reinforce the beneficial effects induced by anandamide and make it an attractive candidate for the management of those pathologies with neuroinflammation as one of their hallmarks.     

Zaprinast, an inhibitor of cGMP-selective phosphodiesterases, enhances the secretion of TNF-alpha and IL-1beta and the expression of iNOS and MHC class II molecules in rat microglial cells

Proinflammatory cytokines produced by activated glial cells may in turn augment the immune/inflammatory reactions of glial cells through autocrine and paracrine routes. The NO/cGMP signaling represents one of the reactions of activated glial cells. We investigated whether the production of proinflammatory cytokines by glial cells is affected by NO-dependent downstream cGMP signaling. In primary cultures of mixed astrocytes and microglial cells, zaprinast (0.1 mM), an inhibitor of cGMP-selective phosphodiesterases, enhanced the basal and LPS (1.0 microg/ml)-induced secretion of TNF-alpha and IL-1beta. Zaprinast also enhanced NO production induced by LPS or IFN-gamma (100 U/ml), and in microglial cell cultures, but not in astrocyte cultures, zaprinast enhanced the basal and the IFN-gamma-induced production of the cytokines, TNF-alpha and IL-1beta, and of NO. This upregulation by zaprinast was partially inhibited by KT5823 (1.0 microM), an inhibitor of protein kinase G. The LPS-induced production of TNF-alpha, IL-1beta, and NO was inhibited by ODQ (50 microM), an inhibitor of soluble guanylyl cyclase, and by KT5823. Immunohistochemical analysis of mixed glial cell cultures showed that LPS/IFN-gamma-induced iNOS expression and the enhanced expression of iNOS by zaprinast were restricted to microglial cells. Zaprinast enhanced the IFN-gamma (200 U/ml)-induced expression of MHC Class II molecules in astrocytes and microglial cells in mixed cultures, but did not enhance this IFN-gamma-induced expression in pure astrocytes, which lacked paracrine TNF-alpha from microglial cells. Summarizing, zaprinast, which is associated with cGMP/protein kinase G signaling, may augment central immune/inflammatory reactions, possibly via the increased production of TNF-alpha and IL-1beta by activated microglial cells.     

Reaction of mouse brain oligodendrocytes and their precursors, astrocytes and microglia, to proinflammatory mediators circulating in the cerebrospinal fluid

The response of glial cells to the acute intracerebroventricular administration of interferon-gamma, and of this cytokine combined with the endotoxin lipopolysaccharide or with tumor necrosis factor-alpha, was investigated in the brain of adult mice over a time course of 1 week. Oligodendrocytes were identified by immunocytochemistry, using O4 to label their precursors and 2',3'-cyclic nucleotide 3'-phosphohydrolase as marker of mature cells. Astrocytes were labeled by glial fibrillary acidic protein immunoreactivity and microglial cells by tomato lectin histochemistry. Compared with ovalbumin-injected control cases, all cytokine treatments caused a marked decrease of immunostained mature oligodendrocytes in the brain since 1 day postinjection. O4+ oligodendrocyte precursors increased instead progressively from 2 to 7 days. Astrocytes, markedly activated by cytokine treatments, also exhibited a progressive quantitative increase from 2 days onward. Activation and proliferation of microglial cells were instead most evident at 24 h postinjection. Such glial responses to interferon-gamma injections were especially marked in the periventricular brain parenchyma and were enhanced by coadministration of lipopolysaccharide or tumor necrosis factor-alpha. The findings show that a pulse of proinflammatory mediators in the cerebrospinal fluid affects mature oligodendrocytes, concomitantly with the early appearance of activated microglia, and that such reactions are rapidly followed by an increase of oligodendrocyte precursors paralleled by astrocytic activation. The data, which allowed dissecting the events elicited in glial cell populations by inflammatory mediators via the cerebrospinal fluid, indicate that these molecules elicit in vivo a toxic effect on mature oligodendrocytes and a stimulation of their precursors in the adult brain.     

Analysis of the presynaptic signaling mechanisms underlying the inhibition of LTP in rat dentate gyrus by the tyrosine kinase inhibitor,genistein

A great deal of recent evidence points to a role for tyrosine kinase in expression of LTP. Data have been presented that are consistent with the idea that tyrosine phosphorylation of proteins occurs in both the presynaptic and postsynaptic areas. In this study, we set out to investigate the role that tyrosine kinase might play presynaptically to modulate release of glutamate in an effort to understand the mechanism underlying the persistent increase in release that accompanies LTP in perforant path-granule cell synapses. We report that LTP was associated with increased calcium influx and glutamate release. LTP was also associated with an increase in phosphorylation of the alpha-subunit of calcium channels and ERK in synaptosomes prepared from dentate gyrus, and these effects were inhibited when LTP was blocked by the tyrosine kinase inhibitor, genistein. LTP was accompanied by increased protein synthesis and increased phosphorylation of CREB in entorhinal cortex, effects that were also blocked by genistein. We conclude that tetanic stimulation leads to enhanced tyrosine phosphorylation of certain presynaptically located proteins that modulate glutamate release and contribute to expression of LTP.     

Thrombin-activated microglia contribute to death of dopaminergic neurons in rat mesencephalic cultures: dual roles of mitogen-activated protein kinase signaling pathways

This study evaluated the role of thrombin-activated microglia in the neurodegeneration of mesencephalic cultures. Immunocytochemical and biochemical evidence indicated that in co-cultures consisting of rat cortical microglia and mesencephalic neurons, thrombin led to nonselective loss of mesencephalic neurons. Accompanying neurodegeneration, microglial activation was obvious, evidenced by expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-1beta, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) and by increasing production of TNF-alpha and nitric oxide (NO). In mesencephalic neurons treated with conditioned media (CM) taken from thrombin-activated microglia, the number of dopaminergic neurons was significantly attenuated. The neurotoxicity of the CM was diminished when it was derived from microglia co-treated with thrombin and either an extracellular signal-regulated kinase 1/2 (ERK1/2) pathway inhibitor (PD98059) or a p38-mitogen-activated protein kinase (p38-MAPK) inhibitor (SB203580). Moreover, jun N-terminal kinase (JNK) and p38-MAPK were activated in mesencephalic neurons treated with CM of thrombin-activated microglia. Inhibition of JNK and p38-MAPK rescued the dopaminergic neurons. Collectively, these results indicate that thrombin-activated microglia induce neurodegeneration in cultured mesencephalic neurons and that the MAPKs actively participate in both microglial activation and neurodegeneration. The present data carefully suggest that microglial activation triggered by thrombin may be involved in the neuropathological processes of dopaminergic neuronal cell death that occur in Parkinson's disease.     

Low-density lipoprotein receptor-related protein mediates in PC12 cell cultures the inhibition of nerve growth factor-promoted neurite outgrowth by pregnancy zone protein and alpha2-macroglobulin

Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein closely related to human alpha(2)-macroglobulin (alpha(2)M). It has been demonstrated that monoamine-activated forms of human and rat alpha(2)M and rat alpha(1)M can bind to TrkA and, respectively, inhibit and stimulate NGF-promoted neurite outgrowth, Trk phosphorylation, and intracellular signal transduction in PC12 cells. However, the effect of PZP on neurons is unknown, and the molecular mechanism of neuroinhibition by monoamine-activated alpha(2)M is still unclear. In this report, we show that methylamine-activated PZP (MA-PZP), like MA-alpha(2)M, inhibits in a dose-dependent way the NGF-promoted neurite extension and TrkA phosphorylation in PC12 cells. On the other hand, normal PZP (N-PZP) had little or no effect. In addition, the inhibitory effect of activated alpha-macroglobulins (alphaMs) was reversible upon its removal from the cell culture. In addition, PZP, as well as alpha(2)M, is neuroinhibitory without being directly cytotoxic. It is known that the activated alphaMs bind to the multiligand receptor termed low-density lipoprotein receptor-related protein (LRP) and that the receptor-associated protein (RAP) specifically blocks uptake of all known LRP ligands. To investigate the potential role of LRP in neuromodulation by activated PZP/alpha(2)M, the effect of RAP on the neuroinhibitory activities of these alphaMs was also studied. Data presented here show that RAP blocked the neurite- and Trk-inhibitory activities of both MA-PZP and MA-alpha(2)M, whereas RAP itself had no neuromodulatory effect. Hence, we conclude that these data suggest that the LRP receptor and its alphaM ligands may play a role in regulating Trk receptors.     

Transforming growth factor-β1 inhibits the proliferation of rat astrocytes induced by serum and growth factors

A number of cytokines and growth factors may affect astrocyte proliferation and functions. Transforming growth factor-β1 (TGF-β1) is a pleiotropic cytokine which exerts multiple effects on growth and differentiation of different cell types. TGF-β1 is present in low amounts in the normal brain. TGF-β1 gene expression, however, is increased in the central nervous system (CNS) in several pathological conditions. In this study we examined the in vitro effects of TGF-β1 on the proliferative response of rat astrocytes to serum and growth factors. Astrocyte cultures were established from the cerebellum and cortex of newborn Lewis rats. The proliferative response of these cultures to serum and growth factors [platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), IGF-2, interleukin 1 (IL-1)] was studied by [³H]-thymidine incorporation test in the presence or absence of TGF-β1. TGF-β1 significantly inhibited the proliferative response of astrocyte cultures to both autologous and heterologous serum. In addition, a strong inhibition of bFGF-, EGF-, and PDGF-induced proliferation was observed. The effect of TGF-β1 on the proliferative response to IL-1 was less evident but still significant. No effect was observed when TGF-β1 was added to IGF-1 and IGF-2 stimulated cultures. These data confirm previous reports showing a down-regulating activity of TGF-β on astrocyte proliferation and suggest that this cytokine may play physiological and pharmacological roles in the regulation of reactive astrocytosis in the CNS. © 1995 Wiley-Liss, Inc.     

Neonatal polyamine depletion by α-difluoromethylornithine: effects on adenylyl cyclase cell signaling are separable from effects on brain region growth

Ornithine decarboxylase (ODC) and the polyamines play an essential role in brain cell replication and differentiation. We administered alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, to neonatal rats on postnatal days 5-12, during the mitotic peak of the cerebellum, a treatment regimen that leads to selective growth inhibition and dysmorphology. In adulthood, cell signaling responses mediated through the adenylyl cyclase pathway were evaluated in order to determine if synaptic dysfunction extends to regions that appear to be otherwise unaffected by DFMO. Total adenylyl cyclase catalytic activity, evaluated with the direct enzymatic stimulant, Mn(2+), was significantly elevated in male rats both in the cerebellum and in brain regions showing no growth retardation (cerebral cortex, brainstem); there were no significant effects in females. In contrast, signaling mediated through the G proteins that couple neurotransmitter receptors to adenylyl cyclase showed a deficit in the DFMO group, as evaluated with the response to fluoride; in males, there was no corresponding increase in activity as would have been expected solely from the enhancement of adenylyl cyclase, and in females, there was actually a significant decrease in the response to fluoride. Again, the deficits were not restricted to the cerebellum. Stimulation of adenylyl cyclase by isoproterenol, a beta-adrenergic receptor agonist that acts through G(s), likewise displayed deficits in both males and females, and without distinction by brain region. These results indicate that the ODC/polyamine pathway plays a role in the development of cell signaling, and hence in neurotransmission, above and beyond its role in cell replication and differentiation. Given the fact that numerous drugs and environmental contaminants have been shown to alter ODC and the polyamines in the developing brain, our findings suggest that changes in brain region growth or structure are inadequate to predict the targeting of specific neurotransmitter or signaling pathways, and that gender-selective functional defects may be present despite the absence of morphological differences.     

Role of ornithine decarboxylase and the polyamines in nervous system development: Short-term postnatal administration of α-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase

The role of ornithine decarboxylase (ODC) and the polyamines in development of central and peripheral catecholaminergic neurons was examined through the use of α-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC. Short-term postnatal administration of DFMO (500 mg/kg daily on days 1–6) to neonatal rats resulted in effective inhibition of ODC and depletion of both putrescine and spermidine in brain, heart and kidney; after cessation of DFMO administration, polyamine levels returned to normal by 10–13 days of age. There were no signs of generalized toxicity of short-term DFMO treatment, as body weight gains were largely unaffected over the course of study (through weaning). However, development of peripheral sympathetic neurons was severely retarded by DFMO, with persistent and profound deficits of both cardiac and renal norepinephrine; the catecholamine deficiencies were unrelated to effects on end-organ growth, as cardiac weights were essentially normal whereas kidney weights were adversely affected by DFMO. Development of the adrenal medulla, a peripheral catecholaminergic tissue which displays approximately the same developmental profile as do sympathetic neurons but which does not develop axonal projections, was not slowed by DFMO treatment; similarly, central noradrenergic and dopaminergic neurons, which undergo the majority of axonal outgrowth and synaptogenesis during the second to third postnatal week (just after the period in which polyamines returned to control levels), developed normally as assessed by measurements of transmitter levels, tyrosine hydroxylase activity and synaptosomal uptake of [³H]norepinephrine or [³H]dopamine. Extension of the period of DFMO treatment and consequent depletion of polyamines into the period in which central synaptogenesis occurs does, however, produce slowing of development of brain catecholamine neuronal projections. Thus, the ODC/polyamine system appears to play a critical postnatal role in catecholamine systems specifically undergoing active synaptogenesis.     

Heterogeneity and potentiation of AMPA type of glutamate receptors in rat cultured microglia

alpha-amino-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor in rat cultured microglia were analyzed precisely using flop- and flip-preferring allosteric modulators of AMPA receptors, 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetamide (PEPA) and cyclothiazide (CTZ), respectively. Glutamate (Glu)- or kainite (KA)-induced currents were completely inhibited by a specific blocker of AMPA receptor, LY300164, indicating that functional Glu-receptors in cultured microglia are mostly AMPA receptor but not KA receptor in many cells. Glu- and KA-induced currents were potentiated by PEPA and CTZ in a concentration-dependent manner. The ratio of the potentiation by PEPA to the potentiation by cyclothiazide varied with cells between 0.1 and 0.9, suggesting cell-to-cell heterogeneity of AMPA receptor subunits expressed in microglia. Quantitative RT-PCR revealed that GluR1-3 mainly occurred in the flip forms, which agreed with the stronger potentiation of receptor currents by CTZ vs. PEPA. Finally, the potentiation of microglial AMPA receptors by PEPA and CTZ inhibited the Glu-induced release of tumor necrosis factor-alpha (TNF-alpha) unpredictably. The increase in TNF-alpha release by Glu or KA required extracellular Na+ and Ca2+ ions but not mitogen-activated protein kinase (MAPK), suggesting the effects of PEPA and CTZ were not due to the inhibition of MAPK. These results suggest that potentiation of microglial AMPA receptors serves as a negative feedback mechanism for the regulation of TNF-alpha release and may contribute to the ameliorating effects of allosteric modulators of AMPA receptors.     

Cytokine-induced cell death in human oligodendroglial cell lines. II: Alterations in gene expression induced by interferon-gamma and tumor necrosis factor-alpha

Cytokines, such as interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), can initiate dual effects resulting in either cell growth or cell death. In this study, the human oligodendroglial cell lines HOG and MO3.13 were used as a model to study the molecular mechanisms of cytokine-induced cell death in human oligodendrocytes. We have previously shown that TNF-alpha and IFN-gamma induce apoptosis in both oligodendroglial cell lines within 72 hr. In the present study, the cell death pathways operating within these cells were further investigated at the gene expression level. Both cell lines express a broad repertoire of caspases and apoptosis-related genes. Some of these genes are specifically up-regulated by cytokine treatment; e.g., caspase-1 is up-regulated by IFN-gamma. In addition to direct cytotoxic effects, IFN-gamma and TNF-alpha also enhance the expression of Fas, TNFR1, and MHC class I molecules in both cell lines. This suggests that cytokines can make oligodendrocytes more vulnerable to different cell death pathways in an inflammatory environment. cDNA microarray analysis of the HOG cell line revealed that TNF-alpha induces genes that regulate apoptosis, survival, inflammation, cell metabolism, and cell signaling. The data suggest that oligodendroglial cells activate both death and survival pathways upon cytokine challenges. However, the survival pathways seem to be unable to compete with the death signal after more than 24 hr of cytokine treatment. These results may contribute to the development of therapeutic strategies aimed at interfering with cytokine-induced cell death of oligodendrocytes in patients with multiple sclerosis.     

GM-CSF and M-CSF modulate beta-chemokine and HIV-1 expression in microglia

Significant numbers of patients with acquired immunodeficiency syndrome (AIDS) develop CNS infection primarily in macrophages and microglial cells. Therefore, the regulation of human immunodeficiency virus type 1 (HIV-1) infection and activation of the brain mononuclear phagocytes subsequent to infection are important areas of investigation. In the current report, we studied the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage-CSF (M-CSF) in the expression of antiviral beta-chemokines and HIV-1 p24 in cultures of primary human fetal microglia. We found that stimulation with GM-CSF or M-CSF induced macrophage inflammatory proteins (MIP-1alpha and MIP-1beta) and augmented RANTES expression, after HIV-1 infection of microglia. This was not due to the effect of GM-CSF on viral expression because GM-CSF was neither necessary nor stimulatory for viral infection, nor did GM-CSF enhance the expression of env-pseudotyped reporter viruses. Blocking GM-CSF-induced microglial proliferation by nocodazole had no effect on beta-chemokine or p24 expression. The functional significance of the GM-CSF-induced beta-chemokines was suggested by the finding that, in the presence of GM-CSF, exogenous beta-chemokines lost their anti-HIV-1 effects. We further show that although HIV-1-infected microglia produced M-CSF, they failed to produce GM-CSF. In vivo, GM-CSF expression was localized to activated astrocytes and some inflammatory cells in HIV-1 encephalitis, suggesting paracrine activation of microglia through GM-CSF. Our results demonstrate a complex interplay between CSFs, chemokines, and virus in microglial cells and may have bearing on the interpretation of data derived in vivo and in vitro.     

Ampakine CX546 increases proliferation and neuronal differentiation in subventricular zone stem/progenitor cell cultures

Ampakines are chemical compounds known to modulate the properties of ionotropic α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)-subtype glutamate receptors. The functional effects attributed to ampakines involve plasticity and the increase in synaptic efficiency of neuronal circuits, a process that may be intimately associated with differentiation of newborn neurons. The subventricular zone (SVZ) is the main neurogenic niche of the brain, containing neural stem cells with brain repair potential. Accordingly, the identification of new pharmaceutical compounds with neurogenesis-enhancing properties is important as a tool to promote neuronal replacement based on the use of SVZ cells. The purpose of the present paper is to examine the possible proneurogenic effects of ampakine CX546 in cell cultures derived from the SVZ of early postnatal mice. We observed that CX546 (50 μm) treatment triggered an increase in proliferation, evaluated by BrdU incorporation assay, in the neuroblast lineage. Moreover, by using a cell viability assay (TUNEL) we found that, in contrast to AMPA, CX546 did not cause cell death. Also, both AMPA and CX546 stimulated neuronal differentiation as evaluated morphologically through neuronal nuclear protein (NeuN) immunocytochemistry and functionally by single-cell calcium imaging. Accordingly, short exposure to CX546 increased axonogenesis, as determined by the number and length of tau-positive axons co-labelled for the phosphorylated form of SAPK/JNK (P-JNK), and dendritogenesis (MAP2-positive neurites). Altogether, this study shows that ampakine CX546 promotes neurogenesis in SVZ cell cultures and thereby may have potential for future stem cell-based therapies.     

Immunohistochemical localization of adrenergic receptors in the rat organ of corti and spiral ganglion

Alpha(1)-, beta(1)-, and beta(2)-adrenergic receptors (ARs), which mediate responses to adrenergic input, have been immunohistochemically identified within the organ of Corti and spiral ganglion with polyclonal antibodies of established specificity. Alpha(1)-AR was immunolocalized to sites overlapping supranuclear regions of inner hair cells as well as to nerve fibers approaching the base of inner hair cells, most evident in the basal cochlear turn. A similar preponderance across cochlear turns for alpha(1)-AR in afferent cell bodies in the spiral ganglion pointed to type I afferent dendrites as a possible neural source of alpha(1)-AR beneath the inner hair cell. Foci of immunoreactivity for alpha(1)-AR, putatively neural, were found overlapping supranuclear and basal sites of outer hair cells for all turns. Beta(1)- and beta(2)-ARs were immunolocalized to sites overlapping apical and basal poles of the inner and outer hair cells, putatively neural in part, with immunoreactive nerve fibers observed passing through the habenula perforata. Beta(1)- and beta(2)-ARs were also detected in the cell bodies of Deiters' and Hensen's cells. Within the spiral ganglion, beta(1)- and beta(2)-ARs were immunolocalized to afferent cell bodies, with highest expression in the basal cochlear turn, constituting one possible neural source of receptors within the organ of Corti, specifically on type I afferent dendrites. Beta(1)- and beta(2)-ARs in Hensen's and Deiters' cells would couple to Galphas, known to be present specifically in the supporting cells. Overall, adrenergic modulation of neural/supporting cell function within the organ of Corti represents a newly considered mechanism for modifying afferent signaling.