Astrocytes in culture express the full-length Trk-B receptor and respond to brain derived neurotrophic factor by changing intracellular calcium levels: effect of ethanol exposure in rats

Neuroscience Letters 288 (2000) 53±56

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Astrocytes in culture express the full-length Trk-B receptor and respond to brain derived neurotrophic factor by changing intracellular calcium levels: effect of ethanol exposure in rats Eva Climent a, MarõÂa Sancho-Tello b, Rosa MinÄana a, Domingo Barettino c, Consuelo Guerri a,* a

Instituto de Investigaciones CitoloÂgicas (FVIB), Amadeo de Saboya 4, 46010-Valencia, Spain b Department of Biologia Celular, University of Valencia, Valencia, Spain c Instituto de Biomedicina, CSIC, Jaume Roig 7, 46010-Valencia, Spain Received 21 February 2000; received in revised form 18 May 2000; accepted 19 May 2000

Abstract Although cultured astroglial cells were reported to express exclusively the truncated non-catalytic Trk B receptor for brain-derived neurotrophic factor (BDNF), we detect here, using a sensitive ribonuclease protection assay, mRNAs for both truncated (TrkB±T) and the full length catalytic (TrkB±¯) form of BDNF receptor in developing cortical astrocytes and neurons in culture. Cortical neurons and immature astroglia, such as radial glia and proliferating astrocytes, express both the protein and mRNAs for TrkB-¯ and TrkB-T, whereas the differentiation of astrocytes leads to a decrease in the trkB-¯ mRNA, being the truncated TrkB the predominant receptor in differentiating and con¯uent astrocytes. The levels of TrkB-¯ expression in proliferating and differentiating astrocytes and neurons correlates with the cell response to BDNF, monitored by the rise in intracellular [Ca 21]i. Foetal exposure to ethanol alters astroglial development and delays the reduction in trkB-¯ mRNA levels observed with differentiation of astrocytes. These results demonstrate that immature astrocytes are able to express the catalytic Trk B receptors and to respond to BDNF with the activation of conventional signal transduction pathways. The results suggest that this signalling pathway is more activated in ethanolexposed cells. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Astrocytes; Cell culture; Neurotrophins; Brain-derived neurotrophic factor; TrkB receptors; Prenatal ethanol exposure; Ethanol toxicity

Brain derived neurotrophic factor (BDNF) is a member of the neurotrophin family of growth factors regulating neuronal survival, outgrowth and differentiation in the developing nervous system [1]. In the mature nervous system, BDNF is also involved in speci®c aspects of activity-dependent synaptic plasticity [22] and appears to protect neurons against different types of brain insults [12]. Such BDNF actions appear to be mediated through the activation of signaling pathways coupled to the high-af®nity receptor TrkB, a receptor that exists in two alternatively spliced forms, one full-length tyrosine kinase-containing isoform (TrkB±¯) and the truncated tyrosine kinase-lacking isoform (TrkB±T) [1,15]. The role of TrkB-T may be a negative regulator of BDNF action by competing with TrkB-¯ [5]. * Corresponding author. Tel.: 134-96-339-12-53; fax: 134-96360-14-53. E-mail address: guerri@ochoa.®b.es (C. Guerri).

Both forms of TrkB receptors are widely distributed throughout the central nervous system (CNS) both during development and in the adult brain. TrkB-T increases with central nervous system (CNS) maturation and glial development [7]. Glial cells predominantly express TrkB-T [3,6,12,20], although TrkB-¯ is expressed in reactive astrocytes after chronic CNS insult [12] and is strongly up-regulated in the glial scar following CNS lesions [2,6]. The ability of reactive astrocytes to express TrkB-¯ raises the question of whether the astroglial proliferation observed after brain injury resembles that occurring during brain development. In the present study we show that normal proliferating cortical astrocytes in culture express both proteins and mRNAs for TrkB-¯ and TrkB-T, whereas differentiating astrocytes predominantly express the truncated isoform. Since we have shown in previous studies that ethanol exposure during development causes astroglial damage, we have also investigated whether this treatment

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E. Climent et al. / Neuroscience Letters 288 (2000) 53±56

affects the expression levels of trkB-¯ and trkB-T in astrocytes from foetuses of alcohol-fed rats. Primary cultures of astrocytes from 21-day-old foetuses obtained from control and alcohol-fed rats were prepared from brain hemispheres as previously described [18].The cultures were 98% pure as judged by immuno¯uorescence using anti-glial ®brillary acidic protein (GFAP). Neuronal cells (MAP±2 and neuro®lament ± 68 positive cells) were absent from these cultures [18]. Cultures of radial glia and neurons were carried out as previously described [21,10]. The cultures were 85±90% pure as judged by immuno¯uorescence using anti-Rat 401 (marker of radial glial) and anti MAP-2 and anti neuro®lament-68 (markers of neurons). For measurement of intracellular free Ca 21 concentration and immuno¯uorescence analysis, cells were plated on 16 mm glass coverslips. In order to determine the expression of full-length (¯) and truncated (T) TrkB receptor genes in astrocytes in culture,

Fig. 1. trkB mRNA levels in cultured cells. (A) Representative autoradiogram of RNase protection assay illustrating the levels of full-length (¯) and truncated (T) trkB mRNAs in primary culture of radial glia, astrocytes and neurons at different days after plating. (B) Histogram illustrating the relative levels of mRNAs encoding the TrkB-¯ and TrkB-T receptors obtained by densitometric analysis of the bands (Sigma Gel 1.0 image analysis software). The values obtained for trkB mRNA levels were normalized to cyclophilin levels. Control: untreated cells; PEE: prenatal ethanol exposed astrocytes. Each bar is an average of ®ve separate determinations from three different cultures (^SD). *P , 0:05 signi®cantly different from day 4. **P , 0:005 signi®cantly different from control group, as determined by two-way ANOVA test.

we used a sensitive ribonuclease protection assay (RPAII kit, Ambion, Austin, TX). The Trk B riboprobe is a HincIISca I 391-bp fragment from rat trkB c-DNA [15] and protects two fragments of 391 pb (trkB kinase) and 238 pb (trkB truncated) [11]. Cyclophilin probe, used as loading control, contains a 126-bp NcoI fragment from rat cyclophilin cDNA [4] (Ortiz and Barettino, unpublished). Using this procedure to determine trkB-¯ and trkB-T expression in astrocytes and in radial glia (the main astrocytic precursor cells) we were able to recognize and distinguish both fulllength and truncated trkB mRNAs in the same sample due to the different lengths of the protected mRNA fragments after RNase digestion. Indeed, radial glia and proliferating astrocytes (4 days of culture) [9] express both trkB-¯ and trkB-T mRNAs (Fig.1). However, as the astrocytes differentiate [9], the levels of trkB-¯ but not of trkB-T mRNA decrease, and the mRNA for trkB-T becomes predominant in differentiating and con¯uent (30 days, data not shown) astrocytes. Thus, the pattern of trkB mRNAs was also analyzed in cultured cortical neuronal cells to compare it with that obtained in astrocyte cultures. Fig. 1 shows that cortical neurons express both forms of trkB mRNA receptors, although densitometric analysis of the bands reveals that while the ratio trkB-T/ trkB-¯ mRNA is ,1 for neurons, it is ,1.3 and 3 in radial glia and in proliferating astrocytes, respectively (Fig.1B). We have also characterized the protein expression pattern of TrkB isoforms in astrocytes by Western blotting using a monoclonal antibody which recognized both full-length and truncated TrkB receptors (Transduction Laboratories). Western blot analysis of astrocytes at day 4 of culture revealed two immunoreactive bands, one of ,140 kDa and another of ,95 kDa, corresponding with the full-length and truncated TrkB receptors, respectively (Fig. 2) However, as astrocytes differentiate, the 140 kDa band progressively disappeared (day 7), being the truncated TrkB (95 kDa) the predominant form expressed in 14-day astrocytes, in agreement with the results of mRNA analyzed. Astrocytes from fetuses of chronic ethanol-fed rats (prenatally exposed to ethanol or PEE astrocytes) when cultured in the absence of ethanol, exhibited delayed proliferation and differentiation [8,18]. We presently show that in PEE astrocytes there is no decrease in trkB-¯ mRNA

Fig. 2. Western blot analysis of TrkB-¯ and TrkB-T in cortical astrocytes at 4, 7 and 14 days of culture. Each line contains 30 mg of total protein from the cell lysate. MW, molecular weight standard. As a control we use a cortical hemispheres homogenate from 5-day old rat (P5).

E. Climent et al. / Neuroscience Letters 288 (2000) 53±56

between days 4 and 14, and that the levels of trkB-¯ mRNA at day 14 are higher than in control astrocytes. Densitometric analysis of the autoradiographic bands revealed a decrease in the ratio trkB-T/trkB-¯ mRNA in PEE astrocytes (,2.4 and 3,at 4 and 14 days of culture) with respect to the ratio in control cells (,3 and 8, at 4 and 14 days). When control astrocytes were cultured in the presence of 50 mM ethanol, a reduction in trkB-T/trkB-¯ ratio was also observed (data not shown). To correlate trkB-¯ expression with BDNF response, we measured the changes in intracellular calcium concentration, [Ca 21]i, triggered by addition of BDNF (50 ng/ml). For this experiments, cells (astrocytes or neurons) were loaded for 1 h with 10 mM Fluo-3 AM (Molecular Probes) and changes in intracellular calcium concentration [Ca 21]i were monitored using confocal laser cytometry (ACAS570, Meridiam Instruments). We found that while 43% of the proliferating astrocytes (day 4) responded to BDNF by a rapid rise in intracellular calcium concentrations, in differentiating astrocytes (day 14) only 19% responded to this neurotrophin (Fig. 3). Furthermore, we observed a heterogeneity in the intensity of Ca 21 response to BDNF among the responding proliferating and differentiating astrocytes. Thus, while some cells display a low BDNF response (increasing their [Ca 21]i#1.5±fold above their basal levels), other cells have a higher response ([Ca 21]i . 1.5 above basal levels) (Fig. 3A,B). This heterogeneity in the response was also observed in cortical neurons in culture, where 76% of the cells responded to BDNF and 50% of them displayed a high intensity response. These results suggest that both the number of responding cells as well as the intensity of the response to BDNF are correlated with the expression of the catalytic form of the TrkB receptor among the different subpopulations of neurons and astrocytes. In line with this interpretation, the high trkB-¯ expression or lower trkB-T/ trkB-¯ ratio in PEE astrocyte coincides with a higher percentage of cells responding to BDNF (48 and 33% of astrocytes at 4 and 14 days of culture, respectively) than in control astrocytes (Fig. 3B). Numerous studies have demonstrated that astroglial cells synthesize and secrete neurotrophins (e.g. 23), but there have been few indications that these cells are also targets for neurotrophin actions, including BDNF, for which its catalytic full length TrkB receptor was reported not to be present in these cells [3,6,12,20]. However, our results show that immature astroglia (e.g. radial glia and growing astrocytes) expresses the catalytic TrkB-¯ receptor, and that this expression decreases with differentiation, whereas the expression of the truncated form of the receptor remains unabated in con¯uent astrocytes. The function of TrkB-T remains controversial, although this form of the receptor, when coexpressed with TrkB-¯, was found to act as a dominant negative receptor, inhibiting BDNF response [5]. In our studies the cells proved sensitive to BDNF, as shown by changes in cell calcium mobilization, and the response to BDNF appears to correlate with the level of the full-length

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receptor, both in astrocytes and in cortical neurons in culture. Thus, our ®ndings would appears to support the view that only the catalytic TrkB-¯ receptor is able to elicit BDNF responses in these cells, however we cannot exclude the possibility that TrkB-T receptor could be able to activate glial signal transduction [19]. In this respect, it is interesting that expression of TrkB-¯ has been observed in reactive astrocytes following brain injury [14] raising the question of whether glial cell proliferation following brain injury corresponds to the proliferation that occurs during develop-

Fig. 3. Calcium mobilization in cultured cells after BDNF addition. The ¯uorescent emission was analyzed at wavelengths of .515 nm. Basal Ca 21 levels was recorded every 20 s for 2±3 min before adding BDNF and recordings continued for 5±6 min thereafter. (A) Representative [Ca 21]i responses to BDNF (50 ng/ml) in two individual cells. Normalized Ca 21 levels are shown as a function of time. In cell 1 the rise of [Ca 21]i was .1.5-fold the basal level, while in cell 2 there was a 1.5 fold increase. (B) Histogram representing the percentage of cells responding to 50 ng/ml BDNF. Astrocytes and neurons at different days of culture, from control or prenatal exposure to alcohol (PEE) rats, were obtained as describe in the methods. Individual cells responding to BDNF showed a relative increase in their basal [Ca 21]i level of ,1.5 (low response) or .1.5-fold (high response) rise in [Ca 21]i. Approximately 80±100 cells of three different cultures were analyzed in each group. The response of astrocytes to BDNF is signi®cantly different (*P , 0:001 by two±way ANOVA) between day 4 and day 14 control astrocytes. The effect of PEE treatment on the BDNF response is considered signi®cantly different (**P , 0:001 by two±way ANOVA) from control group.

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ment and maturation of astrocytes in primary culture. In fact, growing and reactive astrocytes are able to synthesize and secrete neurotrophic factors [23] and to express speci®c cell adhesion molecules [17] in¯uencing neuronal survival and neurite outgrowth. The present ®ndings also show that in vivo ethanol exposure during fetal development, which affects astrogliogenesis (see review, [8]), delays the decrease of trkB-¯ mRNA levels in differentiating astrocytes, suggesting an immature state of the cells. These ®ndings are consistent with previous results indicating that ethanol exposure during fetal development alters the differentiation of radial glial cells and their transformation into astrocytes and affects astroglia maturation in vivo and in vitro [8]. In fact, in vivo ethanol exposure during brain development induces a wide array of adverse effects, including defects in cell proliferation, neuronal migration, cell loss in different brain regions and synaptic development [16]. Increasing evidence suggests that ethanol interferes with the production and action of neurotrophic factors [13,23], which are necessary for survival, growth and function of the developing neurons. Indeed, recent studies demonstrate that neurotrophic factors are able to afford protection against ethanol-induced cytotoxicity in different neuronal populations [13] and the present results indicate that astrocytes exposed to alcohol are able to respond more actively to BDNF-induced signaling pathways due to their immature state. These results suggest a potential bene®cial effect of BDNF against ethanol-induced toxicity in astrocytes and some preliminary results from our laboratory support this hypothesis.

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