P. Dell'Albani

Expression of Neurotrophins and Their Receptors in Primary Astroglial Cultures: Induction by Cyclic AMP-Elevating Agents

Abstract: By northern blot analysis and ribonuclease protection assay, we observed the presence of a high level of trkB mRNA in primary brain cultures devoid of neuronal cells and highly enriched in glial fibrillary acidic protein‐positive astroglial cells prepared from newborn rat cerebral hemispheres, cerebral cortex, hippocampus, and striatum. In primary astroglial cultures, the more abundant trkB transcripts code for the truncated receptor without tyrosine kinase activity; probes specific for the full‐length trkB mRNA did not detect any signal in northern blot analysis. By the sensitive ribonuclease protection assay, we could show the presence of trkC mRNA in cultured astrocytes, whereas no trkA mRNA was detected. We confirmed the presence of relatively high levels of nerve growth factor and neurotrophin‐3 mRNA, and very low basal level of brain‐derived neurotrophic factor mRNA. Moreover, we demonstrated that another member of the neurotrophin family, neurotrophin‐4, is also expressed in cultured astroglial cells. In view of the fact that many functional receptors for conventional neurotransmitters or neuropeptides present on astroglial cells may act via the adenylate cyclase system, we studied also the effect of agents able to increase the intracellular cyclic AMP concentration. A sharp increase in the trkB mRNA level was observed after treatment of primary astroglial cultures with dibutyryl cyclic AMP, 8‐bromo‐cyclic AMP, or the phosphodiesterase inhibitor, 3‐isobutyl‐1‐methylxanthine. On the contrary, trkC mRNA levels were unaffected by treatment with cyclic AMP‐elevating agents. All the neurotrophin mRNAs examined, except neurotrophin‐4, were increased by 3‐isobutyl‐1‐methylxanthine treatment. Therefore, in cultured astroglial cells, gene expression of neurotrophins and trkB is regulated by activation of the cyclic AMP‐second messenger system. This process may take part in the neuronal‐glial interactions during the normal neuronal activity or after injury events.

JAK/STAT signaling pathway mediates cytokine‐induced iNOS expression in primary astroglial cell cultures

The production of nitric oxide by the calcium‐independent inducible nitric oxide synthase (iNOS) in glial cells has been implicated in the neuropathogenesis of various diseases. It is well known that in response to lipopolysaccharide (LPS) and cytokines, such as IFN‐γ, glial cells are induced to synthesize large amount of nitric oxide (NO) (Bolaños et al., 1996 ; Nicoletti et al., 1998 ). The signaling transduction pathways for iNOS transcription in astroglial cells have however not yet been established. Because IFN‐γ receptor chains are associated with Janus tyrosine kinases (JAK1 and JAK2) (Darnell et al., 1994 ), we analyzed the involvement of the JAK/STAT signal transduction pathway in iNOS expression. Our study shows increased JAK2 and STAT1α/β tyrosine phosphorylation in primary astroglial cell culture after treatment with IFN‐γ and LPS. A temporal correlation was observed between JAK2 and STAT1α/β tyrosine phosphorylation, the appearance of interferon‐regulatory factor‐1 (IRF‐1) mRNA and the iNOS expression. Inhibition experiments showed that JAK2 and STAT1α/β tyrosine phosphorylation were necessary for IFNγ‐mediated iNOS induction in astroglial cells. We conclude that JAK2 and STAT1α/β tyrosine phosphorylation is an early event involved in the expression of iNOS in astroglial cells. J. Neurosci. Res. 65:417–424, 2001.

Excitatory Amino Acids Stimulate Inositol Phospholipid Hydrolysis and Reduce Proliferation in Cultured Astrocytes

Abstract: Excitatory amino acids stimulated inositol phospholipid hydrolysis in primary cultures of astrocytes, as reflected by an increased formation of [3H]inositol monophosphate ([3H]InsP) in the presence of 10 mM Li+. Quisqualate was the most potent activator of inositol phospholipid hydrolysis, followed by glutamate and ibotenate. Kainate exhibited low activity, whereas N‐methyl‐D‐aspartate (NMDA) and α‐amino‐3‐hydroxy‐5‐methylisoxazolepropionate (AMPA) were inactive. The increase in [3H]InsP formation induced by glutamate was potentiated after 12‐h exposure to the proliferative agent epidermal growth factor (EGF), suggesting that activation of the mitotic cycle leads to an enhanced coupling of glutamate recognition sites with phospholipase C. To study how glutamate receptors are involved in regulating cell proliferation, we have measured [methyl‐3H]thymidine incorporation in cultured astrocytes. Excitatory amino acids reduced thymidine incorporation with a pharmacological profile similar to that observed for the stimulation of inositol phospholipid hydrolysis. Quisqualate acted as a potent antiproliferative agent, both under basal conditions and in cells stimulated to proliferate by addition of EGF or phorbol 12‐tetradecanoate 13‐acetate. Glutamate and ibotenate reduced [methyl‐3H]‐thymidine incorporation at high concentrations, whereas kainate, AMPA, and NMDA were virtually inactive. The action of quisqualate on both inositol phospholipid hydrolysis and thymidine incorporation was attenuated by 2‐amino‐4‐phosphonobutyrate, which acted as a weak agonist/competitive antagonist. Other excitatory amino acid receptor antagonists were not effective. Inhibition of [methyl‐3H]thymidine incorporation by quisqualate required a lag time of about 4 h and, in cells synchronized to proliferate, occurred when the drug was added during the transition between G0 and G1, but not during the S phase of the mitotic cycle. This suggests that an inducible factor may be involved in the antiproliferative effect of excitatory amino acids. Accordingly, activation of quisqualate receptors led to a rapid and transient increase in mRNA levels of the early inducible gene, c‐fos. These results suggest that activation of a specific class of “quisqualate‐preferring”excitatory amino acid receptors reduces proliferation of astrocytes in primary cultures.

Growth Conditions Differentially Regulate the Expression of α‐Amino‐3‐Hydroxy‐5‐Methylisoxazole‐4‐Propionate (AMPA) Receptor Subunits in Cultured Neurons

We have studied the expression of a‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptor subunits in cultured cerebellar granule cells [7 days in vitro (DIV)] grown in medium containing different concentrations of K± (10, 25, or 40 mM) with or without 100 μM N‐methyl‐D‐aspartate (NMDA; added once after 2 DIV). All these conditions are known to influence maturation and survival of granule cells, as well as the functional expression of NMDA receptors during development in culture. The expression of both glutamate receptor (GluR) subunit 1 mRNA and receptor protein was low in cultures grown in 10 mM K± (K10) and increased dramatically in cultures grown in 25 mM K± (K25), with intermediate levels found in cultures grown in K10 and chronically exposed to NMDA (K10 ± NMDA). In cultures grown in 40 mM K± (K40), the expression of GluR1 mRNA and receptor protein was lower than in K25 but still higher than in K10. GluR2 and ‐3 subunits were differently regulated by growth conditions, with their expression being higher in K10 and progressively reduced to the lowest levels in K40 (both mRNA and receptor proteins). GluR4 mRNA levels did not differ between K10 and K25, although they were reduced by chronic exposure to NMDA. To test how the differential expression of the various subunits affects the functional activity of AMPA receptors, we have measured AMPA‐stimulated 4SCa2± influx and 40‐[3H]phorbol 12, 13‐dibutyrate binding in intact cells. Both functional parameters increased along with the K± concentration and were maximal in K40, in coincidence with the lowest expression of the GluR2 subunits. These results indicate that functional diversity of AMPA receptors can be generated by the degree of chronic depolarization and/or exposure to NMDA in neurons developing in primary culture.

Metabotropic Glutamate Receptors in Cultured Cerebellar Granule Cells: Developmental Profile

Abstract: Excitatory amino acid (EAA)‐induced polyphosphoinositide (PPI) hydrolysis was studied during the development in culture of cerebellar granule cells. The developmental pattern was similar using metabotropic glutamate (Glu) receptor (mGluR) agonists, including L‐Glu, quisqualate, and trans‐(±)‐1‐amino‐1,3‐cyclopentanedicarboxylic acid: The stimulation of [3H]inositol monophosphate ([3H]‐InsP) formation was low at 2 days in vitro (DIV), but the response increased steeply, reaching a peak at 4 DIV, followed by a progressive decline. In contrast, carbamylcholine‐induced PPI hydrolysis exhibited a plateau after a pronounced increase during the first week in vitro. At 6 DIV, but not at 4 DIV, when the activity peaked, PPI hydrolysis elicited by Glu was reduced by the N‐methyl‐d‐aspartate (NMDA) receptor antagonist MK‐801, indicating that in cultured granule cells, NMDA receptors contribute to [3H]‐InsP formation and that this component of the response develops relatively late. Accordingly, NMDA‐induced [3H]‐InsP formation, estimated under Mg2+‐free conditions, increased markedly from very low values at 2 DIV to a plateau at 8–10 DIV. The developmental pattern of EAA‐induced PPI hydrolysis was paralleled by changes in the level of an mRNA for a specific mGluR subtype (mGluR1 mRNA). RNA blot analysis performed with the pmGR1 cDNA probe revealed that the hybridization signal in RNA extracts from cultures at 1 DIV was very weak, but mGluR mRNA levels increased dramatically between 1 and 3 DIV, followed by a progressive decrease, so that by 15 DIV the mRNA levels were only ∼10% of the values at 3 DIV. These observations indicate that the functional expression of the mGluR is subject to developmental regulation, which critically involves receptor mRNA levels.

Induction of Primary Response Genes by Excitatory Amino Acid Receptor Agonists in Primary Astroglial Cultures

Abstract: We have characterized the genomic response of astroglial cells to excitatory amino acids by using selective agonists and antagonists for the various receptor subtypes and by analyzing different primary response genes, such as members of the Fos (c‐fos and fosB) and Jun (c‐jun, junB, and junD) families, zif/268, and c‐myc. A rapid and transient elevation of mRNA levels for c‐fos, fosB, c‐jun, junB, and zif/268 was observed after addition of glutamate to cultured astrocytes, whereas junD and c‐myc expression was not affected. The level of AP‐1 DNA binding activity, as measured by the electrophoretic mobility shift assay, also increased after addition of glutamate to cultured astrocytes. Glutamate‐induced c‐fos expression was not affected by the N‐methyl‐d‐aspartate receptor antagonists MK‐801 and D‐2‐amino‐5‐phosphonopentanoate, by the kainate/α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptor antagonist 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX), or by the broad‐spectrum antagonist kynurenate. Kainate and AMPA were also effective in inducing primary response gene expression, and their actions were antagonized by kynurenate and DNQX but not by MK‐801. 1S,3R‐1‐Aminocyclopentane‐1,3‐dicarboxylic acid, a selective agonist for the metabotropic glutamate receptor, induced primary response gene expression, but its action was not antagonized by different glutamate antagonists, including L‐2‐amino‐3‐phosphonopropionate. In conclusion, our data suggest that cultured astrocytes express both kainate/AMPA ionotropic receptors and metabotropic receptors coupled to the rapid and coordinated activation of different classes of transcriptional factor genes.

OLIGODENDROGLIAL SURVIVAL FACTORS, PDGF-AA AND CNTF, ACTIVATE SIMILAR JAK/STAT SIGNALING PATHWAYS

Platelet‐derived growth factor (PDGF) and ciliary neurotrophic factor (CNTF) have pleiotropic actions on many cell types. In the presence of these factors, oligodendroglia respond by enhanced survival when deprived of trophic factors or in the presence of the cytotoxic cytokine, tumor necrosis factor‐α (TNF‐α). To determine whether these two oligodendroglial survival factors converge in their signaling cascades, we examined their JAK/STAT pathways in enriched oligodendrocyte (OL) progenitors and in the progenitor OL cell line, central glia‐4 (CG‐4). Cytokine pathways such as JAK/STAT have been characterized extensively in hematopoietic cells; however, it is increasingly evident that the same cytokines that play a role in hematopoiesis also play a role during development and injury of the central nervous system. This is the first study that clearly defines the presence and activation of JAK/STAT proteins in OL progenitors and compares the signal transduction pathway of two well‐known oligodendroglial survival factors. In this study, we report that PDGF‐ and CNTF‐induced OL progenitors responded with a rapid tyrosine phosphorylation of JAK1, JAK2, STAT1α/β, and STAT3. We feel that these identified JAK/STAT signaling molecules play a large role in the cellular response to these factors. Because both PDGF and CNTF enhance OL progenitor survival, these JAK/STATs may play a role in regulating this important cellular process. J. Neurosci. Res. 54:191–205, 1998.

Metabotropic glutamate receptor expression in cultured rat astrocytes and human gliomas

In order to confirm the existence of metabotropic glutamate receptors in astroglial cultures and to provide information on different receptor subtypes, the expression of different mOIuRs was analysed in cultures highly enriched in rat astroglial cells. mRNA levels for mGluR1, 2, 3, 4, 7 were undetectable by Northern blot analysis in primary type-1 astroglial cultures derived from total cerebral hemispheres, cerebral cortex and striatum. Interestingly, these cultures expressed a low, but detectable, level of mGluR5 mRNA. The more sensitive technique Reverse Transcription-Polymerase Chain Reaction (RT-PCR) confirmed the presence of mGluR5 transcript in cultured astrocytes and, in addition, revealed the presence of mGluR3 mRNA. The lack of expression of mGluR5 in CG-4 cells, a rat cell line able to differentiate in type-2 astrocytes or oligodendrocytes depending on the culture conditions, suggested that the presence of mGluR5 was not a general feature of cells of glial origin. Moreover, all the examined mGluR transcripts were undetectable by RT-PCR in CG4 cells. In order to confirm the possible expression of mGluR5 in cell of glial origin we examined the mRNA levels for this receptor in tissue samples from human gliomas obtained after surgical resection of the tumors: only 1 sample (grade II astrocytoma), out of 8 examined, showed the presence of mGluR5 mRNA. In conclusion our data show that the only cloned metabotropic receptor linked to phosphoinositide hydrolysis, whose expression is detectable in cultured type-1 astrocytes, is mGluR5. It remains to be established if the low level of expression of mGluR3 could be responsible for the group II metabotropic glutamate receptor activity previously observed in cultured astroglial cells.

Changes in gene expression of AMPA-selective glutamate receptor subunits induced by status epilepticus in rat brain☆

In the present investigation we address the question of whether one of the responses to increased neuronal activity is a modification of the expression of the different subunits of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-selective glutamate receptors (GluR-1, GluR-2, GluR-3). Thus, we used two different models of generalized status epilepticus, as widespread elevated neuronal activity, to study in vivo responses of the AMPA receptor mRNA expression in rat forebrain. By Northern blot analysis and in situ hybridization, we show that one of the delayed responses to LiCl/pilocarpine-induced status epilepticus is a dramatic change in the mRNA level of some subunits of AMPA-selective glutamate receptors. These effects, which appear between 6 and 12 h after the drug treatment, are subunit and brain region specific. The most striking example of differential expression of the three examined GluR mRNAs can be observed in the dentate gyrus of the hippocampus. In this specific brain subregion an increase of GluR-3 mRNA level is induced 12 h after LiCl/pilocarpine treatment, while a clear decrease in GluR-1 mRNA level and no significant change in GluR-2 mRNA level can be observed in the same area under these experimental conditions. Both the GluR-1 decrease and the GluR-3 increase are transient effects and a return to basal level can be observed after 48-72 h. In the CA1 layer of the hippocampus, a parallel decrease of both GluR-1 and GluR-3 expression is found 12-24 h after drug treatment, followed by a recovery of the expression to control values at 48 h. In kainate-induced epilepsy we could reproduce the late increase (12-24 h) in GluR-3 mRNA in the dentate gyrus; however, under this experimental condition, no clear decrease of GluR-1 expression can be observed in this area. A general decrease in mRNA level for the AMPA receptor subunits (GluR-1-3) in the hippocampal layers, in particular in CA3 and CA4 subfields, was also observed. In conclusion the results reported in the present paper reveal a specific regulation of GluR gene expression in the granule cells of the hippocampal dentate gyrus and stimulate further investigation on the functional role of the GluR-3 subunit in the receptor-channel complex.

The nicotinic acetylcholine receptor agonist " -epibatidine increases FGF-2 mRNA and protein levels in the rat brain

In a previous work, we showed that acute intermittent nicotine treatment up-regulates the level of fibroblast growth factor-2 (FGF-2) mRNA in brain regions of tel- and mesencephalon of rats suggesting that neuroprotective effect of (-)nicotine may, at least in part, involve an activation of the neuronal FGF-2 signalling. The present experiments were designed to extend the study on the nicotinic receptor mediated up-regulation of FGF-2 mRNA levels to the use of the potent nicotinic acetylcholine receptor (nAChR) agonist (+/-)-epibatidine. The (+/-)-epibatidine treatment led to a strong and long lasting up-regulation of FGF-2 mRNA expression in the cerebral cortex, in the hippocampal formation, in the striatum and in the substantia nigra. This FGF-2 mRNA induction, already statistically significant at 4 h, peaked at 12 h from treatment and was only partially returned towards normal levels at 48 h, the last time point examined. Using Western blot analysis it was found that the epibatidine-induced upregulation of FGF-mRNA is accompaned by an increase of FGF-2 protein level at the 20-h time-interval. These (+/-)-epibatidine effects on FGF-2 expression were antagonized by the non-competitive nAChR antagonist mecamylamine, indicating an involvement of nicotinic receptors. In the same brain areas examined, no changes were observed in the fibroblast growth factor receptor-1 (FGFR-1) mRNA levels, in brain-derived neurotrophic factor (BDNF) and in glial cell line-derived neurotrophic factor (GDNF) mRNA levels. In view of the neurotrophic function of FGF-2, these results, together with previous ones, could further help to understand the molecular mechanisms mediating the previously observed neuroprotective effects of (-)nicotine.