Monoamine oxidase B expression is selectively regulated by dexamethasone in cultured rat astrocytes

BRAIN RESEARCH ELSEVIER

Brain Research 711 (1996) 175-183

Research report

Monoamine oxidase B expression is selectively regulated by dexamethasone in cultured rat astrocytes Pia Carlo a,*, Elisabetta Violani b Meris Del Rio ~, Marjut Olasmaa a, Sabrina Santagati c Adriana Maggi c, Giovanni B. Picotti Institute of Pharmacology, School of Medicine, Unil,ersity of Genoa, Viale Benedetto XV 2, 1-16132 Genoa, Italy h Department of Pharmacology, School of Medicine, Unicersitv of Milan, 1-20129 Milan, Itah c Institute of Pharmacological Sciences, Molecular Pharmacology Laboratory, 1-20133 Milan, Itah, Accepted 17 October 1995

Abstract

The influence of dexamethasone on monoamine oxidase (MAO) A and B expression and activity was investigated in primary cultures of rat type 1 astrocytes cultured under serum free, defined conditions. Dexamethasone treatment resulted in a dose- and time-dependent induction of MAO-B, but not of MAO-A, activity. The selective MAO-B increase was substantially reduced by the antagonist RU 486, thus suggesting a glucocorticoid receptor-mediated action of the hormone. Kinetic analysis showed an increase in Vm~× of MAO-B with no change in apparent Kr,,. The dexamethasone-induced selective rise in MAO-B activity appeared to be due to enhanced enzyme synthesis, since MAO-B mRNA was markedly increased by dexamethasone treatment and the recovery of MAO-B activity after its irreversible inhibition by deprenyl was more pronounced in the presence than in the absence of the hormone. Furthermore, the dexamethasone effect was abolished by the protein synthesis inhibitors actinomycin D or cycloheximide. The present study demonstrates that dexamethasone is able to selectively induce MAO-B in type 1 astrocytes and leads to speculation of a possible role for glucocorticoids in the increase in brain MAO-B associated with neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Keywords: Astrocyte, Type l; Monoamine oxidase expression; Monoamine oxidase (MAO) A mRNA; Monoaminc oxidasc (MAO) B mRNA; Dexamethasone; RU 486; Deprenyl; Actinomycin D; Cycloheximidc

1. Introduction

Monoamine oxidase (MAO, EC 1.4.3.4) plays a primary role in the metabolism of biogenic and xenobiotic amines. This mitochondrial enzyme exists in two forms, M A O - A and MAO-B, which show different substrate specificity, tissue distribution and sensitivity to inhibitors [7,24,55]. The two isoenzymes are encoded by separate, highly homologous genes and are independently regulated [2,21,58]. Even if knowledge about the regulation of MAO activity is at present rather limited, a line of evidence suggests that glucocorticoid hormones can differentially regulate M A O - A and -B. A role for glucocorticoid hormones in the regulation of the M A O - A isoenzyme has been demonstrated in studies with different types of cultured cells from peripheral tissues, including human skin fibroblasts [13], bovine adrenal endothelial cells [56] and

* Corresponding author. Fax: (39) (10) 353-8849. 0006-8993/96/$15.00 ~) 1996 Elsevier Science B.V. All rights reserved SSDI 0006-89~)3(95)0135 3-9

rat P C I 2 cells [57]. On the other hand, regulation of MAO-B by glucocorticoids has not been demonstrated in studies carried out on either bovine adrenal chromaffin cells, which exclusively express MAO-B activity [56], or cultured human fibroblasts [13]. In the central nervous system (CNS), the oxidative deamination by MAO is a key process in the control of amine neurotransmitter concentrations, and thus of physiological functions mediated by monoaminergic (catecholaminergic and serotonergic) pathways. In addition, altered levels of MAO have been implicated in the etiology of several psychiatric and neurodegenerative disorders [47]. On the other hand, glucocorticoid hormones are known to regulate many CNS functions in which monoamines exert an important role, including responses to stress, adaptation and mood [11,35], and to affect development and aging of the nervous system [18,42,43]. However, little is known about the interactions at the molecular level between glucocorticoids and M A O isoenzymes in defined types of neurons or glia.

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P. Carlo et al. /Brain Research 711 (1996) 175-183

The ability of glia to metabolize amine neurotransmitters is firmly established and over 90% of brain MAO is found extraneuronally [38]. MAO activity in astrocyte cultures has been shown to correlate with the enzyme activity in homogenates of adult rat brain [19,20]. There is also evidence that glucocorticoid receptors are expressed in cultured astrocytes and that they display binding properties and structure similar to those found in brain [9,51]. Although a number of glial responses to glucocorticoid hormones have been reported [36], a regulatory role of these hormones on astroglial MAO has not, to our knowledge, been examined so far. This prompted us to use primary cultures of rat type 1 astrocytes as a model system to investigate whether the expression and activity of glial MAO-A and -B are under the control of glucocorticoid hormones.

2. Materials and methods 2.1. Materials

Pregnant Sprague-Dawley albino rats were obtained from Charles River (Calco, Italy). Disposable plastic tissue culture flasks were purchased from Falcon (Becton Dickinson, Bedford, MA, USA). Heat-inactivated fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), Nutrient mixture Ham F12 (F12), human transferrin, bovine insulin, putrescine dihydrochloride, fatty acid free bovine serum albumin, dexamethasone, charcoal, actinomycin D, cycloheximide, 5-hydroxytryptamine (5HT) and phenylethylamine (PEA) were from Sigma Chemical Co. (St. Louis, MO, USA). Sodium selenite was obtained from E. Merck (Darmstadt, Germany). Dextran T-70 and oligo dTl0_12 were from Pharmacia (Uppsala, Sweden), trypsin 1:250 from Difco (Detroit, M1, USA), clorgyline and deprenyl from RBI (Natick, MA, USA). RU 38-486 (RU 486, mifepristone) was a gift from Roussel Uclaf (Romainville, France). RT buffer and MuMLV reverse transcriptase were purchased from HT Biotechnology (Cambridge, UK), RNAzol from Biotex Laboratories (Houston, USA), deoxynucleotides from Boehringer Mannheim (Mannheim, Germany), PCR buffer was from Promega (Madison, WI, USA), the Taq polymerase (Dynazyme TH 11) was from Finnzymes Oy (Espoo, Finland). The radiochemicals 5-HT [side-chain-2-14C]creatine sulfate (50-62 mCi/mmol) and /3-[ethyl-llaC]PEA hydrochloride (40-60 mCi/mmol) were obtained from Amersham International (Buckinghamshire, UK) and from Du Pont (Hertfordshire, UK) respectively. All other chemicals were of reagent grade. 2.2. Cell cultures

Primary cultures of glial cells were established from Sprague-Dawley male 1- to 2-day-old rats. Mixed glial

cells from cerebral hemispheres as well as purified cultures of type 1 astrocytes were obtained according to the method of McCarthy and de Vellis [34] with small modifications. The brains were rapidly excised, cleaned of their meningeal membranes and mechanically dissociated in basal medium (DMEM/F12, 1:1, 50 I U / m l penicillin, 50 /zg/ml streptomycin) supplemented with 20% heat-inactivated FBS. The concentration of phenol red in the medium was kept at 1.7/xM, since concentrations above 10 /xM have been shown to possess a weak estrogenic activity [4,22], which in turn has been demonstrated to affect MAO activity [30,56]. To remove still undispersed tissue, cell suspensions were filtered through a 70 /xm nylon mesh, counted and plated at a density of approximately 4 × 105 cells/cm 2 into 75 cm 2 tissue culture flasks and incubated at 37°C in a water-saturated 5% CO2-air atmosphere. Unattached cells were removed 3 - 4 days later by rinsing with DMEM/F12. To avoid any influence of steroids contained in the FBS, the serum was treated with charcoal-dextran as previously described [50]. After 14 days in culture, glial cells formed a confluent monolayer of type 1 astrocytes with few oligodendrocytes scattered across the surface. To select for type 1 astrocyte the flasks were shaken at 250 rpm overnight at 37°C on a rotary platform. Type 1 astrocytes still adherent to the bottom of the flask were harvested by 0.04% trypsin-0.02% EDTA in Puck's solution and diluted 1:10 in basal medium containing 10% dextran-charcoal treated FBS. The cells were plated at 5 × 10 4 cells/cm 2 in the same medium. The day after (day 16 of culture) the medium was removed, purified astrocytes were washed twice with basal medium and chemically defined medium (CDM) was added [6,53]. CDM consisted of basal medium including 100 /xM putrescine, 30 nM sodium selenite, 5 /zg/ml bovine insulin, 1 0 / z g / m l human transferrin and 5 0 / z g / m l fatty acid free bovine serum albumin. Astrocyte cultures prepared as above contained over 90% type 1 astrocytes as identified by immunocytochemical staining with antibody to glial fibrillary acidic protein (GFAP). Drug treatments were started on day 16 of culture. Stock solutions (1 mM) of dexamethasone and RU 486 were prepared in absolute ethanol, stored at - 2 0 ° C and diluted in CDM to the appropriate final concentration. CDM or steroid-containing CDM were changed every 3 days. 2.3. Determination o f MAO activity

MAO-A and -B activities were determined by the radiometric method of Wurtman and Axelrod [54] with slight modifications as previously described [8] using 5-HT or PEA as substrates. Cells were scraped off the flasks with the aid of a cell scraper and collected by centrifugation at 1500 × g for 10 min at 4°C and stored at -70°C. Cells were then iysed by osmotic shock in a 10 mM phosphate buffer (pH 7.4) and passed through a 25-gauge needle. Homogenate aliquots corresponding to 30 /xg of protein

P. Carlo et al. / Brain Research 711 (1996) 175-183

were incubated with 14C-labeled substrates (25 nCi) in a final volume of 150 /xl 0.4 M phosphate buffer. Radioactive substrates were diluted with non-labeled substrates to yield a final concentration of 2 × 10 -4 M 5-HT or 2 X 10 -5 M PEA. Reactions were carried out at 37°C for 10 min in a water bath and stopped by the addition of 200 /zl 1 N HCI. The deaminated radioactive products were extracted into 2.5 ml diethylether or n-heptane, respectively. After centrifugation, the radioactivity was measured by scintillation spectrometry. The enzyme activity of MAO-A and MAO-B was expressed as picomoles of product per milligram of protein per minute. For kinetic analysis, samples were incubated with various concentrations of [14C]PEA (2.5-20 /zM) under otherwise identical conditions. Protein content was determined by the method of Lowry et al. [29] with crystalline bovine serum albumin as standard. 2.4. RNA extraction

Total RNA was extracted from cells by RNAzol following the technical instructions. The extracted RNA was analyzed by measuring the absorbance at 260, 280 and 320 nm. All the preparations had an A260/A280 ratio of 1.8 or higher. 2.5. cDNA synthesis

A water solution of total RNA (1 / x g / 2 /xl) was incubated at 65°C for 5 min, then 1 /xg of oligo dTlo_12 was added together with 1 /xl of RT buffer and 5 U of MuMLV reverse transcriptase in a final volume of 10 /xl. The reaction was carried out for 60 min at 37°C and terminated by heat inactivation of the enzyme at 90°C for 30 s. 2.6. PCR synthesis o f MAO-A and M A O - B cDNA

Five microliters of the 1:10 dilution of the cDNA reaction mixture was combined with 10 /zl of 10x PCR buffer, 200 tzM deoxynucleotides, 2.5 U of Taq polymerase, and the synthetic primers to a final volume of 100 /zl. For MAO-B cDNA amplification the following primers were used: (1) 5'-ATGCCTI'rGTGCTTGT-3'; and (2) 5'ACAGAGTAAATCCACA-3' at the final concentration of 0.5 /xM. For MAO-A cDNA amplification the specific primers were: (1) 5'-CAGAGCT'I'CCAGCAG-3'; and (2) 5'-GCTFAGCAAGTCGATC-3' at the final concentration of 0.5 /~M. As an internal control, GAPDH cDNA was co-amplified by the addition of the following set of primers: (1) 5'-CCACCCATGGCAAATTCCATGGCA-3'; and (2) 5'-TCTAGACGGCAGGTCAGGTCCACC-3', at the concentration of 5 and 2 /zM in the amplification reaction of MAO-B and MAO-A, respectively. The amplification was

177

carried out for 30 cycles in the Hybaid DNA thermal cycler (Teddington, UK). The thermal profile of the DNA amplification was as follows: denaturation, 1 min at 95°C; primer annealing, 30 s at 52°C; extension, 30 s at 72°C. At the 30th cycle, the reaction was terminated by the addition of 50 /zl of DNA dye (50% ( v / v ) glycerol, 0.25% ( w / v ) bromophenol blue, and 0.25% ( w / v ) xylene cyanol). Ten /zl of the incubation mixture were then loaded onto a 3% agarose gel, to separate the amplified DNA species (GAPDH DNA, 600 bp; MAO-B DNA, 300 bp; MAO-A DNA, 234 bp). The bands corresponding to the amplified DNA were then visualized by ethidium bromide staining. To ensure the absence of contamination, samples where the cDNA had been omitted were run in parallel. No amplification products were observed in these samples. Stained gels were photographed with Polaroid type 667 film. The photographic negatives were scanned by the AppleScanner laser densitometer (Apple Computer, Cupertino, CA, USA) and data analyzed by AppleScan, ImageFolder, and Cricket Graph programs on a Macintosh computer (Apple Computer). 2. 7. Statistical analysis

Values are expressed as means + S.E.M. The statistical significance of differences among experimental groups was evaluated by a one- or two-way ANOVA with Tukey multiple range test at 95 and 99% for post hoc comparison. Lineweaver-Burk plots for kinetic analysis were fitted by linear regression.

3. Results

Dose-response curves for MAO-A and -B activities in type 1 astrocytes exposed for 6 days to various concentrations of dexamethasone are shown in Fig. 1. The hormone treatment caused a clear dose-dependent increase in MAOB activity, without affecting MAO-A. Within the range of 10-9_10 6 M dexamethasone, no significant effect on cell proliferation was observed. A decrease in DNA amount per flask was only found when cells were treated with the highest concentration of steroid (10-5 M, data not shown). The effect of dexamethasone (10 -s M) on MAO-B activity was antagonized by 70% after simultaneous administration of the specific antagonist RU 486 (5 × 10 -s M, Table 1). Neither MAO-A nor MAO-B activity was modified by the antagonist alone. Fig. 2 shows the time course for MAO-A and -B activities in the presence or absence of 10 -s M dexamethasone. In untreated astrocytes, MAO-B activity increased continuously from day 3 onward reaching a plateau on day 12. When cells were exposed to dexamethasone, the increase in MAO-B activity was significantly enhanced in respect to untreated cells from day 6 to the end of the experiment. In contrast, MAO-A activity increased to a

P. Carlo et al. / B r a i n Research 711 (1996) 175-183

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Concentration (M) Fig. 1. Effect of increasing concentrations of dexamethasone on MAO-A ( O ) and MAO-B ( Q ) activities in type 1 astrocytes. Primary astroglial cultures were established in medium (DMEM/F12) containing 20% FBS and grown for 2 weeks in medium supplemented with 10% dextrancharcoal-treated FBS. Purified type 1 astrocytes were maintained in CDM containing no hormone (controls) or various concentrations of dexamethasone for 6 days. Each point represents the mean 5: S.E.M. of 3 sets of data from 3 separate astrocyte cultures. Statistical significance by one-way ANOVA with Tukey multiple range test: * * P < 0.01, * P < 0.05 vs control values.

much lesser extent in untreated cells and was not modified by the hormone. Kinetic analysis of MAO-B activity after 6 days of 10 -8 M dexamethasone treatment showed an about 2-fold increase in Vmax with no change in K m (Fig. 3), indicating that the hormone caused an increase in the number of active MAO-B molecules, without apparent changes in the kinetic properties of the enzyme. This finding led us to hypothesize an effect of dexamethasone on MAO-B synthesis. Therefore the effect of dexamethasone on MAO-B mRNA was assessed by quantitation with a competitive

Fig. 2. Time course of the effect of dexamethasone on MAO-A ( O ) and MAO-B ( O ) activities in type 1 astrocytes. On day 0, type 1 astrocytes were exposed to CDM containing no hormone (controls, dotted lines) or 10 8 M dexamethasone (solid lines). Samples of treated and untreated cells were collected at 3-day intervals along 15 days. Each point represents the mean_+S.E.M, of 3 sets of data from 3 separate astrocyte cultures. Statistical significance by two-way ANOVA with Tukey multiple range test: * * P < 0.01, * P < 0.05 vs control values at the corresponding time.

PCR method. As shown in Fig. 4, MAO-B mRNA was markedly induced by 10 -8 M dexamethasone ( + 4 2 9 % and + 362% vs controls, on day 3 and 6, respectively). In parallel assessments, MAO-A mRNA did not show any change. Further proof that dexamethasone acted by increasing de novo synthesis of MAO-B was obtained after irre4

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MAO-A ( p m o l / m g % protein/min)

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1/S (/zM) Fig. 3. Lineweaver-Burk plot of PEA deamination by MAO-B in type I astrocytes. MAO-B activity of type 1 astrocytes was measured after 6 days of treatment either in the absence ( O ) or in the presence ( 0 ) of 10 -8 M dexamethasone. Each point represents a single valuc from a representative experiment. The kinetic parameters were: controls, K m 1 0 . 0 5 ~ M , Vma x 1 5 5 8 p m o l / m g protein/rain; dexamethasone-treated cells, K m 9.73 ~M, Vm,x 2884 p m o l / m g protein/min.

P. Carlo et al. / Brain Research 711 (1996) 175-183

versible blockade of the existing MAO pool by combined treatment with clorgyline and deprenyl, two selective inhibitors of MAO-A and MAO-B, respectively. As shown in Fig. 5, at 2 h following the addition of inhibitors (10 s M) MAO-A and -B activities were decreased by more than 90%. Twenty-four hours after the removal of inhibitors, a greater recovery in MAO-A than in MAO-B activity was observed, indicating a faster turnover rate for the MAO-A isoenzyme. In the presence of 10 -s M dexamethasone, the recovery of MAO-B activity was more than doubled ( + 126% vs dexamethasone-untreated cells), while MAO-A recovery was unaffected by the hormone. To investigate further whether the effect of dexamethasone required transcriptional and translational activities, cells were exposed for 24 h to the RNA synthesis inhibitor actinomycin D (0.5 /xg/ml) or to the protein synthesis inhibitor cycloheximide (1 /xg/ml), in the presence or absence of the steroid. These treatments determined a greater decrease in MAO-A ( - 3 8 % and - 3 7 % ) than in MAO-B activity ( - 1 2 % and - 1 4 % ) , again indicating a different turnover of the two isoenzymes (Fig. 6). As

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expected, the presence of the inhibitors prevented the effect of dexamethasone on MAO-B synthesis.

4. Discussion

In the present study, dexamethasone was shown to elicit a dose- and time-dependent induction of MAO-B activity in type 1 astrocytes cultured under serum-free, defined conditions, without affecting MAO-A. The stimulatory effect of the hormone on MAO-B activity was markedly reduced by the antagonist RU 486 [16], thus suggesting that the MAO-B response resulted essentially from glucocorticoid receptor-mediated events. Several findings in this study, i.e. the increase in Vm~x of MAO-B, the more rapid recovery of enzyme activity after irreversible inhibition, as well as suppression of the hormone effect by protein synthesis inhibitors, consistently indicated that the observed induction of MAO-B activity was due to enhanced synthesis of new enzyme molecules. This was further strengthened by the fact that the dexamethasone-induced

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Fig. 4. Ethidium bromide-stained DNA agarose gels (upper panel) and densitometric analysis of the amplified products (lower panel). On day 0, type 1 astrocytes were exposed to CDM containing no hormone or 10 -8 M dexamethasone (Dex), and on days 3 and 6 treated and untreated cells were collected. Following harvest the mRNA was extracted and cDNA prepared as specified in Materials and methods. A set of primers for the constitutively expressed enzyme GAPDH was used in each reaction as an internal control for the amount of mRNA transcribed and amplified. The MW marker is pBR322 DNA HaelII cut. The size of amplification products is 600 bp for GAPDH, 300 bp for MAO-B and 234 bp for MAO-A. The data of densitometric analysis were normalized using the internal control.

P. Carlo et al. / Brain Research 711 (1996) 175-183

180

increase in MAO-B activity was preceded by an enhancement in the level of MAO-B mRNA. It remains though to be demonstrated whether this effect was due to increased MAO-B m R N A stability or to changes in the transcription rate of the MAO-B gene. The sequence of the rat M A O - A and -B promoters is not yet available. However, sequence analysis of the human MAO-B promoter [58] has not revealed the presence of hexamer pairs typical of most glucocorticoid response elements [3,52]. Therefore, only a more detailed analysis of the effect of glucocorticoids on MAO-B promoter by transient transfection assay will allow a conclusion to be made as to whether MAO-B promoter represents a primary target for glucocorticoid in astrocytes. To our knowledge this is the first report on the effect of glucocorticoids on astrocyte MAO. Indeed, the present knowledge on glucocorticoid induction of MAO activity stems from studies on cultured cells from peripheral tissues, i.e. skin fibroblasts, adrenomedullary endothelium and chromaffin cells, or PC12 cells [12,13,56,57]. In contrast to the present findings in astrocytes, these studies demonstrated changes in MAO-A, but not in MAO-B,

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Fig. 6. Actinomycin D and cycloheximide prevent the effect of dexamethasone on MAO-A (upper panel) and MAO-B (lower panel) activities in type 1 astrocytes. Control cultures (C) and cultures treated for 24 h with 0.5 /xg/ml actinomycinD (Act D) or with 1 /zg/ml cycloheximide (CH) were incubated in the presence (hatched columns) or absence (open columns) of 10-8 M dexamethasone (Dex). Each column represents the mean + S.E.M. of triplicate determinations from a representative experiment. Statistical significance by two-way ANOVA with Tukey multiple range test: * P < 0.05 vs control values.

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Fig. 5. Effect of dexamethasone on the recovery of MAO-A (upper panel) and MAO-B (lower panel) activities after irreversible enzyme inhibition in type 1 astrocytes. MAO activity of type 1 astrocytes was assayed immediately before (A), after a 2 h exposure to 10 - s M clorgyline and 10 -8 M deprenyl (B), and then 24 h later in the absence (C) or presence of 10 -8 M dexamethasone (D). Each column represents the mean+ S.E.M. of 3 sets of data from 3 separate astrocyte cultures. Statistical significance by two-way ANOVA with Tukey multiple range test: * * P < 0.01 vs dexamethasone-untreated cultures at the corresponding time.

activity and showed that glucocorticoid hormones acted by increasing the number of active M A O - A molecules. However, only in one study on cultured skin fibroblasts has a direct effect of dexamethasone on M A O - A m R N A been reported [46]. The apparent discrepancy between the above data and the present results might, at least in part, be explained by the fact that the two forms of MAO are heterogeneously distributed in various tissues [44,55], and it is conceivable that cells from different sources possess different gene activation patterns which may reflect different functional needs. Therefore, it is not surprising that the two isoforms of MAO show different responses to the same stimulus in different cellular systems. Indeed, following the cloning of cDNAs for M A O - A and -B [2,21,23,27], characterization of the promoter regions for the two human isoenzymes has shown a substantially different organization [58]. This has not only provided a basis for the different tissue- and cell-specific expression of the two MAO forms, but should also lead to a better understanding of different responses of M A O - A and -B genes to hormonal and microenvironmental stimulation. For example,

P. Carlo et al. / Brain Research 711 (1996) 175-183

the AP-1 binding site is found in the MAO-B, but not in the MAO-A, promoter, whereas cAMP response elementlike sequence is present in both promoters, but at different locations [58]. Glial cells have been shown to co-express type II glucocorticoid (GR) and to a minor extent the type I mineralocorticoid receptors (MR) [5,51]. Following the early observation that hydrocortisone induced glutamine synthetase in mouse primary astrocyte cultures [26], evidence is now rapidly accumulating on glucocorticoidsensitive gene products in astrocytes. In primary rat astrocyte cultures, the effects of corticosterone on transcriptional regulation of GFAP mRNA [41], as well on regulation of 10 hormone responsive mRNA species, including glutamine synthetase and glucocortin mRNAs [36], have been strongly suggested to be mediated through binding to the GR receptor. The induction of astrocyte MAO-B expression shown in this study also appears to be essentially due to GR activation, since dexamethasone, which binds preferentially to GR receptors, was active at relatively low concentrations. Moreover, the hormone effect was substantially decreased by RU 486, a potent antagonist which binds to the cytosolic GR receptor with even higher affinity than dexamethasone [25]. Further experiments will be needed to determine specific responses, if any, of astrocyte MAO to mineralocorticoid agonists. The knowledge provided here could be of value, especially because MAO-B is the predominant form of the enzyme in the human brain [15], being specifically localized in astrocytes and serotonergic neurons [28,44,49]. Moreover, the role of astrocyte MAO-B has received special attention, because of its possible involvement in neurodegenerative disorders, such as Alzheimer's [37,45], Huntington's [32] and Parkinson's [39] diseases. MAO-B has been reported to increase with age in the brain of both humans [40] and rats [14] and this increase is further enhanced in several regions of CNS in the above-mentioned pathologies. Interestingly, elevated urinary free cortisol concentrations have been found in patients with dementia of the Alzheimer type [31], and on the same line, high morning cortisol plasma levels have been observed in such patients [33]. The increase in MAO-B activity in ageing and even to a greater extent in neurodegenerative diseases is generally regarded as a result of a proliferative reaction of glial cells, rich in MAO-B, secondary to a neuronal loss. This degenerative process may be a consequence of an oxidative damage by overproduction of H 2 0 2 from the oxidative deamination catalyzed by MAO-B, without a concomitant increase of detoxifying processes [48]. On the basis of our observations, it is tempting to speculate that excessive circulating concentrations of glucocorticoids could contribute to the enhanced MAO-B activity observed in the brain, as well as in a peripheral tissue, such as blood platelets, of Alzheimer's patients [1,10,17].

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Acknowledgements We are indebted to Dr. D. Martini of Roussel Uclaf for providing us with the glucocorticoid receptor antagonist RU 38-486. We are grateful to Silvio Negro for his excellent secretarial help. This work was supported by grants from the Italian Ministry for University, Scientific and Technological Research and from the National Research Council (Progetto Finalizzato Invecchiamento to G.B.P. and Progetto Strategico Nucleotidi Antisenso to A.M.). M.O. was a recipient of a training grant from the European Economic Community (Biomed I Programme).

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